Nucleic acid-based vaccines encoding escherichia coli fimH antigenic polypeptides

Abstract

The present disclosure relates to encoding RNA encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH. The present disclosure also relates to compositions and vaccines comprising the encoding RNA. Furthermore, the present disclosure relates to kits, in particular kits comprising the encoding RNA, or a composition, or a vaccine. The present disclosure also relates to methods for treating or preventing conditions caused by Escherichia coli.

Description

Sequence Listing

[0001] This application contains an ST.26 sequence listing in an XML file format submitted electronically (generated on 2023-05-19), which is incorporated herein by reference in its entirety. Other sequences shorter than 10 specifically defined nucleotides or shorter than 4 specifically defined amino acids are disclosed in Table 13. Technical Field

[0002] The present disclosure relates to RNA encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH. The present disclosure also relates to a pharmaceutical composition, a vaccine, a kit or a kit set suitable for the treatment and / or prevention of a disease, in particular a urinary tract infection (UTI). Background Art

[0003] Uropathogenic Escherichia coli (UPEC) is a subgroup of extraintestinal pathogenic Escherichia coli (ExPEC) that causes the vast majority of urinary tract infections (UTIs) and is the leading cause of bacteremia in adults and the second most common cause of neonatal meningitis. UTIs are usually treated with antibiotics, but the emergence of multidrug-resistant pathogens has highlighted the need for effective vaccines to prevent both uncomplicated and complicated UTIs (Flores-Mireles AL et al., Nat Rev Microbiol. 2015 May;13(5):269-84).

[0004] The tip-localized adhesin FimH (type 1 fimbrial D-mannose-specific adhesin) of type 1 pili allows ExPEC to colonize the bladder epithelium during UTI by binding to mannosylated receptors on the urothelial surface (Mulvey MA et al., Science. 1998 Nov 20;282(5393):1494-7).

[0005] Full-length FimH consists of two domains connected by a 5-amino acid linker: an N-terminal lectin domain (FimHL), which binds mannose on urothelial cell receptors, and a C-terminal pilin domain (FimHP). The pilin domain (FimHP) has an (Ig)-like fold, but lacks the seventh C-terminal β-strand. The absence of the strand creates deep grooves along the surface of FimHP and exposes its hydrophobic core, explaining the instability of FimH when expressed without a chaperone. In the chaperone-subunit complex, FimHP interacts non-covalently with the donor strand of the chaperone FimC in the periplasm or the donor strand of the subsequent subunit (FimG) of the assembled pilus, in a process called donor strand complementation or donor strand exchange, respectively, which simultaneously stabilizes the pilus subunit and closes its interaction surface.

[0006] The lectin domain (FimHL) is known to adopt two conformations with different mannose binding affinities - a high affinity conformation, also known as the relaxed (R) state, and a low affinity conformation, also known as the tense (T) state. The in vivo conformation of FimH is influenced by flow conditions, shear stress conditions are known to induce the high mannose binding conformation, and also depends, at least in part, on in vivo interactions with the FimH binding proteins FimG or FimC.

[0007] Antibodies that bind to FimH inhibit colonization and promote bacterial clearance by inhibiting bacterial adhesion to the urinary tract (Langermann S et al., Science. 1997 Apr 25; 276(5312): 607-11). In particular, monoclonal antibodies against the low-affinity conformation of FimHL have been shown to provide better inhibition of bladder adhesion (Tchesnokova et al., Infect Immun. 2011 Oct; 79(10): 3895-904). The exudation of serum IgG in the urogenital tract appears to be the reason for the inhibition of bacterial adhesion.

[0008] Therefore, FimH is considered a promising vaccine antigen. However, commercial-scale preparation of FimH is challenging because FimH needs to be prepared in sufficient quantities and in a conformation that can induce functional antibodies.

[0009] A clinical trial testing a four-dose regimen of FimH complexed with its molecular chaperone FimC (FimHC) and formulated with the adjuvant PHAD has been reported (Eldridge GR et al., Hum Vaccin Immunother. 2021 May 4; 17(5): 1262-1270). Although FimC appears to inhibit FimH degradation, providing the FimHC complex involves a significant manufacturing burden.

[0010] Alternative strategies for recombinant preparation of FimH in a functional conformation have also been reported, such as engineering of the mannose pocket (Kisiela DI et al., Proc Natl Acad Sci US A. 2013 Nov 19;110(47):19089-94), complexing FimH with a recombinant donor peptide of FimG (Sauer MM et al., Nat Commun. 2016 Mar 7;7:10738), or mammalian cell expression of FimH stabilized by a FimG donor peptide.

[0011] Therefore, there remains a need to overcome the challenges posed by recombinant preparation of FimH-based vaccines and to provide immunogenic compositions capable of inducing rapid and robust immune responses against ExPEC. Summary of the invention

[0012] In a first aspect of the invention, there is provided an encoding RNA comprising at least one untranslated region (UTR); and at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli ("E. coli", "Ec") type 1 fimbriae D-mannose specific adhesin (FimH). In one embodiment, the E. coli FimH comprises an amino acid sequence identical to or having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, or an immunogenic fragment or immunogenic variant thereof.

[0013] In some embodiments, the coding sequence additionally encodes one or more than one other peptide or protein element, and the one or more than one other peptide or protein element is selected from: donor chain peptide, signal peptide, antigen aggregation domain or transmembrane domain. In one embodiment, the other peptide or protein element is a donor chain peptide, and optionally the coding sequence encodes the following elements in the direction from N-terminus to C-terminus: an antigenic polypeptide selected from or derived from Escherichia coli FimH; and a donor chain peptide.

[0014] In one embodiment, the donor strand peptide comprises or consists of an amino acid sequence of SEQ ID NO: 338 or a variant thereof, optionally wherein the variant of SEQ ID NO: 338 has 1 to 5 single amino acid mutations, such as 1, 2, 3 or 4 single amino acid mutations compared to SEQ ID NO: 338. In one embodiment, the coding sequence additionally encodes a peptide linker element, and optionally the coding sequence encodes the following elements in the direction from the N-terminus to the C-terminus: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker element; and a donor strand peptide. In one embodiment, the peptide linker element comprises or consists of SEQ ID NO: 352.

[0015] In one embodiment, the antigenic peptide is in a low mannose binding affinity conformation.

[0016] In certain embodiments, the coding sequence additionally encodes an antigen aggregation domain, and optionally the antigen aggregation domain is selected from or derived from ferritin or lumazine synthase. In some other embodiments, the amino acid sequence of the antigen aggregation domain is identical to any one of the amino acid sequences SEQ ID NO: 457 to SEQ ID NO: 459, SEQ ID NO: 443, SEQ ID NO: 444 or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is a fragment or variant thereof.

[0017] In certain embodiments, the coding sequence additionally encodes a signal peptide, and optionally the signal peptide is immunoglobulin E (IgE) or immunoglobulin kappa (IgK), or is derived from IgE or Ig K. In some other embodiments, the amino acid sequence of the signal peptide is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NO: 394, SEQ ID NO: 395, or is a fragment or variant thereof.

[0018] In some embodiments, the coding sequence optionally encodes the following elements in the N-terminal to C-terminal direction: (a) signal peptide, antigenic polypeptide; (b) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide; (c) antigen aggregation domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide; (d) signal peptide, antigen aggregation domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide; (e) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, antigen aggregation domain; or (f) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, transmembrane domain.

[0019] In some embodiments, the coding sequence encodes an amino acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, SEQ ID NO: 498 to SEQ ID NO: 520, SEQ ID NO: 1277, or an immunogenic fragment or immunogenic variant thereof. In some embodiments, the coding sequence comprises a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 187 to SEQ ID NO: 246, SEQ ID NO: 257 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant thereof.

[0020] In some embodiments, the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methyl pseudouridine (m1ψ), optionally wherein substantially all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methyl pseudouridine (m1ψ) nucleotides. In some embodiments, the coding sequence is a codon-modified coding sequence, wherein the amino acid sequence encoded by at least one codon-modified coding sequence is optionally unmodified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence, optionally wherein at least one codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage-adaptive coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof.

[0021] In one embodiment, the coding RNA is an mRNA, optionally comprising or consisting of a sequence according to SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: SEQ ID NO: 1194 to SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1250, SEQ ID NO: 1251 to SEQ ID NO: 1252, SEQ ID NO: 1253 to SEQ ID NO: 1254, SEQ ID NO: 1255 to SEQ ID NO: 1256, SEQ ID NO: 1257 to SEQ ID NO: 1258 1276, or a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof.

[0022] In a second aspect, a pharmaceutical composition comprising the RNA encoding the present disclosure is provided. In some embodiments, the pharmaceutical composition further comprises a lipid-based carrier, wherein the lipid-based carrier is a lipid nanoparticle (LNP).

[0023] In a third aspect, a vaccine comprising the encoding RNA or pharmaceutical composition of the present disclosure is provided.

[0024] In a fourth aspect, a kit or kit set is provided, comprising the coding RNA, pharmaceutical composition and / or vaccine of the present disclosure, optionally comprising a liquid carrier for dissolution, and optionally, technical instructions providing information on administration and dosage of the components.

[0025] In other aspects, the coding RNA, pharmaceutical composition, vaccine or kit or kit set of the present disclosure is provided, which is used as a medicament. In one embodiment, the coding RNA, pharmaceutical composition, vaccine or kit or kit set of the present disclosure is used to treat or prevent one or more than one symptom associated with urinary tract infection (UTI) in a subject in need thereof.

[0026] In other aspects, the coding RNA, pharmaceutical composition, vaccine or kit or kit set of the present disclosure is provided, which is used as a medicament. In one embodiment, the coding RNA, pharmaceutical composition, vaccine or kit or kit set of the present disclosure is used to treat or prevent a disease caused by Escherichia coli.

[0027] In other aspects, a method of treating or preventing a disorder is provided, wherein the method comprises administering an effective amount of the encoding RNA, pharmaceutical composition, vaccine, or kit or kit set of the present disclosure to a subject in need thereof. In one embodiment, the method induces antibodies capable of inhibiting bacterial adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1: (A) (B): shows that mRNA constructs encoding different designs of E. coli FimH antigens are expressed and partially secreted by mammalian cells using Western blot analysis. The experiments were performed as described in Example 2.1.

[0029] Figure 2: (A) to (F) show that formulated mRNA constructs encoding different designs of E. coli FimH antigens induce humoral immune responses in mice. Serum and urine IgG titers were assessed by ELISA as described in Example 2.2.

[0030] Figure 3 : shows the CD4+ T cell responses and CD8+ T cell responses induced by vaccination of mice with mRNA constructs encoding different designs of E. coli FimH antigens as described in Example 2.4.

[0031] Figure 4: (A) to (C): shows the dose response of rats after vaccination to formulated mRNA constructs encoding different designs of E. coli FimH antigen. Serum and urine IgG titers were assessed by ELISA as described in Example 3.1.

[0032] Figure 5: (A) (B): shows that mRNA constructs encoding the E. coli FimH antigen containing uridine, ψ or m1ψ are expressed and partially secreted by mammalian cells using Western blot analysis. The experiments were performed as described in Example 4.1.

[0033] Figure 6: (A) to (C): shows that formulated mRNA constructs encoding E. coli FimH antigen design containing uridine, ψ or mlψ induce humoral immune responses in rats. Serum and urine IgG titers were assessed by ELISA as described in Example 2.2.

[0034] definition

[0035] For clarity and readability, the following definitions are provided. Any technical features mentioned in these definitions can be read in each embodiment of the present invention. Additional definitions and explanations can be specifically provided in the context of these embodiments.

[0036] Percentages in the context of numbers are to be understood as relating to the total of the respective item. In other cases, percentages are to be understood as percentages by weight (wt.-%), unless the context indicates otherwise.

[0037] about : The term "about" is used when the determinants or values ​​do not need to be exactly the same, i.e., do not need to be 100% identical. Thus, "about" means that the determinants or values ​​can deviate from 1% to 20%, for example, from 1% to 10%; in particular, from 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. It is known to those skilled in the art that, for example, certain parameters or determinants can be slightly changed based on the method of how the parameters are determined. For example, if certain determinants or values ​​are defined herein to have a length of, for example, "about 100 nucleotides", the length can deviate from 1% to 20%. Thus, it is known to those skilled in the art that in specific embodiments herein, the length can differ by 1 to 20 nucleotides. Thus, a length of "about 100 nucleotides" can cover a sequence of 80 to 120 nucleotides.

[0038] Adaptive immune response : The term "adaptive immune response" as used herein will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to an antigen-specific response of the immune system (adaptive immune system). Antigen specificity allows the generation of responses directed against specific pathogens or pathogen-infected cells. The ability to initiate these targeted responses is usually maintained in vivo by "memory cells" (B cells).

[0039] antigen : The term "antigen" as used herein will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to a substance that can be recognized by the immune system, for example, the adaptive immune system, and can, for example, trigger an antigen-specific immune response by forming antibodies and / or antigen-specific T cells that are part of an adaptive immune response. Typically, an antigen can be or can include a peptide or protein that can be presented to a T cell by MHC. Fragments, variants, and derivatives of a peptide or protein that comprises at least one epitope are also understood to be antigens.

[0040] Antigenic peptide, polypeptide or protein : The term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to a peptide, protein derived from a (antigenic or immunogenic) protein that stimulates the body's adaptive immune system to provide an adaptive immune response. Therefore, an antigenic / immunogenic peptide or protein comprises at least one (as defined herein) epitope or (as defined herein) antigen of the protein from which it is derived.

[0041] Cationic : Unless a different meaning is clear from the specific context, the term "cationic" means that the respective structure carries a positive charge, whether permanently or non-permanently, but in response to certain conditions such as pH. Therefore, the term "cationic" includes "permanently cationic" and "capable of being cationized". The term "permanently cationic" means, for example, that the respective compound or group or atom carries a positive charge at any pH value or hydrogen ion activity of its environment. Usually, the positive charge is generated by the presence of quaternary nitrogen atoms. When a compound carries many such positive charges, it can be called permanently polycationic.

[0042] Cationic: The term "cationizable" as used herein refers to a compound, group or atom that is positively charged at a lower pH value of its environment, and uncharged at a higher pH value of its environment. In addition, in a non-aqueous environment where the pH value cannot be determined, a compound, group or atom that is cationizable is positively charged at a high hydrogen ion concentration and uncharged at a low hydrogen ion concentration or low hydrogen ion activity. This depends on the individual properties of the compound that is cationizable or that is polycationizable, in particular the pKa of each cationizable group or atom, at which pH or hydrogen ion concentration the compound is charged or uncharged. In a dilute aqueous environment, the proportion of compounds, groups or atoms that are positively charged can be estimated using the so-called Henderson-Hasselbalch equation known to those skilled in the art. For example, in some embodiments, if the compound or moiety is capable of cationization, it is suitable that it is positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, such as 9 or below, 8 or below, 7 or below, such as at a physiological pH, such as about 7.3 to 7.4, i.e., under physiological conditions, especially the physiological salt conditions of cells in vivo. In other embodiments, it is suitable that the compound or moiety capable of cationization is mainly neutral at physiological pH, such as about 7.0 to 7.4, but becomes positively charged at lower pH values. In some embodiments, the pKa range of the compound or moiety capable of cationization is from about 5 to about 7.

[0043] Coding sequence / coding region : The term "coding sequence" or "coding region" and the corresponding abbreviation "cds" as used herein will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to a sequence of multiple nucleotide triplets that can be translated into a peptide or protein. A coding sequence in the context of the present disclosure can be an RNA sequence consisting of a number of nucleotides that can be divisible by three, which begins with a start codon, and which, for example, ends with a stop codon.

[0044] Derived from: The term "derived from" as used in the full text of this specification in the context of nucleic acids, i.e. for a nucleic acid "derived from" (another) nucleic acid, refers to a nucleic acid derived from (another) nucleic acid and the nucleic acid derived therefrom having, for example, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. It will be appreciated by those skilled in the art that the calculation of sequence identity is typically for nucleic acids of the same type, i.e. DNA sequences or RNA sequences. Therefore, it will be appreciated that if DNA is "derived from" RNA, or if RNA is "derived from" DNA, then in the first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by replacing the uracil (U) in the entire sequence with thymine (T)), or conversely, the DNA sequence is converted to the corresponding RNA sequence (particularly by replacing T in the entire sequence with U). Then, the sequence identity of the DNA sequence or the sequence identity of the RNA sequence is determined. For example, a nucleic acid "derived from" a nucleic acid also refers to a modified nucleic acid compared to the nucleic acid from which it is derived, for example to further increase RNA stability and / or to extend and / or promote protein production. In the context of amino acid sequences (e.g., antigenic peptides or proteins), the term "derived from" refers to an amino acid sequence derived from (another) amino acid sequence having, for example, at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence from which it is derived.

[0045] Donor peptide : The term "donor chain peptide" as used throughout this specification refers to the portion of the FimC polypeptide or FimG polypeptide that interacts with FimHP in vivo or in vitro and completes the atypical Ig fold of FimHP by occupying the groove and extending parallel to the C-terminal F chain of the subunit.

[0046] Snippet: The term "fragment" as used throughout this specification in the context of a nucleic acid sequence (e.g., RNA or DNA) or an amino acid sequence can generally be a shorter portion of a full-length sequence such as a nucleic acid sequence or an amino acid sequence. Therefore, a fragment is generally composed of a sequence identical to the corresponding segment in the full-length sequence. In the context of the present disclosure, a specific fragment of a sequence is composed of nucleotides or amino acids of a continuous segment of an entity, such as nucleotides or amino acids of a continuous segment of an entity in a molecule derived from the fragment, which represents at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the entire (i.e., full-length) molecule (e.g., a viral protein) derived from the fragment. The term "fragment" as used throughout this specification in the context of a protein or peptide can generally include a sequence of a protein or peptide as defined herein, wherein the sequence of the protein or peptide is truncated at its N-terminus and / or C-terminus compared to the amino acid sequence of the original protein with respect to its amino acid sequence. The term "fragment" as used throughout this specification in the context of an RNA sequence may generally include an RNA sequence whose 5' and / or 3' ends are truncated compared to a reference RNA sequence. Thus, such truncation may occur at the amino acid level or, respectively, at the nucleic acid level. Thus, the sequence identity of such a fragment as defined herein may, for example, refer to all proteins or peptides as defined herein or all (encoding) nucleic acid molecules of such a protein or peptide. A fragment of a protein or peptide may contain at least one epitope of these proteins or peptides.

[0047] (Sequence) identity: The term "identity" as used throughout this specification in the context of a nucleic acid sequence or an amino acid sequence will be recognized and understood by a person of ordinary skill in the art, and is, for example, intended to refer to the percentage of two sequences being identical. In order to determine the percentage of two sequences being identical, sequences can be compared to be compared with each other afterwards, the sequences being, for example, nucleic acid sequences or amino acid (aa) sequences as defined herein, such as aa sequences encoded by nucleic acid sequences as defined herein or aa sequences themselves. Therefore, for example, the position of the first sequence can be compared with the corresponding position of the second sequence. If a certain position in the first sequence is occupied by the same residue as a certain position in the second sequence, the two sequences are identical at that position. If this is not the case, the sequences are different at that position. If, compared to the first sequence, an insertion occurs in the second sequence, a gap can be inserted in the first sequence to allow further comparison. If, compared to the first sequence, a deletion occurs in the second sequence, a gap can be inserted in the second sequence to allow further comparison. Then, the percentage of two sequences being identical is a function of the number of identical positions divided by the total number of positions including those positions occupied only in one sequence. The percentage of two sequences being identical can be determined using an algorithm, for example, an algorithm integrated in the BLAST program. Sequence identity can be determined by using the EMBOSS Water sequence alignment tool of the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_water / , using gap open=12, gap extend=1 and for protein sequence matrix=BLOSUM62 or for DNA / RNA sequence matrix=fullDNA parameters, or by using the EMBOSS Needle sequence alignment tool of the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / , using default parameters (e.g. gap open=10, gap extend=0.5, end gap penalty=false, end gap open=10, end gap extend=0.5 and for protein sequence matrix=BLOSUM62 or for DNA / RNA sequence matrix=fullDNA). Unless otherwise stated, when the application refers to the sequence identity of a specific reference sequence, the identity is intended to refer to calculation over the full length of the reference sequence.

[0048] Immunogen, immunogenicity : The term "immunogen" or "immunogenicity" will be recognized and understood by those of ordinary skill in the art and is, for example, intended to refer to a compound capable of stimulating / inducing an (adaptive) immune response. An immunogen may be a peptide, polypeptide or protein.

[0049] Immune response The term "immune response" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to the specific response of the adaptive immune system to a particular antigen (the so-called specific immune response or adaptive immune response) or the non-specific response of the innate immune system (the so-called non-specific immune response or innate immune response), or a combination thereof.

[0050] Lipidoid : Lipidoids, also called lipidoid compounds, are lipid-like compounds, i.e. amphiphilic compounds having lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoid compounds.

[0051] Nucleic acid, nucleic acid molecule : The term "nucleic acid" or "nucleic acid molecule" as used herein will be recognized and understood by those of ordinary skill in the art. The term "nucleic acid" or "nucleic acid molecule" refers in particular to a DNA (molecule) or an RNA (molecule). The term is used synonymously with the term polynucleotide. For example, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid" or "nucleic acid molecule" also encompasses modified nucleic acids (molecules), such as base-modified, sugar-modified or backbone-modified DNA or RNA (molecules) as defined herein.

[0052] Nucleic acid sequence, DNA sequence, RNA sequence : The terms "nucleic acid sequence", "DNA sequence" and "RNA sequence" will be recognized and understood by those of ordinary skill in the art, and refer, for example, to the specific and individual arrangement order of their nucleotide sequences.

[0053] RNA species : In the context of the present disclosure, the term "RNA species" is not limited to representing a single molecule, but is understood to include a collection of substantially identical RNA molecules. Therefore, it may involve a plurality of substantially identical RNA molecules.

[0054] RNA: The term "RNA" is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer composed of nucleotide monomers. These nucleotides are typically adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or their analogs, which are linked to each other along a so-called backbone. The backbone is typically formed by phosphodiester bonds between the sugar, i.e., ribose, of a first monomer and the phosphate portion of a second adjacent monomer. The specific order of monomers, i.e., the order of bases linked to the sugar / phosphate backbone, is called the RNA sequence. Typically, RNA can be obtained by transcription of a DNA sequence, e.g., in a cell or in vitro. In the context of the present disclosure, RNA can be obtained by RNA in vitro transcription. Alternatively, RNA can be obtained by chemical synthesis.

[0055] RNA in vitro transcription : The term "RNA in vitro transcription" or "in vitro transcription" refers to the process of RNA synthesis in a cell-free system in vitro. RNA can be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which is typically a linear DNA template (e.g., linearized plasmid DNA or PCR product). The promoter used to control RNA in vitro transcription can be any promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or Syn5 RNA polymerase. In one embodiment of the invention, before RNA in vitro transcription is performed on the DNA template, the DNA template is linearized with a suitable restriction enzyme. Reagents commonly used in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6 or Syn5); ribonucleotide triphosphates (NTPs) of the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analog as defined herein; optionally, a modified nucleotide as defined herein; a DNA-dependent RNA polymerase (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase or Syn5 RNA polymerase) that is capable of binding to the promoter sequence within the DNA template; optionally, a ribonuclease (RNase) inhibitor for inactivating any potential contaminating RNases; optionally, pyrophosphatase; MgCl2; a buffer (TRIS or HEPES) for maintaining a suitable pH value, which can also contain an antioxidant (e.g., DTT) and / or a polyamine such as spermidine.

[0056] Variant (of a sequence): The term "variant" as used in the full text of this specification in the context of nucleotide sequence will be recognized and understood by those of ordinary skills, and is, for example, intended to refer to a variant of a nucleotide sequence derived from another nucleotide sequence. For example, compared with the nucleotide sequence derived from the variant, the variant of the nucleotide sequence can show the disappearance, insertion, addition and / or displacement of one or more nucleotides. The variant of the nucleotide sequence can have at least 50%, 60%, 70%, 80%, 90% or 95% identity with the nucleotide sequence of the variant. The variant is a functional variant, i.e., the variant retains at least 50%, 60%, 70%, 80%, 90% or 95% or more than 95% function of the sequence derived from it. The "variant" of the nucleotide sequence can have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity on the section of at least 10, 20, 30, 50, 75 or 100 nucleotides of the nucleotide sequence.

[0057] The term "variant" used in the context of proteins or peptides of this specification is, for example, intended to refer to a protein variant or peptide variant having an amino acid sequence that is different from the original sequence in one or more than one mutation / displacement, such as one or more than one displacement, insertion and / or deletion of the amino acid. Suitably, these fragments and / or variants have the same or comparable specific antigenicity (immunogenic variant, antigenic variant). Insertion and displacement are possible, particularly in those sequence positions that do not cause the three-dimensional structure to change or do not affect the binding region. The modification of the three-dimensional structure by insertion or deletion can be easily determined, for example, using CD spectra (circular dichroism spectra) to determine. A "variant" of a protein or peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity in a segment of at least 10, 20, 30, 50, 75 or 100 amino acids of the protein or peptide. Alternatively, a "variant" of a protein or polypeptide may have from 1 to 20 single amino acid mutations, such as from 1 to 10 single amino acid mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19 or 20 single amino acid mutations compared to the protein or peptide. By mutation we mean or include substitutions, insertions or deletions. In one embodiment, a variant of a protein comprises a functional variant of the protein, which in the context of the present disclosure means that the variant exerts substantially the same or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity of the protein from which it is derived. DETAILED DESCRIPTION

[0058] When referring to the "SEQ ID NO" of other patent applications or patents, the sequences, such as amino acid sequences or nucleic acid sequences, are expressly incorporated herein by reference. For the "SEQ ID NO" provided herein, the information provided under the "feature key", i.e., "source" (for nucleic acids or proteins) or "misc_feature" (for nucleic acids) or "REGION" (for proteins) (in the sequence listing according to the WIPO ST.26 standard) is also expressly included in its entirety herein. When "SEQ ID NO" is mentioned in the context of an RNA sequence, a person skilled in the art will understand and be able to obtain the RNA sequence from the mentioned SEQ ID NO, as well as in the case of providing a DNA sequence. When "SEQ ID NO" is mentioned in the context of a DNA sequence, a person skilled in the art will understand and be able to obtain the respective DNA sequence from the mentioned SEQ ID NO, as well as in the case of providing an RNA sequence.

[0059] The inventors have overcome the challenge of producing recombinant polypeptides of E. coli by administering RNA vaccines encoding antigenic polypeptides that are E. coli FimH or derived from E. coli FimH. The inventors have also overcome the challenge of eliciting a rapid and robust immune response against E. coli FimH.

[0060] 1: RNA encoding Escherichia coli antigenic polypeptide

[0061] In a first aspect, there is provided an encoding RNA comprising at least one untranslated region (UTR); and at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli type 1 fimbriae D-mannose-specific adhesin (FimH).

[0062] It must be noted that the specific features and embodiments described in the context of the first aspect of the disclosure, i.e., the RNA of the disclosure, are equally applicable to the second aspect (the composition of the disclosure), the third aspect (the vaccine of the disclosure), the fourth aspect (the kit or kit set of the disclosure), or other aspects including medical uses and methods of treatment.

[0063] The term "coding RNA" as used herein will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to an RNA comprising a coding sequence ("cds"), wherein the cds comprises a plurality of nucleotide triplets, wherein the cds can be translated into a peptide or protein (e.g., when administered to a cell or organism).

[0064] The Escherichia coli FimH disclosed in the present invention can be selected from or derived from any strain of Escherichia coli, such as any strain of Escherichia coli J96, Escherichia coli 536, Escherichia coli CFT073, Escherichia coli UMN026, Escherichia coli CLONE D i14, Escherichia coli CLONE D i2, Escherichia coli IA139, Escherichia coli NA114, Escherichia coli IHE3034, Escherichia coli 789, Escherichia coli F11 and Escherichia coli UTI89.

[0065] In one embodiment, the E. coli FimH of the present disclosure comprises or consists of or is derived from an amino acid sequence of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256. In one embodiment, the E. coli FimH comprises an amino acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256. In one embodiment, the E. coli FimH is an immunogenic fragment or immunogenic variant of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256. In one embodiment, the E. coli FimH comprises an amino acid sequence that is identical, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 177. In one embodiment, the E. coli FimH comprises an amino acid sequence that is identical, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 247.

[0066] In some embodiments, the glycosylation site in the encoded amino acid sequence is mutated / replaced, which refers to the encoded amino acid that can be glycosylated, for example, the encoded amino acid that can be glycosylated after the encoding RNA is translated when administered in vivo is replaced with a different amino acid. Therefore, at the nucleic acid level, the codon encoding a known amino acid or an amino acid predicted to be N-glycosylated or O-glycosylated is replaced by an amino acid that is not glycosylated or not easily glycosylated, for example, by serine (S, Ser), aspartic acid (D, Asp), alanine (A, Ala) or glutamine (Q, Gln).

[0067] N-glycosylation and / or O-glycosylation may be determined using any suitable method known in the art, for example using the NetNGlyc 1.0 and NetOGlyc 4.0 servers (available at https: / / services.healthtech.dtu.dk / service.php?NetOGlyc-4.0 and https: / / services.healthtech.dtu.dk / service.php?NetOGlyc-4.0) using default settings.

[0068] In one embodiment, the antigenic polypeptide does not comprise (or is modified to not comprise) a glycosylation site at one or more positions selected from position 28, position 91, position 228, position 249 and position 256 relative to SEQ ID NO: 177 or at positions of SEQ ID NO: 178 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256 corresponding to those positions of SEQ ID NO: 177. In one embodiment, the polypeptide comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 177: N28S, N91D, N249D, N256D, or at positions of SEQ ID NO: 178 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256 corresponding to those positions of SEQ ID NO: 177, such as one, two, three or four of those amino acid substitutions.

[0069] In various embodiments, the antigenic polypeptide comprises at least one amino acid substitution or mutation to lock the FimH lectin domain into a low mannose binding affinity conformation. In some embodiments in this context, the antigenic polypeptide comprises an amino acid selected from valine (V, Val), isoleucine (I, Ile), leucine (L, Leu), glycine (G, Gly), methionine (M, Met) and alanine (A, Ala) at a position corresponding to position 165 of SEQ ID NO: 177, or at a position corresponding to the position of SEQ ID NO: 178 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256 of SEQ ID NO: 177. In one embodiment, the polypeptide comprises an F165V substitution of SEQ ID NO: 177 or an F165V substitution at a position of SEQ ID NO: 178 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256 corresponding to that position of SEQ ID NO: 177. Such mutations locking the FimH lectin domain into a low mannose binding affinity conformation have been reported in WO2021144369, the contents of which are incorporated herein by reference.

[0070] In some embodiments, the encoded FimH comprises one or more amino acid replacements selected from the group consisting of N28S, V48C, L55C, N91S, N249Q, N256D, F165V of SEQ ID NO: 177, or amino acid replacements at positions corresponding to these positions of SEQ ID NO: 177 in SEQ ID NO: 178 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256; for example, the encoded FimH comprises amino acid replacements of N28S, N91S, N249Q; N28S, N91S, N249Q, N256D; N28S, V48C, L55C, N91S, N249Q, or F165V of SEQ ID NO: 177, or amino acid replacements at positions corresponding to these positions of SEQ ID NO: 178 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: Amino acid substitutions at positions in 256 corresponding to these positions in SEQ ID NO: 177.

[0071] In some embodiments, the encoded FimH comprises one or more than one at a position selected from F1, P12, G14, G15, G16, A18, P26, V27, V28, Q32, N33, L34, V35, R60, S62, Y64, G65, L68, F71, T86, L107, Y108, L109, V112, S113, A115, G116, V118, A119, A127, L129, Q133, F144, V154, V155, V156, P157, T158, V163 of SEQ ID NO: 247 or at a position corresponding to SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 248 to SEQ ID NO: 256. Amino acid substitutions at these positions of 247.

[0072] In some embodiments, the encoded FimH comprises one or more amino acid substitutions selected from the group consisting of F1I; F1L; F1V; F1M; F1Y; F1W; P12C; G14C; G15A; G15P; G16A; G16P; A18C; P26C; V27A; V27C; V28C; Q32C; N33C; L34C; L34N; L34S; L34T; L34D; L34E; L34K; L34R; V35C; R60P; S62C; Y64C; G65A; L68C; F71C; 186, SEQ ID NO: 248 to SEQ ID NO: 256, or amino acid replacements at positions corresponding to these positions of SEQ ID NO: 247, or any combination thereof.

[0073] In some embodiments, the encoded FimH comprises an amino acid replacement selected from the group consisting of G15A and G16A; P12C and A18C; G14C and F144C; P26C and V35C; P26C and V154C; P26C and V156C; V27C and L34C; V28C and N33C; V28C and P157C; Q32C and Y108C; N33C and L 109C; N33C and P157C; V35C and L107C; V35C and L 109C; S62C and T86C; S62C and L 129C; Y64C and L68C; Y64C and A 127C; L68C and F71C; V112C and T158C; S113C and G116C; S113C and T158C; V118C and V156C; A119C and V155C; L34N and V27A; L34S and V27A; L34T and V27A; L34D and V27A; L34E and V27A; L34K and V27A; L34R and V27A; A119N and V27A; A119S and V 27A; A119T and V27A; A119D and V27A; A119E and V27A; A119K and V27A; A119R and V27A; G15A and V27A; G16A and V27A; G15P and V27A; G16P and V27A; G15A, G16A and V27A; G65A and V27A; V27A and Q133K; and G15A, G16A, V27A and Q133K, or amino acid replacements at positions corresponding to these positions of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 248 to SEQ ID NO: 256.

[0074] In one embodiment, the encoded FimH comprises the amino acid substitutions G15A, G16A and V27A of SEQ ID NO: 247, or the amino acid substitutions G15A, G16A and V27A at positions corresponding to these positions of SEQ ID NO: 247 of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 248 to SEQ ID NO: 256.

[0075] Design of specific antigens

[0076] According to various embodiments, the coding sequence of the RNA encodes an antigenic polypeptide selected from or derived from E. coli FimH as defined herein, and one or more than one other peptide or protein element. In some embodiments, one or more than one other peptide or protein element is heterologous.

[0077] Suitably, other peptide or protein elements can stabilize FimH subunits and / or shield their interaction surfaces (e.g., via donor chain peptides). In addition, other peptide or protein elements can promote secretion of the encoded antigenic peptide or protein of the present disclosure (e.g., via a secretory signal sequence). In addition, other peptide or protein elements can promote anchoring of the encoded antigenic peptide or protein of the present disclosure in the plasma membrane (e.g., via a transmembrane element), or promote the formation of antigenic complexes (e.g., via a multimerization domain or an antigen aggregation domain).

[0078] Suitably, the coding sequence additionally encodes one or more than one peptide or protein element selected from a donor chain peptide, a signal peptide, an auxiliary epitope, an antigen aggregation domain or a transmembrane domain. In some embodiments, the coding sequence encodes one or more than one other peptide or protein element and a peptide linker.

[0079] Donor peptide

[0080] In some embodiments, the encoding RNA encodes an antigenic protein selected from or derived from E. coli FimH and additionally encodes a donor strand peptide.

[0081] In some embodiments, the coding sequence encodes the following elements in the N-terminal to C-terminal direction: an antigenic polypeptide selected from or derived from E. coli FimH; and a donor strand peptide.

[0082] In one embodiment, the donor strand peptide comprises or consists of an amino acid sequence of SEQ ID NO: 338 or SEQ ID NO: 339 or a variant thereof. In one embodiment, the variant of SEQ ID NO: 338 or SEQ ID NO: 339 has 1 to 5 single amino acid mutations, such as 1, 2, 3 or 4 single amino acid mutations, compared to SEQ ID NO: 338 or SEQ ID NO: 339.

[0083] In one embodiment, the donor strand peptide comprises or consists of SEQ ID NO: 338. Particularly suitable in the context of the present disclosure is that the donor strand peptide comprises or consists of SEQ ID NO: 338, such that the polypeptide of the present disclosure is in a low mannose binding affinity conformation.

[0084] Peptide linker

[0085] In protein constructs consisting of several elements, the protein elements are often separated by peptide linkers, which can be beneficial because they allow the correct folding of the single elements and thus the correct function of each element.

[0086] When used in the context of the present disclosure, such a linker is particularly useful when encoded by a nucleic acid encoding at least two protein elements, for example, by a nucleic acid encoding an antigenic polypeptide and at least one other peptide or protein element. In this case, the linker is usually located on the polypeptide chain between the target polypeptide and at least one other protein element. When the coding sequence encodes more than one other peptide or protein element, the linker can be suitably located between each other peptide or protein element. At the nucleic acid level, the coding sequence of such a linker is usually located in the reading frame, 5' or 3' of the coding sequence of the target polypeptide or target protein, or between the coding regions of the individual peptide or protein elements.

[0087] In one embodiment, the peptide linker comprises or consists of 2 to 20 amino acids, 4 to 15 amino acids, or 5 to 10 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. The peptide linker is, for example, composed of small non-polar amino acids (e.g., glycine) or polar amino acids (e.g., serine or threonine). The small size of these amino acids provides flexibility and allows the activity of the connected functional domains, as described by Chen et al. (Adv Drug Deliv Reb. 2013; 65(10): 1357-1369). Incorporation of serine (S, Ser) or threonine (T, Thr) can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing the interaction between the linker and the protein portion. Rigid linkers usually maintain the distance between protein domains and they can be based on helical structures and / or they have proline-rich sequences.

[0088] The typical sequence of a flexible linker is composed of repeated amino acid glycine (G, Gly) and serine (S, Ser). For example, the linker can have the following sequence: GS, GSG, SGG, SG, GGS, SGS, GSS, SSG. In some embodiments, the same sequence is repeated multiple times (e.g., twice, three times, four times, five times, or six times) to produce a longer linker. In other embodiments, a single amino acid residue such as S or G can be used as a linker.

[0089] At the nucleic acid level, particularly at the RNA level, any nucleotide sequence portion encoding any joint used in the disclosure can be used. Due to the degeneracy of the genetic code, for most of the polypeptides of SEQ ID NO: 352 to SEQ ID NO: 358, it is conceivable that more than one specific nucleotide sequence encodes a respective polypeptide list. Although each such nucleic acid can be used generally in the context of the disclosure, it is suitable to select the nucleic acid sequence of the coded polypeptide sequence to be codon-optimized according to the general guidance provided in this specification sheet.

[0090] In some embodiments, the coding sequence encodes an antigenic protein selected from or derived from E. coli FimH, a donor strand peptide, and a peptide linker.

[0091] In one embodiment, the coding sequence encodes the following elements in the N-terminal to C-terminal direction: an antigenic polypeptide selected from or derived from E. coli FimH; a peptide linker; and a donor strand peptide.

[0092] When the coding sequence encodes the following elements in the direction from N-terminus to C-terminus: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker; and a donor chain peptide, it is particularly suitable that the peptide linker comprises or consists of:

[0093] (i) PGDGN [SEQ ID NO: 352], or a variant or fusion thereof, or

[0094] (ii) GGGGSGG [SEQ ID NO: 353], or a variant or fusion thereof, or

[0095] (iii) GGGGSGGGGSGGGGS [SEQ ID NO:354], or a variant or fusion thereof, or

[0096] (iv) SGG [SEQ ID NO: 355], or a variant or fusion thereof, or

[0097] (v) SGGM [SEQ ID NO: 356], or a variant or fusion thereof, or

[0098] (VI) GGSGGSGGSGGSGGG [SEQ ID NO:357], or a variant or fusion thereof, or

[0099] (vii) GGSGGSGGSGGS [SEQ ID NO: 358], or a variant or fusion thereof.

[0100] In one embodiment, the peptide linker is a variant of any one of SEQ ID NO: 352 to SEQ ID NO: 358, optionally wherein the variant has 1 to 5 single amino acid mutations, such as 1, 2, 3 or 4 single amino acid mutations compared to SEQ ID NO: 352 to SEQ ID NO: 358.

[0101] In one embodiment, the peptide linker comprises or consists of SEQ ID NO: 352. It may be particularly suitable in the context of the present disclosure that the peptide linker positioned between the antigenic polypeptide and the donor strand peptide comprises or consists of SEQ ID NO: 352, such that the polypeptide of the present disclosure is locked into a low mannose binding affinity conformation.

[0102] Conformation of antigenic peptides

[0103] In one embodiment, the antigenic polypeptide selected from or derived from E. coli FimH is in a low mannose binding affinity conformation or a tense (T) state, e.g., having a K of about 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM or 1 mM. d In one embodiment, the mannose binding affinity is K d ≈300 µM or above (i.e., no detectable mannose binding affinity), as disclosed in Kisiela DI et al., Proc Natl Acad Sci US A. 2013 Nov 19;110(47):19089-94 and Sauer MM et al., J Am Chem Soc. 2019 Jan 16;141(2):936-944. It is known in the art that the high mannose binding affinity conformation or relaxed (R) state of FimH corresponds to, for example, K d Mannose binding affinity <1.2µM.

[0104] Mannose binding can be determined using any suitable method known in the art, for example, surface plasmon resonance can be used to verify the binding, binding specificity and binding constant of FimH constructs to Man-BSA and Glc-BSA (negative control), see, for example, Rabbani S et al., J Biol Chem. 2018 Feb 2;293(5):1835-1849, which is incorporated herein by reference.

[0105] The conformation of FimH can also be evaluated by using any suitable method known in the art, such as surface plasmon resonance, to detect the binding of conformational antibodies. Exemplary antibodies can recognize epitopes that cover the mannose binding pocket of FimH to varying degrees, such as antibodies that bind to epitopes that cover the mannose binding pocket, such as epitopes that are limited to a loop of the mannose binding pocket. Exemplary antibodies are antibodies disclosed in WO2016183501 or in Kisiela DI et al., Proc Natl Acad Sci US A. 2013 Nov 19; 110(47): 19089-94, Kisiela DI et al., PLoS Pathog. 2015 May 14; 11(5): e1004857, which are incorporated herein by reference. In one embodiment, the conformational antibody has a variable heavy chain (VH) sequence of SEQ ID NO: 173 and a variable light chain (VL) sequence of SEQ ID NO: 174. In one embodiment, the conformational antibody has a variable heavy chain (VH) sequence of SEQ ID NO: 175 and a variable light chain (VL) sequence of SEQ ID NO: 176.

[0106] Signal peptide

[0107] In some embodiments, the coding sequences of the present disclosure encode at least one antigenic polypeptide selected from or derived from E. coli FimH, and additionally encode a signal peptide.

[0108] Suitably, the signal peptide is selected from or derived from FimH, FimC, immunoglobulin kappa (IgK), immunoglobulin E (IgE), tissue plasminogen activator (TPA or HsPLAT), or human serum albumin (HSA or HsALB), or MHC class I lymphocyte antigen (HLA-A2).

[0109] In some embodiments, the signal peptide is selected from or derived from IgE, IgK, FimH, FimC, TPA, HSA or HLA-A2, wherein the amino acid sequence of the signal peptide is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NO: 394 to SEQ ID NO: 400, or is a fragment or variant of any of these sequences.

[0110] In some embodiments, the signal peptide is heterologous. In some embodiments, the signal peptide is selected from or derived from IgE or IgK, wherein the amino acid sequence of the signal peptide is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NO: 394, SEQ ID NO: 395, or is a fragment or variant of any of these sequences.

[0111] In the embodiment in which the coding sequence of the present disclosure additionally encodes a signal peptide, it is particularly suitable for producing a fusion protein comprising an N-terminal signal peptide and a C-terminal peptide or protein selected from or derived from Escherichia coli FimH, optionally comprising a peptide linker and a donor chain peptide, wherein the C-terminal peptide or protein selected from or derived from Escherichia coli FimH is, for example, a peptide or protein lacking an endogenous N-terminal secretion signal peptide, such as SEQ ID NO: 247 to SEQ ID NO: 256 and SEQ ID NO: 1277.

[0112] Constructs comprising an N-terminal signal peptide can ideally improve the secretion of E. coli FimH (which is encoded by the encoding RNA of the first aspect). Therefore, when the encoding RNA of the first aspect is administered, the improved secretion of E. coli FimH can be beneficial for inducing a humoral immune response against the encoded E. coli FimH antigenic protein.

[0113] Other suitable signal peptides can be selected from the amino acid sequence listing of SEQ ID NO: 1 to SEQ ID NO: 1115 and SEQ ID NO: 1728 according to the published PCT patent application WO2017081082, which is incorporated herein by reference, or fragments or variants of these sequences, wherein the secretory signal peptide is fused at the N-terminus to an antigenic polypeptide lacking an endogenous secretory signal sequence selected from or derived from Escherichia coli FimH or an immunogenic fragment or immunogenic variant thereof.

[0114] Suitable examples of constructs comprising an N-terminal signal sequence are SEQ ID NO: 498 to SEQ ID NO: 520. The corresponding nucleic acid sequence for each of the constructs listed above can be found in Table 1.

[0115] Antigen aggregation domain or multimerization domain

[0116] In various embodiments, the coding sequences of the present disclosure encode an antigenic polypeptide selected from or derived from E. coli FimH and additionally encode an antigenic aggregation domain or a multimerization domain.

[0117] Suitably, the antigen aggregation domain (multimerization domain) is selected from or derived from ferritin or leucovorin synthase (LS, LumSynth).

[0118] In the embodiment in which the coding sequence of the present disclosure additionally encodes an antigen aggregation domain, it is particularly suitable for generating a fusion protein comprising an antigenic polypeptide selected from or derived from Escherichia coli FimH, which optionally comprises a donor chain peptide and a (first) peptide linker, and which further comprises an antigen aggregation domain and an optional (second) peptide linker. Constructs comprising antigen aggregation domains can enhance antigen aggregation and thus promote immune responses, for example, by promoting immune responses through multiple binding events occurring simultaneously between aggregated antigens and host cell receptors (see Lopez-Sagaseta, Jacinto et al., "Self-assembling protein nanoparticles in the design of vaccines". Computational and structural biotechnology journal 14 (2016): 58-68). In addition, such constructs may additionally comprise an N-terminal signal sequence (as defined above).

[0119] LumSynth (LS) is an enzyme with the property of forming particles, widely present in a variety of organisms, and involved in riboflavin biosynthesis. Jardine et al. reported that they attempted to optimize candidate vaccines by introducing LumSynth (LS) to enhance the immunoreactivity of recombinant gp120 against HIV infection (Jardine, Joseph et al., "Rational HIV immunogen design to target specific germline B cellreceptors". Science 340.6133 (2013): 711-716). Constructs containing LumSynth allow the formation of multimeric nanoparticles, particularly 60-mer nanoparticles, displaying antigenic polypeptides, to optimize B cell activation.

[0120] In some embodiments, dioxotetrahydropterin synthase is used to promote antigen aggregation, and thus can promote immune responses to coding sequences encoding E. coli FimH antigens.

[0121] In some embodiments, the antigen aggregation domain (multimerization domain) is selected from or derived from dioxotetrahydropterin synthase (LS, LumSynth), wherein the amino acid sequence of the antigen aggregation domain is identical to any one of the amino acid sequences (SEQ ID NO: 443, SEQ ID NO: 444) or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or is a fragment or variant of any of these amino acid sequences.

[0122] Ferritin is a protein whose main function is to store iron in cells. Almost all living organisms produce ferritin, which is composed of 24 subunits self-assembled in a quaternary structure with octahedral symmetry. Its self-assembly into nanoparticles is very suitable for carrying and exposing antigens.

[0123] In some embodiments, ferritin is used to promote antigen aggregation and thus can promote immune responses to RNA encoding the E. coli FimH antigen.

[0124] In some embodiments, the antigen aggregation domain (multimerization domain) is ferritin or is derived from ferritin, wherein the amino acid sequence of the antigen aggregation domain is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences (SEQ ID NO: 457 to SEQ ID NO: 459), or is a fragment or variant thereof.

[0125] In some embodiments, the coding sequence encodes the following elements in the N-terminal to C-terminal direction: an optional signal peptide, an antigenic aggregation domain selected from or derived from dioxotetrahydropterin synthase or ferritin as defined herein, a (second) peptide linker, and an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally further comprising a (first) peptide linker and a donor chain peptide as defined herein.

[0126] In some alternative embodiments, the coding sequence encodes the following elements in the N-terminal to C-terminal direction: an optional signal peptide, an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally comprising a (first) peptide linker and a donor chain peptide, a (second) peptide linker, and an antigenic aggregation domain as defined herein that is appropriately selected from or derived from dioxotetrahydropterin synthase or ferritin.

[0127] In one embodiment, the (first) peptide linker comprises or consists of any one of SEQ ID NO: 352 to SEQ ID NO: 354 or a variant thereof, optionally wherein the variant has 1 to 5 single amino acid mutations, such as 1, 2, 3 or 4 single amino acid mutations compared to SEQ ID NO: 352 to SEQ ID NO: 354. In one embodiment, the (first) peptide linker comprises or consists of SEQ ID NO: 352. In one embodiment, the (second) peptide linker comprises or consists of any one of SEQ ID NO: 355 to SEQ ID NO: 358 or a variant thereof, optionally wherein the variant has 1 to 5 single amino acid mutations, such as 1, 2, 3 or 4 single amino acid mutations compared to SEQ ID NO: 355 to SEQ ID NO: 358. In one embodiment, the (second) peptide linker comprises or consists of SEQ ID NO: 355.

[0128] Suitable examples of constructs comprising heterologous antigen aggregation domains are SEQ ID NO: 507 to SEQ ID NO: 510, SEQ ID NO: 512 to SEQ ID NO: 520. The corresponding nucleic acid sequences for each of the constructs listed above can be found in Table 1.

[0129] In various embodiments, the coding sequence of the present disclosure encodes an antigenic polypeptide selected from or derived from E. coli FimH, and additionally encodes a transmembrane domain. In one embodiment, the transmembrane domain is heterologous. The heterologous transmembrane domain promotes membrane anchoring of the encoded E. coli FimH antigenic polypeptide, and thereby can enhance the immune response (particularly the cellular immune response).

[0130] Suitably, the transmembrane domain is or is derived from an influenza HA transmembrane domain, e.g. derived from influenza A HA H1N1, more particularly e.g. derived from H1N1 / A / Netherlands / 602 / 2009, HA, aa521 to aa566, GenBank Accession No.: ACQ45338.1, (SEQ ID NO: 478).

[0131] Other suitable transmembrane domains are derived from human immunodeficiency virus 1, Env, aa19 to aa35, BAF32550.1, AB253679.1; human immunodeficiency virus 1, Env, aa515 to aa536, BAF32550.1, AB253679.1; human immunodeficiency virus 1, Env, aa680 to aa702, BAF32550.1, AB253679.1; equine infectious anemia virus, Env, aa450 to aa472, AAC03762.1, AF016316.1; equine infectious anemia virus, Env, aa614 to aa636, AAC03762.1, AF016316.1; equine infectious anemia virus, Env, aa79 8 to aa819, AAC03762.1, AF016316.1; murine leukemia virus, Env, aa601 to aa623, AAA46526.1, M93052.1; mouse mammary tumor virus, Env, aa457 to aa479, BAA03768.1, D16249.1; mouse mammary tumor virus, Env, aa624 to aa646, NP_056883.1, NC_001503.1; vesicular stomatitis virus, G, aa477 to aa499, CAA24525.1, V01214.1; rabies virus, G, aa460 to aa479, AEV43288.1, JN234423.1 (Env: envelope glycoprotein; G: glycoprotein).

[0132] In the embodiment in which the coding sequence of the present disclosure additionally encodes a heterologous transmembrane domain, it is particularly suitable for generating a fusion protein comprising an N-terminal peptide or a protein comprising an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally comprising a (first) peptide linker between a donor chain peptide and an antigenic polypeptide and a donor chain peptide (as defined above); and a C-terminal heterologous transmembrane domain, and optionally a (second) peptide linker between the N-terminal peptide and the C-terminal peptide (as defined above). Constructs comprising heterologous transmembrane domains can promote membrane anchoring of antigens, and thus can promote immune responses, particularly cellular immune responses, of RNA encoding antigenic polypeptides. In addition, such constructs can additionally comprise an N-terminal secretion signal sequence (as defined above). Alternatively, the transmembrane domain can be located at the N-terminus.

[0133] Other transmembrane elements / domains can be selected from the amino acid sequence listing of SEQ ID NO: 1228 to SEQ ID NO: 1343 according to patent application WO2017081082, or fragments or variants thereof, WO2017081082 is incorporated herein by reference.

[0134] In some embodiments, the coding sequence encodes the following elements in the N-terminal to C-terminal direction: a secretory signal peptide, an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally comprising a (first) peptide linker and a donor chain peptide, a (second) peptide linker and a heterologous transmembrane domain.

[0135] A suitable example of a construct comprising a heterologous transmembrane element is SEQ ID NO: 511. The corresponding nucleic acid sequence of the construct can be found in Table 1.

[0136] In various embodiments of the invention, the coding sequence encodes the following elements, for example, in the N-terminal to C-terminal direction:

[0137] a) a signal peptide, an antigenic polypeptide as defined herein;

[0138] b) a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, a donor peptide;

[0139] c) an antigen aggregating domain, a peptide linker, an antigenic polypeptide as defined herein, a peptide linker, a donor chain peptide;

[0140] d) a signal peptide, an antigen aggregation domain, a peptide linker, an antigenic polypeptide as defined herein, a peptide linker, a donor chain peptide;

[0141] e) a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, a donor peptide, a peptide linker, an antigen aggregation domain; or

[0142] f) a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, a donor peptide, a peptide linker, a transmembrane domain.

[0143] In some embodiments, the coding sequence encodes the following elements, for example, in the N-terminal to C-terminal direction, encodes the following elements: a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, and a donor strand peptide; optionally wherein the signal peptide is selected from SEQ ID NO: 394 to SEQ ID NO: 400, optionally wherein the signal peptide is SEQ ID NO: 395; the antigenic polypeptide is selected from SEQ ID NO: 247 to SEQ ID NO: 256, optionally wherein the antigenic polypeptide is SEQ ID NO: 247; the peptide linker is selected from SEQ ID NO: 352 to SEQ ID NO: 354, optionally wherein the peptide linker is SEQ ID NO: 352; the donor strand peptide is selected from SEQ ID NO: 338, SEQ ID NO: 339, optionally wherein the donor strand peptide is SEQ ID NO: 338.

[0144] In some embodiments, the coding sequence encodes the following elements, for example, in the N-terminal to C-terminal direction: a signal peptide, an antigenic polypeptide as defined herein, a (first) peptide linker, a donor strand peptide, a (second) peptide linker; and an antigen aggregation domain; optionally wherein the signal peptide is selected from SEQ ID NO: 394 to SEQ ID NO: 400, optionally wherein the signal peptide is SEQ ID NO: 394; the antigenic polypeptide is selected from SEQ ID NO: 247 to SEQ ID NO: 256, optionally wherein the antigenic polypeptide is SEQ ID NO: 247; the (first) peptide linker is selected from SEQ ID NO: 352 to SEQ ID NO: 354, optionally wherein the (first) peptide linker is SEQ ID NO: 352; the donor strand peptide is selected from SEQ ID NO: 338, SEQ ID NO: 339, optionally wherein the donor strand peptide is SEQ ID NO: 338; the (second) peptide linker is selected from SEQ ID NO: 355 to SEQ ID NO: 358, optionally wherein the (second) peptide linker is SEQ ID NO: 355; the antigen aggregation domain is selected from SEQ ID NO: 443, SEQ ID NO: 444, SEQ ID NO: 457 to SEQ ID NO: 459, optionally wherein the peptide linker is SEQ ID NO: 444 or SEQ ID NO: 459.

[0145] Suitable sequences as defined above are provided in Table 1. Therein, each row corresponds to a suitable sequence. Column A of Table 1 provides a brief description of a suitable antigenic construct. Column B of Table 1 provides the protein (amino acid) SEQ ID NO of the respective antigenic construct. Column C of Table 1 provides the SEQ ID NO of the corresponding wild-type nucleic acid coding sequence or reference nucleic acid coding sequence. Column D of Table 1 provides the SEQ ID NO of the corresponding G / C optimized nucleic acid coding sequence (opt1, gc). Column E of Table 1 provides the SEQ ID NO of the corresponding human codon usage adaptive nucleic acid coding sequence (opt3, human). Column F of Table 1 provides the SEQ ID NO of the further codon-optimized coding sequence (opt4, main or opt5, gc mod).

[0146] It is noteworthy that this description explicitly includes the information provided under the "feature key" of the ST.26 sequence table of the present application, i.e., "source" (for nucleic acid or protein) or "misc_feature" (for nucleic acid) or "REGION" (for protein). Table 3 provides an RNA construct comprising the coding sequence of Table 1, for example, an mRNA sequence comprising the coding sequence of Table 1.

[0147] Table 1: Sequences (amino acid sequences and coding sequences).

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155] Aa: Aquifex aeolicus; Ec: Escherichia coli; Env: envelope glycoprotein; G: glycoprotein; HA: hemagglutinin; Hp: Helicobacter pylori; Hs: Homo sapiens; IgE: immunoglobulin E; IgK: immunoglobulin kappa; LumSynth, LS: dioxotetrahydropterin synthase; Mm: mouse (Mus musculus); TMdomain, TM: transmembrane domain.

[0156] Suitable coding sequences:

[0157] According to some embodiments, the coding RNA of the present disclosure comprises a coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH or fragments and variants thereof as defined herein. In this context, any coding sequence encoding at least one antigenic protein as defined herein or fragments and variants thereof can be understood as a suitable coding sequence and can therefore be included in the nucleic acid of the present disclosure.

[0158] In some embodiments, the encoding RNA of the first aspect comprises or consists of a coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, for example, a coding sequence encoding a fragment of any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, SEQ ID NO: 498 to SEQ ID NO: 520, SEQ ID NO: 1277 or a variant thereof. It should be understood that at the RNA level, any sequence encoding an amino acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, SEQ ID NO: 498 to SEQ ID NO: 520, SEQ ID NO: 1277, or a fragment or variant thereof can be selected and can be accordingly understood as a suitable coding sequence of the present disclosure.

[0159] In some embodiments, the coding sequence encodes an amino acid sequence that is identical to, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 498 to SEQ ID NO: 520, SEQ ID NO: 1277, or an immunogenic fragment or immunogenic variant thereof.

[0160] In some embodiments, the coding sequence encodes an amino acid sequence that is identical to, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 504, SEQ ID NO: 508, and SEQ ID NO: 509, or an immunogenic fragment or immunogenic variant thereof.

[0161] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 187 to SEQ ID NO: 246, SEQ ID NO: 257 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant of any of these sequences.

[0162] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant thereof.

[0163] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising the sequence according to SEQ ID NO: 529, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 554, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 579, SEQ ID NO: 583, SEQ ID NO: 584, SEQ ID NO: 604, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 654, SEQ ID NO: 658, SEQ ID NO: 659, or at least one of the nucleic acid sequences that are identical or have at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any of the 659, or a fragment or variant of any of these sequences.

[0164] In some embodiments, the encoding RNA of the first aspect is an artificial RNA.

[0165] The term "artificial RNA" as used herein is intended to refer to non-natural RNA. In other words, artificial RNA can be understood as non-natural RNA molecules. Such RNA molecules are due to their separate sequences (e.g., coding sequences, UTRs modified with G / C content) and / or due to other modifications, such as structural modifications of nucleotides. Generally, artificial RNA can be designed and / or generated by genetic engineering to correspond to the desired artificial nucleotide sequence. In this context, artificial RNA is a sequence that may not be naturally occurring, i.e., a sequence that has at least one nucleotide different from a wild-type sequence or a reference sequence / naturally occurring sequence (e.g., by codon modification as further described below). The term "artificial RNA" is not limited to representing "a single molecule", but is understood to include an entire or a plurality of substantially identical RNA molecules.

[0166] In some embodiments, the encoding RNA is a modified and / or stabilized RNA.

[0167] According to some embodiments, the coding RNA can therefore be provided as a "stabilized RNA", that is, an RNA showing improved resistance to degradation in vivo and / or an RNA showing improved stability in vivo, and / or an RNA showing improved translatability in vivo. The term "stabilized RNA" refers to a modified RNA that is modified to be more stable in the face of decomposition or degradation, such as by environmental factors or enzymatic digestion, such as degradation by exonucleases or endonucleases, compared to RNA without such modifications. In one embodiment, the stabilized RNA in the context of the present disclosure is stabilized in cells, such as prokaryotic cells or eukaryotic cells, such as in mammalian cells such as human cells. Stabilization can also be performed outside the cell, such as in a buffer solution, etc., for example, for storing a composition comprising stabilized RNA.

[0168] The encoding RNA of the present disclosure may be provided as "stabilized RNA."

[0169] Suitable modifications / adaptations capable of "stabilizing" RNA are described below.

[0170] In some embodiments, the encoding RNA comprises at least one codon-modified coding sequence.

[0171] In some embodiments, at least one coding sequence of the coding RNA is a codon-modified coding sequence. Suitably, the amino acid sequence encoded by at least one codon-modified coding sequence is not modified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence or the reference coding sequence.

[0172] The term "codon-modified coding sequence" relates to a coding sequence that is different in at least one codon (nucleotide triplet encoding one amino acid) compared to a corresponding wild-type coding sequence or a reference coding sequence. Suitably, a codon-modified coding sequence in the context of the present disclosure may show improved in vivo degradation resistance and / or improved in vivo stability, and / or improved in vivo translatability. Codon modification in the broadest sense utilizes the degeneracy of the genetic code, wherein multiple codons can encode the same amino acid and can be used interchangeably to optimize / modify coding sequences for in vivo applications.

[0173] In some embodiments, the coding sequence encoding the RNA is a codon-modified coding sequence, wherein the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage-adaptive coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof.

[0174] In some embodiments, the coding sequence of the encoding RNA has a G / C content of at least about 50%, 55% or 60%. In specific embodiments, at least one coding sequence of the RNA has a G / C content of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71% or 72%.

[0175] When transfected into a mammalian host cell, the coding RNA comprising the codon-modified coding sequence has a stability of 12 hours to 18 hours or greater than 18 hours, such as 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours or greater than 72 hours, and can be expressed by the mammalian host cell (e.g., muscle cell). RNA detection methods are known in the art.

[0176] When transfected into a mammalian host cell, the coding RNA comprising the codon-modified coding sequence is translated into a protein, wherein the amount of protein is at least comparable to the amount of protein obtained by transfecting a naturally occurring coding sequence, or a wild-type coding sequence, or a reference coding sequence into a mammalian host cell, or for example, at least 10% higher, or at least 20% higher, or at least 30% higher, or at least 40% higher, or at least 50% higher, or at least 100% higher, or at least 200% higher.

[0177] In some embodiments, the coding RNA may be modified, wherein the C content of at least one coding sequence may be increased, for example, maximized, compared to the C content of the corresponding wild-type coding sequence or reference coding sequence (referred to herein as a "C-maximized coding sequence"). The generation of a C-maximized nucleic acid sequence may be suitable for use according to the modification method of WO2015062738. In this context, the disclosure of WO2015062738 is incorporated herein by reference.

[0178] In some embodiments, the coding RNA can be modified, wherein the G / C content of at least one coding sequence can be optimized (referred to herein as "G / C optimized coding sequence") compared to the G / C content of the corresponding wild-type coding sequence or reference coding sequence. "Optimization" in this context refers to a coding sequence in which the G / C content is, for example, increased to substantially the highest possible G / C content. The generation of a G / C content-optimized nucleic acid sequence can be performed using a method according to WO2002098443. In this context, the disclosure of WO2002098443 is included in the present disclosure in its entirety. G / C optimized coding sequences are represented by the abbreviations "opt1" or "gc".

[0179] In some embodiments, the coding RNA may be modified, wherein the codons in at least one coding sequence may be adapted to human codon usage (referred to herein as "human codon usage adapted coding sequences"). Codons encoding the same amino acid appear at different frequencies in humans. Therefore, the coding sequence of the RNA is modified so that the frequency of the codons encoding the same amino acid corresponds to the natural frequency of the codon according to human codon usage. For example, for the amino acid Ala, the wild-type coding sequence or the reference coding sequence is adapted, for example, in a manner such that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" is used at a frequency of 0.28, the codon "GCA" is used at a frequency of 0.22, and the codon "GCG" is used at a frequency of 0.10. (See, for example, Table 2 of published PCT patent application WO2021156267, which is incorporated herein by reference). Therefore, this process (as exemplified by alanine) is applied to each amino acid encoded by the coding sequence of the RNA to obtain a sequence adapted to human codon usage. Human codon usage-adapted coding sequences are indicated by the abbreviation "opt3" or "human".

[0180] In some embodiments, the coding RNA may be modified, wherein the G / C content of at least one coding sequence may be modified compared to the G / C content of the corresponding wild-type coding sequence or a reference coding sequence (referred to herein as a "G / C modified coding sequence"). In this context, the term "G / C optimization" or "G / C content modification" relates to a nucleic acid comprising a modified, for example, increased number of guanosine nucleotides and / or cytosine nucleotides compared to the corresponding wild-type coding sequence or a reference coding sequence. This increase in number can be generated by replacing a codon containing adenosine nucleotides or thymusine nucleotides with a codon containing guanosine nucleotides or cytosine nucleotides. Advantageously, the RNA sequence with increased G / C content is more stable or exhibits better expression than a sequence with increased A / U. For example, compared to the G / C content of the coding sequence of the corresponding wild-type nucleic acid sequence or a reference nucleic acid sequence, the G / C content of the coding sequence of the RNA is increased by at least 10%, 20%, 30%, for example, by at least 40% (referred to herein as "opt5" or "gc mod").

[0181] In some embodiments, the coding RNA may be modified, wherein the codon adaptation index (CAI) may be increased or, for example, maximized in at least one coding sequence (referred to herein as "CAI maximization coding sequence"). Suitably, in a relatively rare wild-type nucleic acid sequence or reference nucleic acid sequence, for example, in humans, all of its codons are replaced with respective codons with high frequencies in, for example, humans, wherein the codons with high frequencies encode the same amino acids as the relatively rare codons. Suitably, each amino acid of the encoded protein uses the codon with the highest frequency (see Table 2 of the published PCT patent application WO2021156267, the most frequent human codons are marked with asterisks). Suitably, RNA comprises at least one coding sequence, wherein the codon adaptation index (CAI) of at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. For example, the codon adaptation index (CAI) of at least one coding sequence is 1 (CAI=1). For example, for the amino acid Ala, a wild-type coding sequence or a reference coding sequence may be adapted in a manner such that the most frequent human codon "GCC" is always used for the amino acid. Thus, this procedure can be applied to each amino acid encoded by a nucleic acid coding sequence (such as the example of alanine) to obtain a coding sequence that maximizes the CAI (referred to herein as "opt4" or "main").

[0182] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 197 to SEQ ID NO: 246, SEQ ID NO: 267 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant thereof.

[0183] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant thereof.

[0184] In some embodiments, at least one coding sequence of the RNA of the present disclosure is a G / C optimized coding sequence.

[0185] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 197 to SEQ ID NO: 206, SEQ ID NO: 207 to SEQ ID NO: 216, SEQ ID NO: 237 to SEQ ID NO: 246, SEQ ID NO: 267 to SEQ ID NO: 276, SEQ ID NO: 277 to SEQ ID NO: 286, SEQ ID NO: 307 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant thereof.

[0186] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment or variant thereof.

[0187] In some embodiments, the encoding RNA of the first aspect comprises a coding sequence comprising at least one of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 529, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 554, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 654, SEQ ID NO: 658, SEQ ID NO: 659, or a fragment or variant of any of these sequences.

[0188] In some embodiments, the coding sequence comprises more than one termination codon to allow sufficient translation termination. In a specific embodiment, the coding sequence comprises two or three termination codons to allow sufficient translation termination. These more than one termination codons can optionally be located in a variable reading frame.

[0189] UTR:

[0190] The RNA of the first aspect comprises at least one untranslated region (UTR).

[0191] The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a portion of a nucleic acid molecule that is typically located 5' or 3' of a coding sequence. A UTR cannot be translated into protein. A UTR can be part of an RNA. A UTR can include elements that control gene expression, also referred to as regulatory elements. Such regulatory elements can be, for example, ribosome binding sites, miRNA binding sites, promoter elements, etc.

[0192] In some embodiments, the coding RNA comprises a protein coding region ("coding sequence" or "cds"), and a 5'-UTR and / or a 3'-UTR. It is noteworthy that the UTR may have regulatory sequence elements that determine the conversion, stability and positioning of the RNA. In addition, the UTR may have sequence elements that enhance translation. In medical applications, translating RNA into at least one peptide or protein is essential for therapeutic effects. Certain combinations of 3'-UTR and / or 5'-UTR can enhance the expression of operably linked coding sequences encoding peptides or proteins as defined herein. The RNA molecule with the UTR combination advantageously enables antigenic peptides or proteins to be rapidly and transiently expressed after being administered to a subject, for example, after intramuscular administration. Therefore, the RNA of the present disclosure comprising certain 3'-UTR and / or 5'-UTR combinations is particularly suitable for administration as a vaccine, particularly suitable for administration to the muscle, dermis or epidermis of a subject.

[0193] Suitably, the coding RNA comprises at least one 5'-UTR and / or at least one 3'-UTR. The heterologous 5'-UTR or 3'-UTR may be derived from a naturally occurring gene or may be synthetically engineered. In some embodiments, the RNA comprises at least one coding sequence as defined herein operably linked to at least one (heterologous) 3'-UTR and / or at least one (heterologous) 5'-UTR.

[0194] In some embodiments, the encoding RNA of the present disclosure comprises at least one 3'-UTR.

[0195] The term "3' untranslated region" or "3'-UTR" will be recognized and understood by those of ordinary skill in the art and is, for example, intended to refer to a portion of an RNA molecule that is located 3' (i.e., downstream) of a coding sequence and is not translated into protein. A 3'-UTR may be a portion of a nucleic acid that is located between a coding sequence and an (optional) terminal poly(A) sequence. A 3'-UTR may contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc.

[0196] Optionally, the encoding RNA comprises at least one 3'-UTR, which may be derived from a gene associated with RNA having enhanced half-life (ie, which provides a stable RNA).

[0197] In some embodiments, the 3'-UTR comprises one or more polyadenylation signals, a binding site for a protein that affects the stability of the intracellular localization of the nucleic acid, or one or more miRNAs or binding sites for miRNAs.

[0198] MicroRNAs (or miRNAs) are non-coding RNAs of approximately 19 to 25 nucleotides in length that bind to the 3'-UTR of RNA molecules and downregulate gene expression by reducing RNA stability or inhibiting translation. For example, it is known that microRNA regulates RNA, thereby regulating protein expression, such as protein expression in liver (miR-122), heart (miR-ld, miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7, miR-30c), kidney (miR-192, miR-194, miR-204), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133, miR-206, miR-208) and lung epithelial cells (let-7, miR-133, miR-126). RNA can include one or more than one microRNA target sequence, microRNA sequence or microRNA seed. Such sequences may correspond to any known microRNA, such as those taught in US20050261218 and US20050059005, which are incorporated herein by reference.

[0199] Thus, a miRNA or a binding site for a miRNA as defined above may be removed from or introduced into a 3'-UTR in order to tailor the expression of the RNA to a desired cell type or tissue (eg muscle cells).

[0200] In some embodiments, the coding RNA comprises at least one 3'-UTR, wherein the at least one 3'-UTR comprises a nucleic acid sequence derived from or selected from the 3'-UTR of the following genes, or a homolog, fragment or variant of any one of these genes, the gene being selected from: PSMB3, α-globin, ALB7, CASP1, COX6B1, FIG4, GNAS, NDUFA1 and RPS9.

[0201] In some embodiments, at least one 3'-UTR derived from or selected from PSMB3, α-globin, ALB7, CASP1, COX6B1, FIG4, GNAS, NDUFA1 or RPS9 comprises or consists of a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 67 to SEQ ID NO: 90, SEQ ID NO: 109 to SEQ ID NO: 120, or a nucleic acid sequence of a fragment or variant of any of these.

[0202] In one embodiment, the encoding RNA comprises a 3'-UTR derived from or selected from a PSMB3 gene.

[0203] In one embodiment, the 3'-UTR derived from or selected from PSMB3 comprises a nucleic acid sequence consisting of a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 109 to SEQ ID NO: 120, or a fragment or variant thereof.

[0204] In other embodiments, the encoding RNA comprises a 3'-UTR comprising or consisting of a nucleic acid sequence that is identical to, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, SEQ ID NO: 91 to SEQ ID NO: 108, or a fragment or variant thereof.

[0205] In other embodiments, the coding RNA comprises a 3'-UTR as described in WO2016107877, and the disclosures of 3'-UTR sequences in WO2016107877 are incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NO: 1 to SEQ ID NO: 24 and SEQ ID NO: 49 to SEQ ID NO: 318 in WO2016107877, or fragments or variants of these sequences. In other embodiments, the RNA comprises a 3'-UTR as described in WO2017036580, and the disclosures of 3'-UTR sequences in WO2017036580 are incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NO: 152 to SEQID NO: 204 in WO2017036580, or fragments or variants of these sequences. In other embodiments, the RNA comprises a 3'-UTR as described in WO2016022914, the disclosure of which relating to 3'-UTR sequences in WO2016022914 is incorporated herein by reference. Particularly suitable 3'-UTRs are nucleic acid sequences according to SEQ ID NO: 20 to SEQ ID NO: 36 of WO2016022914, or fragments or variants of these sequences.

[0206] In some embodiments, the encoding RNA of the present disclosure comprises at least one 5'-UTR.

[0207] The term "5' untranslated region" or "5'-UTR" will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to a portion of an RNA that is located 5' (i.e., "upstream") of a coding sequence and is not translated into protein. A 5'-UTR can be a portion of a nucleic acid that is located 5' of a coding sequence. Typically, a 5'-UTR begins at the transcription start site and ends before the start codon of the coding sequence. The 5'-UTR may contain elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements may be, for example, ribosome binding sites, miRNA binding sites, etc. The 5'-UTR may be modified, for example, by the addition of an enzymatic or co-transcriptional 5' cap structure (e.g., for mRNA as defined below).

[0208] Optionally, the encoding RNA comprises at least one 5'-UTR, which may be derived from a gene associated with RNA having enhanced half-life (ie, which provides a stable RNA).

[0209] In some embodiments, the 5'-UTR comprises one or more binding sites for proteins that affect intracellular RNA stability or RNA localization, or one or more miRNAs (as defined above) or binding sites for miRNAs.

[0210] Thus, a miRNA or a binding site for a miRNA as defined above may be removed from or introduced into a 5'-UTR in order to tailor the expression of the nucleic acid to a desired cell type or tissue (eg muscle cells).

[0211] In some embodiments, the coding RNA comprises at least one 5'-UTR, wherein the at least one 5'-UTR comprises a nucleic acid sequence derived from or selected from the 5'-UTR of the following genes, or a homolog, fragment or variant of any one of these genes, the gene being selected from: HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2.

[0212] In some embodiments, at least one 5'-UTR derived from or selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2 comprises or consists of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 32, SEQ ID NO: 65, SEQ ID NO: 66, or a fragment or variant of any of these nucleic acid sequences.

[0213] In one embodiment, the encoding RNA comprises a 5'-UTR derived from or selected from the HSD17B4 gene.

[0214] In one embodiment, the 5'-UTR derived from or selected from HSD17B4 comprises or consists of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 65, SEQ ID NO: 66, or a fragment or variant thereof.

[0215] In other embodiments, the encoding RNA comprises a 5'-UTR comprising or consisting of a nucleic acid sequence that is identical to, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, SEQ ID NO: 33 to SEQ ID NO: 64, or a fragment or variant thereof.

[0216] In other embodiments, the coding RNA comprises a 5'-UTR as described in WO2013143700, and the disclosure of 5'-UTR sequences in WO2013143700 is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences derived from SEQ ID NO: 1 to SEQ ID NO: 1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of WO2013143700, or fragments or variants of these sequences. In other embodiments, the coding RNA comprises a 5'-UTR as described in WO2016107877, and the disclosure of 5'-UTR sequences in WO2016107877 is incorporated herein by reference. Particularly suitable 5'-UTR is according to WO2016107877 SEQ ID NO: 25 to SEQ ID NO: 30 and SEQ ID NO: 319 to SEQ ID NO: 382 nucleic acid sequences, or fragments or variants of these sequences. In other embodiments, RNA comprises 5'-UTR as described in WO2017036580, and the disclosures related to 5'-UTR sequences in WO2017036580 are incorporated herein by reference. Particularly suitable 5'-UTR is according to WO2017036580 SEQ ID NO: 1 to SEQ ID NO: 151 nucleic acid sequences, or fragments or variants of these sequences. In other embodiments, RNA comprises 5'-UTR as described in WO2016022914, and the disclosures related to 5'-UTR sequences in WO2016022914 are incorporated herein by reference. Suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NO: 3 to SEQ ID NO: 19 of WO2016022914, or fragments or variants of these sequences, the disclosure of which is incorporated herein by reference.

[0217] In some embodiments, the encoding RNA of the present disclosure comprises a coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as described herein, and a 3'-UTR and / or 5'-UTR selected from the following 5'-UTR / 3'-UTR combinations (also referred to as "UTR designs"): a-1 (HSD17B4 / PSMB3), a-2 (NDUFA4 / PSMB3), a-3 (SLC7A3 / PSMB3), a-4 (NOSIP / PSMB3), a-5 (MP68 / PSMB3), b-1 (UBQLN2 / RPS9), b-2 (ASAH1 / RPS9), b-3 (HSD17B4 / RPS9), b-4 (HSD17B4 / CASP1), b-5 (NOSIP / COX6B1), c-1 (NDUFA4 / RPS9), c-2 (NDUFA4 / PSMB3), c-3 (SLC7A3 / PSMB3), (NDUFA4 / COX6B1), c-4 (NDUFA4 / NDUFA1), c-5 (ATP5A1 / PSMB3), d-1 (Rpl31 / PSMB3), d-2 (ATP5A1 / CASP1), d-3 (SLC7A3 / GNAS), d-4 (HSD17B4 / NDUFA1), d-5 (Slc7a3 / Ndufa1), e-1 (TUBB4B / RPS9), e-2 (RPL31 / RPS9), e-3 (MP68 / RPS9), e-4 (NOSIP / RPS9), e-5 (ATP5A1 / RPS9), e-6 (ATP5A1 / COX6B1), f-1 (ATP5A1 / GNAS), f-2 (ATP5A1 / NDUFA1), f-3 (HSD17B4 / COX6B1), f-4 (HSD17B4 / GNAS), f-5 (MP68 / COX6B1), g-1 (MP68 / NDUFA1), g-2 (NDUFA4 / CASP1), g-3 (NDUFA4 / GNAS), g-4 (NOSIP / CASP1), g-5 (RPL31 / CASP1), h-1 (RPL31 / COX6B1), h-2 (RPL31 / GNAS), h-3 (RPL31 / NDUFA1), h-4 (Slc7a3 / CASP1), h-5 (SLC7A3 / COX6B1), i-1 (SLC7A3 / RPS9), i-2 (RPL32 / ALB7), i-2 (RPL32 / ALB7), or i-3 (α-globin gene).

[0218] In some embodiments, the encoding RNA comprises a coding sequence encoding an antigenic polypeptide selected from or derived from E. coli FimH as defined herein, and HSD17B4 5'-UTR and PSMB3 3'-UTR (HSD17B4 / PSMB3(a-1)). The inventors have demonstrated that this embodiment is particularly beneficial for inducing an immune response against E. coli FimH.

[0219] In various embodiments, the encoding RNAs of the present disclosure are monocistronic, bicistronic, or polycistronic.

[0220] In some embodiments, the encoding RNAs of the present disclosure are monocistronic.

[0221] The term "monocistronic" will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to an RNA comprising only one coding sequence. The term "bicistronic" or "polycistronic" as used herein is, for example, intended to refer to an RNA comprising two (bicistronic) or more than two (polycistronic) coding sequences.

[0222] In some embodiments, the A / U (A / T) content in the ribosome binding site environment of the RNA is increased compared to the A / U (A / T) content in the ribosome binding site environment of its respective wild-type nucleic acid or reference nucleic acid. This modification improves the efficiency of ribosome binding to RNA, which in turn is beneficial to the efficient translation of RNA into antigenic peptides or proteins.

[0223] Thus, in one embodiment, the encoding RNA comprises a ribosome binding site, also known as a "Kozak sequence", identical to or having at least 80%, 85%, 90%, 95% identity to any one of SEQ ID NO: 128 to SEQ ID NO: 135, or any fragment or variant thereof.

[0224] Poly(N) sequences, histone stem-loops:

[0225] In some embodiments, the encoding RNA comprises at least one poly (N) sequence, such as at least one poly (A) sequence, at least one poly (U) sequence, at least one poly (C) sequence, or a combination thereof.

[0226] In some embodiments, the encoding RNA of the present disclosure comprises at least one poly(A) sequence.

[0227] The terms "poly(A) sequence", "poly(A) tail" or "3'-poly(A) tail" as used herein will be recognized and understood by those of ordinary skill in the art, and are, for example, intended to be a sequence of adenosine nucleotides, typically located at the 3' end of a linear RNA of up to about 1000 adenosine nucleotides. For example, the poly(A) sequence is substantially homopolymeric, for example, a poly(A) sequence of, for example, 100 adenosine nucleotides substantially has a length of 100 nucleotides. In other embodiments, the poly(A) sequence is interrupted by at least one nucleotide that is different from an adenosine nucleotide, for example, a poly(A) sequence of, for example, 100 adenosine nucleotides may have a length of more than 100 nucleotides (it comprises 100 adenosine nucleotides, and additionally, the at least one nucleotide - or a stretch of nucleotides - that is different from an adenosine nucleotide).

[0228] In some embodiments, at least one poly(A) sequence may comprise about 40 to about 500 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, about 40 to about 150 adenosine nucleotides, such as about 60 to about 150 adenosine nucleotides.

[0229] In some embodiments, at least one poly(A) sequence may comprise about 40 to about 500 consecutive adenosine nucleotides, about 40 to about 200 consecutive adenosine nucleotides, about 40 to about 150 consecutive adenosine nucleotides, such as about 60 to about 150 consecutive adenosine nucleotides.

[0230] Suitably, the length of the poly(A) sequence may be at least or even more than about 10, 50, 64, 75, 100, 200, 300, 400 or 500 adenosine nucleotides, such as consecutive adenosine nucleotides.

[0231] In some embodiments, at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), such as about 100 consecutive adenosine nucleotides.

[0232] In other embodiments, the RNA comprises at least one poly(A) sequence comprising about 100 adenosine nucleotides, wherein the poly(A) sequence is interrupted by non-adenosine nucleotides, for example, by about 10 non-adenosine nucleotides (A30-N10-A70).

[0233] Suitably, the poly(A) sequence as defined herein may be directly located at the 3' end of the RNA. In some embodiments, the 3' terminal nucleotide (i.e., the last 3' terminal nucleotide in the polynucleotide chain) is the 3' terminal A nucleotide of at least one poly(A) sequence. The term "directly located at the 3' end" must be understood as being precisely located at the 3' end - in other words, the 3' end of the RNA consists of a poly(A) sequence terminated with A.

[0234] By terminating with an adenosine nucleotide, the RNA of the present disclosure can reduce the induction of interferons such as IFNα if administered, for example, as a vaccine. This is particularly important because the induction of interferons such as IFNα is believed to be a major factor in causing fever in vaccinated subjects.

[0235] Thus, in some embodiments, the encoding RNA of the present disclosure comprises a poly(A) sequence of about 100 consecutive adenosine nucleotides, wherein the poly(A) sequence is located directly at the 3' end of the RNA, optionally wherein the 3' terminal nucleotide is an adenosine.

[0236] In some embodiments, the poly (A) sequence of RNA is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly (A) sequence is obtained in vitro by a commonly used chemical synthesis method, and does not need to be transcribed from a DNA template. In other embodiments, according to the methods and means described in WO2016174271, (after RNA in vitro transcription) using, for example, immobilized poly (A) polymerase, by enzymatic polyadenylation of RNA, a poly (A) sequence is generated, WO2016174271 is incorporated herein by reference.

[0237] In some embodiments, the coding RNA comprises at least one poly(A) sequence obtained by enzymatic polyadenylation, wherein a majority of the RNA molecules comprise about 100 (+ / - 20) to about 500 (+ / - 100) adenosine nucleotides, e.g., about 100 (+ / - 20) to about 200 (+ / - 40) adenosine nucleotides.

[0238] In some embodiments, the encoding RNA comprises at least one poly(A) sequence derived from a template DNA and additionally at least one poly(A) sequence generated by enzymatic polyadenylation, e.g., as described in published PCT patent application WO2016091391, which is incorporated herein by reference.

[0239] In some embodiments, the encoding RNA comprises at least one polyadenylation signal.

[0240] In some embodiments, the coding RNA comprises at least one poly(C) sequence.The poly(C) sequence in the context of the present disclosure may be located in a UTR region, for example in a 3'-UTR.

[0241] The term "poly (C) sequence" as used herein is intended to refer to a cytosine nucleotide sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly (C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In one embodiment, the poly (C) sequence comprises about 30 cytosine nucleotides.

[0242] In some embodiments, the encoding RNA of the present disclosure comprises at least one poly(C) sequence and / or at least one miRNA binding site and / or a histone stem-loop sequence.

[0243] In some embodiments, the encoding RNA of the present disclosure comprises at least one histone stem-loop (hSL) or a histone stem-loop structure. The hSL in the context of the present disclosure may be located in the UTR region, for example in the 3'-UTR.

[0244] The term "histone stem-loop" (hSL) is intended to refer to nucleic acid sequences that form a stem-loop secondary structure, which is primarily found in histone mRNA.

[0245] The histone stem-loop sequence / structure may be suitably selected from the hSL sequences disclosed in WO2012019780, the disclosure of which relating to histone stem-loop sequences / histone stem-loop structures is incorporated herein by reference. The hSL sequences useful in the present disclosure may be derived from formula (I) or (II) of WO2012019780. According to one embodiment, the RNA comprises at least one hSL sequence derived from at least one of formula (Ia) or (IIa) of a specific WO2012019780.

[0246] In some embodiments, the encoding RNA comprises at least one hSL, wherein the hSL comprises or consists of an RNA sequence that is identical to any one of SEQ ID NO: 136, SEQ ID NO: 137, or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment thereof.

[0247] In alternative embodiments, the encoding RNA does not comprise a histone stem-loop as defined herein.

[0248] In some embodiments, the encoding RNA comprises a 3' terminal sequence element. The 3' terminal sequence element represents the 3' end of the RNA. The 3' terminal sequence element may comprise at least one poly (N) sequence as defined herein, and optionally comprises at least one hSL as defined herein.

[0249] In some embodiments, the encoding RNA comprises at least one 3' terminal sequence element comprising or consisting of an RNA sequence that is identical to or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 138 to SEQ ID NO: 172, or a fragment or variant of these sequences.

[0250] In some embodiments, the encoding RNA comprises a 3' terminal sequence element comprising hSL as defined herein, followed by a poly(A) sequence comprising about 100 consecutive adenosines.

[0251] In some embodiments, the encoding RNA comprises a 3' terminal sequence element comprising or consisting of an RNA sequence that is identical to, or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 144, or a fragment or variant thereof.

[0252] In some embodiments, the encoding RNA comprises a 5' terminal sequence element comprising or consisting of an RNA sequence that is identical to or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 121 to SEQ ID NO: 127, or a fragment or variant of these sequences.

[0253] In some embodiments, the encoding RNA comprises a 5' terminal sequence element comprising or consisting of an RNA sequence that is identical to, or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 122, or a fragment or variant thereof.

[0254] Such a 5' terminal sequence element may comprise, for example, a binding site for T7 RNA polymerase. In addition, the first nucleotide of the 5' terminal start sequence may, for example, comprise a 2'O-methylation, such as a 2'O-methylated guanosine or a 2'O-methylated adenosine.

[0255] Cap structure:

[0256] Suitably, the encoding RNA comprises a 5' cap structure which suitably stabilizes the RNA and / or enhances expression of the encoded antigen and / or reduces stimulation of the innate immune system (upon administration to a subject, such as a human subject).

[0257] Thus, in some embodiments, the encoding RNA comprises a 5' cap structure, such as an m7G, cap 0, cap 1, cap 2, a modified cap 0, or a modified cap 1 structure.

[0258] The term "5' cap structure" as used herein will be recognized and understood by those of ordinary skill in the art and is, for example, intended to refer to a 5' modified nucleotide, particularly a guanine nucleotide, located at the 5' end of an RNA, such as an mRNA. For example, the 5' cap structure is attached to the RNA via a 5'-5'-triphosphate bond.

[0259] 5' cap structures suitable for use in the context of the present disclosure are cap 0 (methylation of the first base, such as m7GpppN), cap 1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), cap 2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap 3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap 4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (such as thiophosphate-modified ARCA), inosine, N1-methylguanosine, 2'-fluoroguanosine, 7-deazaguanosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine and 2-azidoguanosine.

[0260] The 5' cap (cap 0 or cap 1) structure can be formed using a cap analog during chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping).

[0261] The term "cap analogue" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a non-polymerizable dinucleotide or trinucleotide having a cap functionality, wherein when it is bound to the 5' end of a nucleic acid molecule, translation or localization is promoted, and / or RNA molecule degradation is inhibited. Non-polymerizable means that the cap analogue is incorporated only at the 5' end, because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent polymerase, particularly a template-dependent RNA polymerase. Examples of cap analogs include, but are not limited to, chemical structures selected from m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG; m7,2'dGpppG; m7,3'OmeGpppG; m7,3'dGpppG and their tetraphosphate derivatives). Other suitable cap analogs are described in WO2008016473, WO2008157688, WO2009149253, WO2011015347, WO2013059475, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017053297, WO2017066782, WO2018075827 and WO2017066797, said disclosures relating to cap analogs are incorporated herein by reference.

[0262] In an embodiment, a cap 1 structure is generated using a trinucleotide cap analog as disclosed in WO2017053297, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017066782, WO2018075827, and WO2017066797. For example, a cap structure that can be derived from the structure disclosed in claims 1 to 5 of WO2017053297 can be suitable for co-transcriptional generation of a cap 1 structure. In addition, any cap structure that can be derived from the structure defined in claim 1 or claim 21 of WO2018075827 can be suitable for generating a cap 1 structure. These disclosures are incorporated herein by reference.

[0263] In some embodiments, a trinucleotide cap analogue as defined herein may be suitably used to co-transcriptionally add a 5' cap structure, particularly in an RNA in vitro transcription reaction as defined herein.

[0264] In some embodiments, the coding RNA, particularly the mRNA, of the present disclosure comprises a cap 1 structure.

[0265] In some embodiments, the Cap 1 structure of the RNA is formed by co-transcriptional capping using the trinucleotide cap analogs m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG.

[0266] In this context, a particularly suitable Cap 1 analogue is m7G(5')ppp(5')(2'OMeA)pG.

[0267] In other embodiments, the Cap 1 structure of the RNA is formed by co-transcriptional capping using the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0268] In an alternative embodiment, the 5' cap structure is formed by enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or cap-dependent 2'-O methyltransferase), thereby generating a structure of Cap 0 or Cap 1 or Cap 2. In this context, the 5' cap structure (Cap 0 or Cap 1) can be added using an immobilized capping enzyme and / or cap-dependent 2'-O methyltransferase using the methods and means disclosed in published PCT patent application WO2016193226, which is incorporated herein by reference.

[0269] In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises a cap structure, e.g., a cap 1 structure as determined by a capping assay.

[0270] To determine whether a cap structure is present, the capping assay described in published PCT application WO2015101416, in particular claims 27 to 46 of published PCT application WO2015101416, can be used. Other capping assays that can be used to determine whether a cap structure of an RNA is present are described in published PCT application WO2020127959. These disclosures are incorporated herein by reference.

[0271] Modified Nucleotides:

[0272] According to various embodiments, the encoding RNA of the present disclosure is a modified RNA, wherein the modification refers to a chemical modification including backbone modification and sugar modification or base modification.

[0273] Modified RNA can include nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications. The backbone modification in the context of the present disclosure is a modification in which the phosphate of the RNA nucleotide backbone is chemically modified. The sugar modification in the context of the present disclosure is a chemical modification of the sugar of the RNA nucleotide. In addition, the base modification in the context of the present disclosure is a chemical modification of the base portion of the RNA nucleotide. In this context, nucleotide analogs or modifications are, for example, selected from nucleotide analogs suitable for transcription and / or translation.

[0274] Thus, in some embodiments, the encoding RNAs of the present disclosure comprise at least one modified nucleotide.

[0275] In some embodiments, at least one modified nucleotide is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine.

[0276] Suitable in this context are pseudouridine (ψ) and N1-methylpseudouridine (m1ψ). N1-methylpseudouridine (m1ψ) is particularly suitable.

[0277] In some embodiments, substantially all uracils, such as substantially 100% of the uracils in the coding sequence (or the entire RNA sequence), have a chemical modification, such as a chemical modification at the 5 position of uracil.

[0278] Incorporation of modified nucleotides such as, for example, pseudouridine (ψ) or N1-methylpseudouridine (m1ψ) into the coding sequence (or the entire RNA sequence) can be advantageous since (if desired) unwanted innate immune responses (upon administration of the RNA) can be modulated or reduced.

[0279] In alternative embodiments, the encoding RNA of the present disclosure does not comprise a modified nucleotide, such as a position of N1-methylpseudouridine (m1ψ) substitution or a position of pseudouridine (ψ) substitution.

[0280] In some embodiments in this context, the encoding RNA of the present disclosure comprises a coding sequence consisting of only G, C, A, and U nucleotides, and thus comprises no modified nucleotides.

[0281] Other RNA features:

[0282] In the context of the present disclosure, the encoding RNA provides a coding sequence encoding an antigenic polypeptide selected from or derived from E. coli FimH as defined herein, which is translated into a (functional) antigen upon administration (e.g., upon administration to a subject, such as a human subject).

[0283] In the context of the present disclosure, the coding RNA can be any type of RNA comprising a coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH. For example, the coding RNA can be any type of single-stranded coding RNA, double-stranded coding RNA, linear coding RNA or circular coding RNA or any combination thereof.

[0284] Optionally, the encoding RNA comprises about 50 to about 20,000 nucleotides, or about 500 to about 10,000 nucleotides, or about 1,000 to about 10,000 nucleotides, or such as about 1,000 to about 5,000 nucleotides, or such as about 2,000 to about 5,000 nucleotides.

[0285] In some embodiments, the encoding RNA is selected from mRNA, encoding self-replicating RNA, encoding circular RNA, encoding viral RNA, or encoding replicon RNA.

[0286] In an embodiment, the coding RNA is a circular RNA. As used herein, "circular RNA" or "circRNA" must be understood as an RNA construct that is connected to form a circle and therefore does not include a 3' end or a 5' end. In some embodiments, the circRNA comprises a coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein.

[0287] In some embodiments, the coding RNA is mRNA.

[0288] Suitable features optionally comprised by the mRNA of the present disclosure are, for example, a 5' cap structure as defined herein, a 5'-UTR as defined herein, a 3'-UTR as defined herein, an hSL as defined herein, a poly(A) sequence as defined herein, and optionally chemical modifications as defined herein.

[0289] In some embodiments, the encoding RNA is in vitro transcribed RNA (eg, in vitro transcribed mRNA).

[0290] In some embodiments, the nucleotide mixture for RNA in vitro transcription comprises modified nucleotides as defined herein. In this context, suitable modified nucleotides can be selected from pseudouridine (ψ) or N1-methyl pseudouridine (m1ψ). Suitably, the uracil nucleotides in the nucleotide mixture are replaced (partially or completely) by pseudouridine (ψ) and / or N1-methyl pseudouridine (m1ψ) to obtain modified RNA (e.g., modified mRNA).

[0291] In some embodiments, the nucleotide mixture for RNA in vitro transcription does not include modified nucleotides as defined herein. In some embodiments, the nucleotide mixture for RNA in vitro transcription only includes nucleotides of guanosine (G), cytidine (C), adenosine (A), and uridine (U), and optionally includes a cap analog as defined herein to obtain unmodified RNA (e.g., unmodified mRNA).

[0292] In some embodiments, the nucleotide mixture (i.e., the ratio of each nucleotide in the mixture) used for the RNA in vitro transcription reaction is optimized for a given RNA sequence, for example, as described in WO2015188933, which is incorporated herein by reference.

[0293] In one embodiment, the coding RNA is lyophilized (e.g., according to WO2016165831 or WO2011069586) to obtain temperature-stable dry RNA. Spray drying or spray freeze drying (e.g., according to WO2016184575 or WO2016184576) can also be used to dry RNA to obtain temperature-stable RNA (powder). These disclosures are incorporated herein by reference.

[0294] In the context of the present disclosure (e.g., for RNA-based vaccines), it may be necessary to provide GMP-grade RNA. GMP-grade RNA is prepared using a preparation process approved by a regulatory agency. In some embodiments, RNA preparation is carried out under current Good Manufacturing Practice (GMP), and a variety of quality control steps are implemented at the DNA and RNA levels, such as the quality control steps selected from the methods described in WO2016180430. In some embodiments, the RNA of the present disclosure is GMP-grade RNA, such as GMP-grade mRNA.

[0295] In some embodiments, the encoding RNA of the present disclosure is purified RNA, optionally purified mRNA.

[0296] The term "purified RNA" or "purified mRNA" as used herein must be understood as having a higher purity than the raw material (such as in vitro transcribed RNA) after certain purification steps (e.g., HPLC, TFF, Oligo d (T) purification, precipitation steps). Typical impurities that are substantially absent from purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, such as RNA polymerase, RNase, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double-stranded RNA (dsRNA)), free nucleotides (modified nucleotides, conventional NTPs, cap analogs), template DNA fragments, buffer components (HEPES, TRIS, MgCl2), etc. Other potential impurities that may be derived, for example, from fermentation processes include bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification processes (organic solvents, etc.). Therefore, in this regard, it is desirable that "RNA purity" is as close to 100% as possible. Thus, "purified RNA" as used herein has a purity greater than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most advantageously 99% or higher. Purity is determined, for example, by analytical HPLC, wherein the percentages provided above correspond to the ratio of the peak area of ​​the target RNA to the total area of ​​all peaks, including peaks representing byproducts. Alternatively, purity is determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0297] Suitably, purification of the encoding RNA of the present disclosure may be performed by (RP)-HPLC, AEX, size exclusion chromatography, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow-through chromatography, oligo(dT) purification and / or cellulose-based purification.

[0298] Optionally, the RNA is purified using RP-HPLC (e.g. as described in WO2008077592) and / or tangential flow filtration (e.g. as described in WO2016193206) and / or oligo d(T) purification (e.g. as described in WO2016180430), e.g. to remove dsRNA, non-capped RNA and / or RNA fragments.

[0299] In an embodiment, the encoding RNA of the present disclosure has a certain RNA integrity.

[0300] The term "RNA integrity" generally describes whether there is an intact RNA sequence. Low RNA integrity may be due to RNA degradation, RNA cleavage, incorrect or incomplete RNA chemical synthesis, incorrect base pairing, incorporation of modified nucleotides or modification of already incorporated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation, or incomplete RNA in vitro transcription, etc. RNA is a fragile molecule that can be easily degraded, which can be caused by, for example, temperature, RNases, pH or other factors (e.g., nucleophilic attack, hydrolysis, etc.), and can reduce RNA integrity, thereby reducing its functionality.

[0301] Those skilled in the art can choose a variety of different chromatography methods or electrophoresis methods to determine RNA integrity. Chromatographic methods and electrophoresis methods (such as capillary gel electrophoresis) are well known in the art. In the case of using chromatography (such as RP-HPLC), the analysis of RNA integrity can be based on measuring the peak area (or "area under the peak") of the expected full-length RNA (RNA with the correct RNA length) in the corresponding chromatogram.

[0302] In embodiments, the coding RNA of the present disclosure has an RNA integrity of about 40% to about 100%. In embodiments, the RNA has an RNA integrity of about 50% to about 100%. In embodiments, the RNA has an RNA integrity of about 60% to about 100%. In embodiments, the RNA has an RNA integrity of about 70% to about 100%. In embodiments, the RNA integrity is, for example, about 50%, about 60%, about 70%, about 80% or about 90%. Suitably, analytical HPLC, such as analytical RP-HPLC, is used to measure RNA.

[0303] In some embodiments, the coding RNA has at least about 50% RNA integrity, such as at least about 60% RNA integrity, such as at least about 70% RNA integrity, such as at least about 80% or about 90% RNA integrity. RNA integrity is suitably determined using analytical HPLC, such as analytical RP-HPLC.

[0304] In some embodiments, the encoding RNA is suitable for intranasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal or subcutaneous administration. In some embodiments, the encoding RNA is suitable for intramuscular administration.

[0305] RNA constructs:

[0306] In some embodiments, the encoding RNA comprises at least the following elements, for example, in the 5' to 3' direction:

[0307] A) a 5' cap structure, e.g. as described in detail herein;

[0308] B) at least one cds encoding an antigenic polypeptide selected from or derived from E. coli FimH as defined herein;

[0309] C) a 5'-UTR and / or a 3'-UTR, e.g. as specifically described herein;

[0310] D) at least one poly(A) sequence, e.g. as specified herein;

[0311] In various embodiments, the coding RNA, such as an mRNA, comprises the following elements, such as in the 5' to 3' direction:

[0312] A) a 5' cap structure, e.g. as described in detail herein;

[0313] B) a 5' initiation element, e.g. as specifically described herein;

[0314] C) optionally, a 5'-UTR, e.g. as specified herein;

[0315] D) a ribosome binding site, e.g. as specified herein;

[0316] E) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein;

[0317] F) 3'-UTR, e.g. as specified herein;

[0318] G) optionally, at least one poly(A) sequence, e.g. as specified herein;

[0319] H) optionally, at least one poly(C) sequence, e.g. as specified herein;

[0320] I) optionally, a histone stem-loop, e.g. as specified herein;

[0321] J) optionally, a 3' terminal sequence element, e.g. as specified herein;

[0322] K) optionally, chemically modified nucleotides, e.g. as specified herein;

[0323] In various embodiments, the coding RNA, such as an mRNA, comprises the following elements, such as in the 5' to 3' direction:

[0324] A) 5' cap structure;

[0325] B) a 5'-UTR, for example, a 5'-UTR selected from or derived from the HSD17B4 gene;

[0326] C) at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein;

[0327] D) a 3'-UTR, for example, selected from or derived from a 3'-UTR of a PSMB3 gene;

[0328] E) optionally, a histone stem-loop; and

[0329] F) A poly(A) sequence, for example comprising about 100 A nucleotides.

[0330] In some embodiments, the mRNA comprises the following elements, for example, in the 5' to 3' direction:

[0331] A) a Cap 1 structure, e.g. as defined herein;

[0332] B) a 5'-UTR as defined herein derived from the HSD17B4 gene;

[0333] C) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, for example wherein the coding sequence comprises the same as SEQ ID NO: 187 to SEQ ID NO: 246, SEQ ID NO: 257 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or at least one of the nucleic acid sequences or fragments thereof that are identical or have at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the 670.

[0334] D) a 3'-UTR as defined herein derived from the 3'-UTR of the PSMB3 gene;

[0335] E) optionally, a histone stem-loop as defined herein;

[0336] F) a poly(A) sequence, for example comprising about 100 A nucleotides;

[0337] G) Optionally, chemically modified nucleotides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0338] In other embodiments, the mRNA comprises the following elements, for example, in the 5' to 3' direction:

[0339] A) a Cap 1 structure, e.g. as defined herein;

[0340] B) a 5'-UTR as defined herein derived from the HSD17B4 gene;

[0341] C) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, wherein the coding sequence comprises a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670, or a fragment thereof, or at least one of its fragments or variants.

[0342] D) a 3'-UTR as defined herein derived from the 3'-UTR of the PSMB3 gene;

[0343] E) optionally, a histone stem-loop as defined herein;

[0344] F) a poly(A) sequence, for example comprising about 100 A nucleotides;

[0345] G) Optionally, chemically modified nucleotides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0346] In some embodiments, the mRNA comprises the following elements in the 5' to 3' direction:

[0347] A) a Cap 1 structure as defined herein;

[0348] B) a 5'-UTR as defined herein derived from the HSD17B4 gene;

[0349] C) at least one cds encoding an E. coli FimH as defined herein or an antigenic polypeptide derived therefrom, for example wherein the coding sequence comprises a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence according to any one of SEQ ID NO: 529, SEQ ID NO: 554, SEQ ID NO: 579, SEQ ID NO: 604, SEQ ID NO: 629, SEQ ID NO: 654, or a fragment thereof, or at least one of its fragments or variants.

[0350] D) a 3'-UTR as defined herein derived from the 3'-UTR of the PSMB3 gene;

[0351] E) optionally, a histone stem-loop as defined herein;

[0352] F) a poly(A) sequence comprising about 100 A nucleotides, for example representing the 3' end;

[0353] G) Optionally, chemically modified nucleotides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0354] In some embodiments, the mRNA comprises the following elements in the 5' to 3' direction:

[0355] A) a Cap 1 structure as defined herein;

[0356] B) a 5'-UTR as defined herein derived from the HSD17B4 gene;

[0357] C) at least one cds encoding an E. coli FimH as defined herein or an antigenic polypeptide derived therefrom, for example wherein the coding sequence comprises a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence according to any one of SEQ ID NO: 533, SEQ ID NO: 558, SEQ ID NO: 583, SEQ ID NO: 608, SEQ ID NO: 633, SEQ ID NO: 658, or a fragment thereof, or at least one of its fragments or variants.

[0358] D) a 3'-UTR as defined herein derived from the 3'-UTR of the PSMB3 gene;

[0359] E) optionally, a histone stem-loop as defined herein;

[0360] F) a poly(A) sequence comprising about 100 A nucleotides, for example representing the 3' end;

[0361] G) Optionally, chemically modified nucleotides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0362] In some embodiments, the mRNA comprises the following elements in the 5' to 3' direction:

[0363] A) a Cap 1 structure as defined herein;

[0364] B) a 5'-UTR as defined herein derived from the HSD17B4 gene;

[0365] C) at least one cds encoding an E. coli FimH as defined herein or an antigenic polypeptide derived therefrom, for example wherein the coding sequence comprises a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence according to any one of SEQ ID NO: 534, SEQ ID NO: 559, SEQ ID NO: 584, SEQ ID NO: 609, SEQ ID NO: 634, SEQ ID NO: 659, or a fragment thereof, or at least one of its fragments or variants.

[0366] D) a 3'-UTR as defined herein derived from the 3'-UTR of the PSMB3 gene;

[0367] E) optionally, a histone stem-loop as defined herein;

[0368] F) a poly(A) sequence comprising about 100 A nucleotides, for example representing the 3' end;

[0369] G) Optionally, chemically modified nucleotides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0370] The RNA sequences are provided in Table 2. Therein, each row represents a specific suitable RNA construct of the present disclosure (as opposed to Table 1), wherein the description of the construct is indicated in Column A of Table 2, and the SEQ ID NO of the amino acid sequence of the respective construct is provided in Column B. The corresponding SEQ ID NO encoding the coding sequence of the respective construct is provided in Table 1.

[0371] Corresponding RNA sequences, in particular mRNA sequences, are provided in columns C and D, wherein column C provides an RNA sequence having the UTR combination "HSD17B4 / PSMB3" and a 3'-end hSL-A100 tail, and wherein column D provides a nucleic acid sequence having the UTR combination "HSD17B4 / PSMB3" and a 3'-end A100 tail.

[0372] Table 2: RNA constructs.

[0373]

[0374]

[0375]

[0376] Ec: Escherichia coli; HA: hemagglutinin; Hs: Homo sapiens; IgE: immunoglobulin E; IgK: immunoglobulin kappa; LumSynth, LS: dioxotetrahydropterin synthase; Mm: mouse; TMdomain, TM: transmembrane domain

[0377] In some embodiments, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: 995, SEQ ID NO: 998 to SEQ ID NO: 1020, SEQ ID NO: 1023 to SEQ ID NO: 1025, SEQ ID NO: 1026 SEQ ID NO: 1194 to SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1250, SEQ ID NO: 1251 to SEQ ID NO: 1252, SEQ ID NO: 1253 to SEQ ID NO: 1254, SEQ ID NO: 1255 to SEQ ID NO: 1256, SEQ ID NO: 1257 to SEQ ID NO: 1258 1270, or a nucleic acid sequence that is identical to, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to, any of the nucleic acid sequences in 1270.

[0378] In other embodiments, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: 995, SEQ ID NO: 998 to SEQ ID NO: 1020, SEQ ID NO: 1023 to SEQ ID NO: 1025, SEQ ID NO: 1026 SEQ ID NO: 1194 to SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1250, SEQ ID NO: 1251 to SEQ ID NO: 1252, SEQ ID NO: 1253 to SEQ ID NO: 1254, SEQ ID NO: 1255 to SEQ ID NO: 1256, SEQ ID NO: 1257 to SEQ ID NO: 1258 1270 or a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof, wherein at least one of the RNA sequences, for example all of the uracil nucleotides, are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.In one embodiment, the encoding RNA comprises or consists of a nucleic acid sequence or fragments or variants thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1271 to SEQ ID NO: 1273, wherein all uracil nucleotides in the RNA sequence are replaced by N1-methyl pseudouridine (m1ψ) nucleotides. In one embodiment, the encoding RNA comprises or consists of a nucleic acid sequence or fragments or variants thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1274 to SEQ ID NO: 1276, wherein all uracil nucleotides in the RNA sequence are replaced by pseudouridine (ψ) nucleotides.

[0379] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 679, SEQ ID NO: 704, SEQ ID NO: 729, SEQ ID NO: 754, SEQ ID NO: 779, SEQ ID NO: 804, SEQ ID NO: 829, SEQ ID NO: 854, SEQ ID NO: 879, SEQ ID NO: 904, SEQ ID NO: 929, SEQ ID NO: 954, SEQ ID NO: 979, SEQ ID NO: 1004, SEQ ID NO: 1029, SEQ ID NO: 1054, SEQ ID NO: 1079, SEQ ID NO: 1104, SEQ ID NO: 1129, SEQ ID NO: 1154, SEQ ID NO: 1179, SEQ ID NO: 1204, SEQ ID NO: 1229, SEQ ID NO: 1254 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally does not comprise chemically modified nucleotides.

[0380] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 683, SEQ ID NO: 708, SEQ ID NO: 733, SEQ ID NO: 758, SEQ ID NO: 783, SEQ ID NO: 808, SEQ ID NO: 833, SEQ ID NO: 858, SEQ ID NO: 883, SEQ ID NO: 908, SEQ ID NO: 933, SEQ ID NO: 958, SEQ ID NO: 983, SEQ ID NO: 1008, SEQ ID NO: 1033, SEQ ID NO: 1058, SEQ ID NO: 1083, SEQ ID NO: 1108, SEQ ID NO: 1133, SEQ ID NO: 1158, SEQ ID NO: 1183, SEQ ID NO: 1208, SEQ ID NO: 1233, SEQ ID NO: 1258 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally does not comprise chemically modified nucleotides.

[0381] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 684, SEQ ID NO: 709, SEQ ID NO: 734, SEQ ID NO: 759, SEQ ID NO: 784, SEQ ID NO: 809, SEQ ID NO: 834, SEQ ID NO: 859, SEQ ID NO: 884, SEQ ID NO: 909, SEQ ID NO: 934, SEQ ID NO: 959, SEQ ID NO: 984, SEQ ID NO: 1009, SEQ ID NO: 1034, SEQ ID NO: 1059, SEQ ID NO: 1084, SEQ ID NO: 1109, SEQ ID NO: 1134, SEQ ID NO: 1159, SEQ ID NO: 1184, SEQ ID NO: 1209, SEQ ID NO: 1234, SEQ ID NO: 1259 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally does not comprise chemically modified nucleotides.

[0382] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 679, SEQ ID NO: 704, SEQ ID NO: 729, SEQ ID NO: 754, SEQ ID NO: 779, SEQ ID NO: 804, SEQ ID NO: 829, SEQ ID NO: 854, SEQ ID NO: 879, SEQ ID NO: 904, SEQ ID NO: 929, SEQ ID NO: 954, SEQ ID NO: 979, SEQ ID NO: 1004, SEQ ID NO: 1029, SEQ ID NO: 1054, SEQ ID NO: 1079, SEQ ID NO: 1104, SEQ ID NO: 1129, SEQ ID NO: 1154, SEQ ID NO: 1179, SEQ ID NO: 1204, SEQ ID NO: 1229, SEQ ID NO: 1254 or a nucleic acid sequence or fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally comprises a pseudouridine (ψ) nucleotide.

[0383] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 683, SEQ ID NO: 708, SEQ ID NO: 733, SEQ ID NO: 758, SEQ ID NO: 783, SEQ ID NO: 808, SEQ ID NO: 833, SEQ ID NO: 858, SEQ ID NO: 883, SEQ ID NO: 908, SEQ ID NO: 933, SEQ ID NO: 958, SEQ ID NO: 983, SEQ ID NO: 1008, SEQ ID NO: 1033, SEQ ID NO: 1058, SEQ ID NO: 1083, SEQ ID NO: 1108, SEQ ID NO: 1133, SEQ ID NO: 1158, SEQ ID NO: 1183, SEQ ID NO: 1208, SEQ ID NO: 1233, SEQ ID NO: 1258 or a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally comprises a pseudouridine (ψ) nucleotide.

[0384] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 684, SEQ ID NO: 709, SEQ ID NO: 734, SEQ ID NO: 759, SEQ ID NO: 784, SEQ ID NO: 809, SEQ ID NO: 834, SEQ ID NO: 859, SEQ ID NO: 884, SEQ ID NO: 909, SEQ ID NO: 934, SEQ ID NO: 959, SEQ ID NO: 984, SEQ ID NO: 1009, SEQ ID NO: 1034, SEQ ID NO: 1059, SEQ ID NO: 1084, SEQ ID NO: 1109, SEQ ID NO: 1134, SEQ ID NO: 1159, SEQ ID NO: 1184, SEQ ID NO: 1209, SEQ ID NO: 1234, SEQ ID NO: 1259 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally comprises a pseudouridine (ψ) nucleotide.

[0385] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 679, SEQ ID NO: 704, SEQ ID NO: 729, SEQ ID NO: 754, SEQ ID NO: 779, SEQ ID NO: 804, SEQ ID NO: 829, SEQ ID NO: 854, SEQ ID NO: 879, SEQ ID NO: 904, SEQ ID NO: 929, SEQ ID NO: 954, SEQ ID NO: 979, SEQ ID NO: 1004, SEQ ID NO: 1029, SEQ ID NO: 1054, SEQ ID NO: 1079, SEQ ID NO: 1104, SEQ ID NO: 1129, SEQ ID NO: 1154, SEQ ID NO: 1179, SEQ ID NO: 1204, SEQ ID NO: 1229, SEQ ID NO: 1254 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this case, the RNA sequence optionally comprises N1-methyl pseudouridine (m1ψ) nucleotides.

[0386] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 683, SEQ ID NO: 708, SEQ ID NO: 733, SEQ ID NO: 758, SEQ ID NO: 783, SEQ ID NO: 808, SEQ ID NO: 833, SEQ ID NO: 858, SEQ ID NO: 883, SEQ ID NO: 908, SEQ ID NO: 933, SEQ ID NO: 958, SEQ ID NO: 983, SEQ ID NO: 1008, SEQ ID NO: 1033, SEQ ID NO: 1058, SEQ ID NO: 1083, SEQ ID NO: 1108, SEQ ID NO: 1133, SEQ ID NO: 1158, SEQ ID NO: 1183, SEQ ID NO: 1208, SEQ ID NO: 1233, SEQ ID NO: 1258 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally comprises N1-methyl pseudouridine (m1ψ) nucleotides.

[0387] In one embodiment, the encoding RNA comprises or consists of the sequence of SEQ ID NO: 684, SEQ ID NO: 709, SEQ ID NO: 734, SEQ ID NO: 759, SEQ ID NO: 784, SEQ ID NO: 809, SEQ ID NO: 834, SEQ ID NO: 859, SEQ ID NO: 884, SEQ ID NO: 909, SEQ ID NO: 934, SEQ ID NO: 959, SEQ ID NO: 984, SEQ ID NO: 1009, SEQ ID NO: 1034, SEQ ID NO: 1059, SEQ ID NO: 1084, SEQ ID NO: 1109, SEQ ID NO: 1134, SEQ ID NO: 1159, SEQ ID NO: 1184, SEQ ID NO: 1209, SEQ ID NO: 1234, SEQ ID NO: 1259 or a nucleic acid sequence or a fragment or variant thereof that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this embodiment, the RNA sequence optionally comprises a 5' end cap 1 structure. In this embodiment, the RNA sequence optionally comprises N1-methyl pseudouridine (m1ψ) nucleotides.

[0388] In other embodiments, the coding RNA of the present disclosure is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), which is identical to the RNA sequence according to any one of SEQ ID NO: 679, SEQ ID NO: 829, SEQ ID NO: 979, SEQ ID NO: 1129, or is a fragment or variant thereof.

[0389] In other embodiments, the coding RNA of the present disclosure is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), which is identical to the RNA sequence according to any one of SEQ ID NO: 683, SEQ ID NO: 833, SEQ ID NO: 983, SEQ ID NO: 1133, or is a fragment or variant thereof.

[0390] In other embodiments, the coding RNA of the present disclosure is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), which is identical to the RNA sequence according to any one of SEQ ID NO: 684, SEQ ID NO: 834, SEQ ID NO: 984, SEQ ID NO: 1134, or is a fragment or variant thereof.

[0391] In one embodiment, the coding RNA of the present disclosure is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, and the RNA sequence is the same as the RNA sequence according to any one of SEQ ID NO: 679, SEQ ID NO: 829, SEQ ID NO: 979, SEQ ID NO: 1129, SEQ ID NO: 1274, or is a fragment or variant thereof.

[0392] In one embodiment, the coding RNA of the present disclosure is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, which is identical to the RNA sequence according to any one of SEQ ID NO: 683, SEQ ID NO: 833, SEQ ID NO: 983, SEQ ID NO: 1133, SEQ ID NO: 1275 or is a fragment or variant thereof.

[0393] In one embodiment, the coding RNA of the present disclosure is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, which is identical to the RNA sequence according to any one of SEQ ID NO: 684, SEQ ID NO: 834, SEQ ID NO: 984, SEQ ID NO: 1134, SEQ ID NO: 1276 or is a fragment or variant thereof.

[0394] In one embodiment, the coding RNA of the present invention is a 5'-capped (cap 1) mRNA, which contains or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides, which is identical to the RNA sequence according to any one of SEQ ID NO: 679, SEQ ID NO: 829, SEQ ID NO: 979, SEQ ID NO: 1129, SEQ ID NO: 1271 or is a fragment or variant thereof.

[0395] In one embodiment, the coding RNA of the present invention is a 5'-capped (cap 1) mRNA, which contains or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides, which is identical to the RNA sequence according to any one of SEQ ID NO: 683, SEQ ID NO: 833, SEQ ID NO: 983, SEQ ID NO: 1133, SEQ ID NO: 1272 or is a fragment or variant thereof.

[0396] In one embodiment, the coding RNA of the present invention is a 5'-capped (cap 1) mRNA, which contains or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides, which is identical to the RNA sequence according to any one of SEQ ID NO: 684, SEQ ID NO: 834, SEQ ID NO: 984, SEQ ID NO: 1134, SEQ ID NO: 1273 or is a fragment or variant thereof.

[0397] 2: Compositions comprising RNA encoding an Escherichia coli FimH antigenic polypeptide

[0398] In a second aspect, a pharmaceutical composition comprising the RNA encoding the first aspect is provided.

[0399] It is noteworthy that the embodiments of the pharmaceutical composition related to the second aspect can similarly continue to be read and understood as suitable embodiments of the vaccine of the third aspect. The embodiments of the vaccine related to the third aspect can also similarly continue to be read and understood as suitable embodiments of the pharmaceutical composition of the second aspect. In addition, the features and embodiments (RNA of the present disclosure) described in the context of the first aspect must continue to be read and must be understood as suitable embodiments of the pharmaceutical composition of the second aspect.

[0400] In the context of the present disclosure, "composition" refers to any type of composition, in which a specified component (e.g., a coding RNA comprising (a) at least one untranslated region (UTR); and (b) a coding sequence encoding an antigenic polypeptide selected from or derived from E. coli FimH operably linked to the UTR) can be incorporated, optionally with any other components, usually with at least one pharmaceutically acceptable carrier or excipient. The composition can be a dry composition, such as a powder, granules or solid lyophilized form. Alternatively, the composition can be in liquid form, and each component can be incorporated independently in a dissolved or dispersed (e.g., suspended or emulsified) form.

[0401] In various embodiments, the encoding RNA of the pharmaceutical composition is selected from the encoding RNA defined in any embodiment of the first aspect.

[0402] In an embodiment, the encoding RNA contained in the pharmaceutical composition is provided in an amount of at least about 100 ng to a maximum of about 500 μg, provided in an amount of at least about 1 μg to a maximum of about 200 μg, provided in an amount of at least about 1 μg to a maximum of about 100 μg, provided in an amount of at least about 5 μg to a maximum of about 100 μg, for example, provided in an amount of at least about 10 μg to a maximum of about 50 μg, specifically provided in an amount of about 1 μg, 2 μg, 3 μg , 4µg, 5µg, 6µg, 7µg, 8µg, 9µg, 10µg, 11µg, 12µg, 13µg, 14µg, 15µg, 20µg, 25µg, 30µg, 35µg, 40µg, 45µg, 50µg, 55µg, 60µg, 65µg, 70µg, 75µg, 80µg, 85µg, 90µg, 95µg or 100µg.

[0403] In one embodiment, the coding RNA of the composition comprises or consists of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: SEQ ID NO: 1194 to SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, SEQ ID NO: 1273 to SEQ ID NO: 1295, SEQ ID NO: 1309 to SEQ ID NO: 1310, SEQ ID NO: 1311 to SEQ ID NO: 1320, SEQ ID NO: 1323 to SEQ ID NO: 1345, SEQ ID NO: 1350 to SEQ ID NO: 1360, SEQ ID NO: 1361 to SEQ ID NO: 1372 NO:1276 or an RNA sequence or fragment or variant thereof that is identical or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0404] In some embodiments, the pharmaceutical composition comprises multiple or at least more than one RNA species.

[0405] In one embodiment, the pharmaceutical composition comprises a first encoding RNA according to the first aspect and a second encoding RNA encoding a polypeptide selected from or derived from Escherichia coli FimC.

[0406] In an embodiment, the second coding sequence encodes a FimC amino acid sequence that is identical to any one of SEQ ID NO: 317, SEQ ID NO: 324, SEQ ID NO: 496, SEQ ID NO: 497, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof. This is particularly suitable when the first coding sequence encodes a FimH amino acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, in particular SEQ ID NO: 498, SEQ ID NO: 500, or an immunogenic fragment or immunogenic variant thereof.

[0407] In an embodiment, the second coding RNA comprises a FimC coding sequence comprising a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 318 to SEQ ID NO: 323, SEQ ID NO: 325 to SEQ ID NO: 330, SEQ ID NO: 521, SEQ ID NO: 522, SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 571, SEQ ID NO: 572, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 646, SEQ ID NO: 647, or at least one of its fragments or variants. This is particularly suitable when the first coding sequence comprises a FimH coding sequence comprising at least one of a nucleic acid sequence that is identical to, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 187 to SEQ ID NO: 246, SEQ ID NO: 257 to SEQ ID NO: 316, SEQ ID NO: 523, SEQ ID NO: 525, SEQ ID NO: 548, SEQ ID NO: 550, SEQ ID NO: 573, SEQ ID NO: 575, SEQ ID NO: 598, SEQ ID NO: 600, SEQ ID NO: 623, SEQ ID NO: 625, SEQ ID NO: 648, SEQ ID NO: 650.

[0408] In an embodiment, the second coding RNA comprises or consists of a sequence according to SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 746, SEQ ID NO: 747, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 796, SEQ ID NO: 797, SEQ ID NO: 821, SEQ ID NO: 822, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 871, SEQ ID NO: 872, SEQ ID NO: 896, SEQ ID NO: 897, SEQ ID NO: 921, SEQ ID NO: 922, SEQ ID NO: 946, SEQ ID NO: 947, SEQ ID NO: 971, SEQ ID NO: 972, SEQ ID NO: NO: 996, SEQ ID NO: 997, SEQ ID NO: 1021, SEQ ID NO: 1022, SEQ ID NO: 1046, SEQ ID NO: 1047, SEQ ID NO: 1071, SEQ ID NO: 1072, SEQ ID NO: 1096, SEQ ID NO: 1097, SEQ ID NO: 1121, SEQ ID NO: 1122, SEQ ID NO: 1146, SEQ ID NO: 1147, SEQ ID NO: 1171, SEQ ID NO: 1172, SEQ ID NO: 1196, SEQ ID NO: 1197, SEQ ID NO: 1221, SEQ ID NO: 1222, SEQ ID NO: 1246, SEQ ID NO: 1247 or a FimC encoding nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant of any of these sequences, optionally wherein at least one uracil nucleotide in the RNA sequence, such as all uracil nucleotides, is replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0409] It is noteworthy that suitable nucleic acid features (e.g. UTRs, cap structures, modifications, codon optimization) disclosed in the context of the FimH encoding RNA sequence of the first aspect may also be applicable and may also be suitable for the RNA sequence of the second aspect encoding FimC.

[0410] The specific features and embodiments of the coding RNA of the first aspect as provided herein may also be applied to the second coding RNA encoding FimC. It is particularly suitable to provide a pharmaceutical composition comprising a first coding RNA encoding FimH and a second coding RNA encoding FimC, so that once the FimH polypeptide and FimC polypeptide encoded by the first coding RNA and the second coding RNA are translated, they can be assembled into a non-covalent complex. This is particularly suitable for stabilizing FimH when the coding sequence of the first RNA encoding FimH does not encode a donor chain peptide.

[0411] The E. coli FimC sequences and constructs are reported in Tables 1 and 2.

[0412] In various embodiments, the RNA encoding the pharmaceutical composition is formulated together with a pharmaceutically acceptable carrier or excipient.

[0413] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is, for example, a liquid base or non-liquid base including a composition for administration. If the composition is provided in liquid form, the carrier can be water, such as pyrogen-free water; isotonic saline or a buffered (aqueous) solution, such as a phosphate, citrate, or other buffered solution. Water or, for example, a buffer, such as an aqueous buffer, including, for example, a sodium salt, a calcium salt, or a potassium salt can be used. According to some embodiments, the sodium salt, calcium salt, or potassium salt may be present in the form of its halide, such as chloride, iodide, or bromide, in the form of its hydroxide, carbonate, bicarbonate, or sulfate, or the like. Examples of sodium salts include NaCl, Na2HPO4, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of optional potassium salts include KCl, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include CaCl2, CaI2, CaBr2, CaCO3, CaSO4, Ca(OH)2.

[0414] In addition, the organic anion of the above-mentioned cation can be in a buffer. Therefore, in embodiments, the pharmaceutical composition can include a pharmaceutically acceptable carrier or excipient, using one or more than one pharmaceutically acceptable carrier or excipient to, for example, increase stability, increase cell transfection, allow to continue or delay, increase the in vivo translation of the encoded antigenic polypeptide, and / or change the in vivo release curve of the encoded antigenic peptide. Except traditional excipients such as any and all solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, excipients of the present disclosure can include but are not limited to lipidoids, liposomes, lipid nanoparticles, polymers, liposome complexes (lipoplex), core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics and combinations thereof. In embodiments, one or more than one compatible solid filler or liquid filler or diluent or encapsulated compound can also be used, which is suitable for use to an object. As used herein, the term "compatible" means that the components of the composition can be mixed with at least one nucleic acid, optionally multiple nucleic acids, of the composition in a manner that does not interact, and the interaction would significantly reduce the biological activity or drug efficacy of the composition under typical conditions of use (e.g., intramuscular or intradermal administration). A pharmaceutically acceptable carrier or excipient must have a sufficiently high purity and a sufficiently low toxicity to make it suitable for administration to a subject to be treated. Compounds that can be used as pharmaceutically acceptable carriers or excipients can be sugars, such as, for example, lactose, glucose, trehalose, mannose and sucrose; starches, such as corn starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; animal fats and oils; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from cocoa; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

[0415] The pharmaceutical compositions of the present disclosure are suitably sterile and / or pyrogen-free.

[0416] Preparation / complexation:

[0417] In some embodiments, the encoding RNA of the pharmaceutical composition is complexed or associated with at least one other compound to obtain a formulated composition. The formulation in this context may function as a transfection reagent. The formulation in this context may also function to protect the RNA from degradation, for example, thereby allowing storage, transportation, etc.

[0418] In some embodiments, the encoding RNA of the pharmaceutical composition is formulated with at least one compound, such as a peptide, protein, lipid, polysaccharide, and / or polymer.

[0419] In some embodiments, the encoding RNA of the pharmaceutical composition is formulated with at least one cationic (cationic or, e.g., ionizable) or polycationic (cationic or, e.g., ionizable) compound.

[0420] In some embodiments, the encoding RNA of the pharmaceutical composition is complexed or associated, or at least partially complexed or partially associated, with one or more than one cationic (cationic or, e.g., ionizable) or polycationic compound.

[0421] The term "cationic or polycationic compound" as used herein will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to refer to a charged molecule that is positively charged at a pH of about 1 to 9, a pH of about 3 to 8, a pH of about 4 to 8, a pH of about 5 to 8, for example, a pH of about 6 to 8, for example, a pH of about 7 to 8, for example, a physiological pH, for example, about 7.2 to about 7.5. Thus, a cationic component, such as a cationic peptide, a cationic protein, a cationic polymer, a cationic polysaccharide, a cationic lipid, can be any positively charged compound or polymer that is positively charged under physiological conditions. A "cationic or polycationic peptide or protein" may contain at least one positively charged amino acid, or more than one positively charged amino acid, such as selected from Arg, His, Lys or Orn. ​​Thus, a "polycationic" component also falls within the scope of exhibiting more than one positive charge under given conditions.

[0422] In some embodiments, the at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combination thereof.

[0423] In some embodiments, the at least one cationic or polycationic compound is selected from cationic or polycationic peptides or proteins.

[0424] In some embodiments, the pharmaceutical composition comprises an encoding RNA as defined herein and a polymer carrier.

[0425] The term "polymer carrier" as used herein will be recognized and understood by those of ordinary skill in the art, and is, for example, intended to refer to a compound that promotes the transport and / or complexing of another compound. A polymer carrier is typically a carrier formed by a polymer. A polymer carrier can be associated with its cargo (e.g., RNA) by covalent or non-covalent interactions. Polymers can be based on different subunits, such as copolymers. Suitable polymer carriers in this context can include, for example, polyethyleneimine (PEI).

[0426] In an embodiment, the pharmaceutical composition comprises at least one RNA complexed or associated with a polymer carrier, and optionally complexed or associated with at least one lipid component as described in WO2017212008, WO2017212006, WO2017212007 and WO2017212009. In this context, the disclosures of WO2017212008, WO2017212006, WO2017212007 and WO2017212009 are incorporated herein by reference. In some embodiments, the lipidoid component of the polymer carrier can be any lipidoid component selected from the table of lipidoid structures of published PCT patent application WO2017212009A1 (pages 50 to 54).

[0427] Formulations in lipid-based vehicles:

[0428] In some embodiments, the pharmaceutical composition comprises a lipid-based carrier.

[0429] In the context of the present disclosure, the term "lipid-based carrier" encompasses a lipid-based delivery system for RNA, which comprises a lipid component. The lipid-based carrier may additionally comprise other components suitable for encapsulating / incorporating / complexing RNA, including cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof.

[0430] In the context of the present disclosure, typical "lipid-based carriers" are selected from liposomes, lipid nanoparticles (LNPs), lipoplexes and / or nanoliposomes. The RNA of the pharmaceutical composition can be fully or partially incorporated or encapsulated in a lipid-based carrier, wherein the RNA can be located in the interior space of the lipid-based carrier, within the lipid layer / membrane of the lipid-based carrier, or associated with the outer surface of the lipid-based carrier. Incorporation of RNA into a lipid-based carrier can be referred to as "encapsulation". "Lipid-based carriers" are not limited to any particular morphology, and include any morphology generated when, for example, a lipid that reduces aggregation is combined with at least one other lipid, for example, in the presence of an aqueous environment of RNA. For example, LNPs, liposomes, lipoplexes, lipoplexes, etc. fall within the scope of the term "lipid-based carrier". Lipid-based carriers can be of different sizes, such as, but not limited to: multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be 50 nm to 500 nm in diameter. Liposomes, a specific type of lipid-based carrier, are characterized by microscopic vesicles with an internal water space that are isolated from the external medium by one or more bilayer membranes. In liposomes, at least one RNA is typically located in the internal water space encapsulated by some or all of the lipid portions of the liposome. The bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin, which contain spatially separated hydrophilic domains and hydrophobic domains. Lipid nanoparticles (LNPs), a specific type of lipid-based carrier, are characterized by microscopic lipid particles with a solid core or a partially solid core. Typically, LNPs do not contain an internal water space isolated from the external medium by a bilayer. In LNP, at least one RNA can be encapsulated or incorporated into the lipid portion of LNP, which is coated by some or all of the lipid portions of LNP. LNP can include any lipid that can form a particle, and RNA can be attached to the particle, or RNA can be encapsulated in the particle. For example, the lipid-based carrier is particularly suitable for intramuscular and / or intradermal administration.

[0431] In some embodiments, the lipid-based carrier of the pharmaceutical composition is selected from a liposome, a lipid nanoparticle, a liposome complex, and / or a nanoliposome.

[0432] In one embodiment, the lipid-based carrier of the pharmaceutical composition is a lipid nanoparticle (LNP). In one embodiment, the lipid nanoparticle of the pharmaceutical composition encapsulates the RNA encoding the present disclosure.

[0433] The term "encapsulated", such as incorporated, complexed, encapsulated, partially encapsulated, associated, partially associated, refers to RNA and one or more than one lipid being substantially stably combined into a lipid-based carrier (e.g., a large complex or assembly), wherein the combination is, for example, free of covalent binding of RNA. The RNA encapsulated by a lipid-based carrier can be completely or partially located within the interior of the lipid-based carrier (e.g., lipid portion and / or internal space) and / or within the lipid layer / membrane of the lipid-based carrier. Since the RNA is, for example, contained within the interior of a lipid-based carrier, encapsulating the RNA into a lipid-based carrier is also referred to herein as "incorporating". Without wishing to be bound by theory, the purpose of incorporating or encapsulating the RNA into a lipid-based carrier may be to protect the RNA from an environment that may contain enzymes, chemicals, or conditions that degrade the RNA. In addition, incorporating the RNA into a lipid-based carrier can promote the uptake of the RNA, and therefore when administered to a cell or object, the therapeutic effect of the RNA can be enhanced.

[0434] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises at least one or more than one lipid selected from at least one aggregation-reducing lipid, at least one cationic lipid, at least one neutral lipid or phospholipid, or at least one steroid or steroid analog, or any combination thereof.

[0435] In one embodiment, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid, a cationic lipid or an ionizable lipid, a neutral lipid or a phospholipid, and a steroid or a steroid analog.

[0436] Cationic lipids:

[0437] In some embodiments, the lipid-based carrier comprises a cationic lipid or an ionizable lipid.

[0438] Cationic lipids or ionizable lipids of lipid-based carriers can be cationizable or ionizable, i.e., they become protonated when the pH is reduced to below the pK of the ionizable groups of the lipid, but gradually become more neutral at higher pH values. At pH values ​​below the pK, the lipids are able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipids comprise zwitterionic lipids, which are positively charged when the pH is reduced.

[0439] In some embodiments, the lipid-based carrier comprises a cationic lipid or an ionizable lipid, which, for example, carries a net positive charge at physiological pH, for example, the cationic or ionizable lipid comprises a tertiary nitrogen group or a quaternary nitrogen group. Thus, in some embodiments, the lipid-based carrier comprises a cationic lipid or an ionizable lipid selected from an amino lipid.

[0440] In other embodiments, the lipid formulation comprises a cationic lipid or ionizable lipid as defined in formula I in paragraph

[00251] of WO2021222801 or a lipid selected from the disclosure of paragraph

[00260] or

[00261] of WO2021222801. In other embodiments, the lipid formulation comprises a cationic lipid or ionizable lipid selected from ATX-001 to ATX-132 such as ATX-0126 disclosed in claim 90 of WO2021183563. The disclosures of WO2021222801 and WO2021183563, in particular the above-mentioned lipids, are incorporated herein by reference.

[0441] In an embodiment, cationic lipids or ionizable lipids can be selected from lipids disclosed in WO2018078053 (i.e., lipids derived from formula I, formula II and formula III of WO2018078053, or lipids as specifically described in claims 1 to 12 of WO2018078053), and the disclosure of WO2018078053 is incorporated herein by reference as a whole. In this context, lipids disclosed in Table 7 of WO2018078053 (e.g., lipids derived from formula I-1 to formula I-41) and lipids disclosed in Table 8 of WO2018078053 (e.g., lipids derived from formula II-1 to formula II-36) are applicable to the context of the present disclosure. Therefore, Formula I-1 to Formula I-41 and Formula II-1 to Formula II-36 of WO2018078053, and the specific disclosures related thereto are incorporated herein by reference.

[0442] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises a cationic lipid selected from or derived from structures III-1 to III-36 of Table 9 of published PCT patent application WO2018078053. Therefore, Formula III-1 to Formula III-36 of WO2018078053, and the specific disclosures related thereto, are incorporated herein by reference.

[0443] In various embodiments, the lipid-based carrier (eg, LNP) of the pharmaceutical composition (eg, component B) of the present disclosure comprises a cationic lipid according to formula (III) or derived from formula (III):

[0444]

[0445] Formula (III) is further defined as:

[0446] One of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, or -NRaC(=O)O-, or a direct bond;

[0447] G1 and G2 are each independently an unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0448] G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0449] Ra is H or C1-C12 alkyl;

[0450] R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0451] R3 is H, OR5, CN, C(=O)OR4, OC(=O)R4 or -NR5C(=O)R4;

[0452] R4 is a C1-C12 alkyl group;

[0453] R5 is H or C1-C6 alkyl; and

[0454] x is 0, 1, or 2.

[0455] In one embodiment, the lipid-based carrier comprises a cationic lipid selected from or derived from Formula III-3:

[0456]

[0457] The lipid of formula III-3 as used herein has the chemical name ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also known as ALC-0315, CAS number 2036272-55-4.

[0458] Other suitable cationic lipids may be selected from or derived from the cationic lipids according to PCT claims 1 to 14 of published patent application WO2021123332 or Table 1 of WO2021123332, and the disclosure relating to claims 1 to 14 or Table 1 of WO2021123332 is incorporated herein by reference.

[0459] Therefore, suitable cationic lipids may be selected from or derived from the cationic lipids of compounds 1 to 27 (C1 to C27) according to Table 1 of WO2021123332.

[0460] In other embodiments, the lipid-based carrier (eg, LNP) of the pharmaceutical composition comprises a lipid-based carrier selected from or derived from (COATSOME ® SS-EC) cationic lipid of SS-33 / 4PE-15 (see C23 in Table 1 of WO2021123332).

[0461] In other embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from HEXA-C5DE-PipSS (see C2 in Table 1 of WO2021123332). In some embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from compound C26 as disclosed in Table 1 of WO2021123332 (see C23 in Table 1 of WO2021123332):

[0462]

[0463] In other embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from 9-heptadecyl-8-{(2-hydroxyethyl)[6-oxo-6-((undecyloxy)hexyl]amino}octanoate, also known as SM-102. Other lipid-based carriers (e.g., LNP) of the pharmaceutical composition comprise a squaramide ionizable amino lipid, such as a cationic lipid selected from formula (M1) and (M2):

[0464] (M1);

[0465] (M2);

[0466] Wherein in claims 1 to 13 of US10392341B2, substituents (such as R1, R2, R3, R5, R6, R7, R 10 , M, M1, m, n, o, l); US10392341B2 is incorporated herein by reference in its entirety.

[0467] Therefore, in some embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from ALC-0315, SM-102, SS-33 / 4PE-15, HEXA-C5DE-PipSS or compound C26 (see C26 in Table 1 of WO2021123332) described above.

[0468] In one embodiment, the lipid-based carrier of the pharmaceutical composition, such as LNP, comprises a cationic lipid selected from or derived from ALC-0315.

[0469] In some embodiments, the lipid-based carriers of the present disclosure comprise two or more than two (different) cationic lipids as defined herein.

[0470] In certain embodiments, a cationic lipid as defined herein, such as cationic lipid ALC-0315, is present in the lipid-based carrier in an amount of about 30 mol % to about 95 mol % relative to the total lipid content of the lipid-based carrier. If more than one cationic lipid is incorporated into the lipid-based carrier, this percentage applies to the combined cationic lipids.

[0471] In embodiments, the cationic lipid is present in the carrier based on lipid with an amount of about 30 % by mole to about 70 % by mole. In one embodiment, the cationic lipid is present in the carrier based on lipid with an amount of about 40 % by mole to about 60 % by mole, as respectively with an amount of about 40 % by mole, 41 % by mole, 42 % by mole, 43 % by mole, 44 % by mole, 45 % by mole, 46 % by mole, 47 % by mole, 48 % by mole, 49 % by mole, 50 % by mole, 51 % by mole, 52 % by mole, 53 % by mole, 54 % by mole, 55 % by mole, 56 % by mole, 57 % by mole, 58 % by mole, 59 % by mole or 60 % by mole. In embodiments, cationic lipid is with the amount of about 47 mol % to about 48 mol %, such as respectively with the amount of about 47.0 mol %, 47.1 mol %, 47.2 mol %, 47.3 mol %, 47.4 mol %, 47.5 mol %, 47.6 mol %, 47.7 mol %, 47.8 mol %, 47.9 mol %, 50.0 mol %, is present in the carrier based on lipid, wherein 47.4 mol % is particularly suitable. In other embodiments, cationic lipid is with the amount of about 55 mol % to about 65 mol %, such as respectively with the amount of about 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol % or 65 mol %, is present in the carrier based on lipid, wherein 59 mol % is particularly suitable.

[0472] In some embodiments, cationic lipid accounts for about 20 mol % to about 70 mol % or 75 mol % or about 45 mol % to about 65 mol % or about 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol % or about 70 mol % ratio of the total lipid present in the carrier based on lipid.In other embodiments, in mole (total mole of the lipid in lipid nanoparticle is 100% as a basis), LNP comprises about 25% to about 75% cationic lipid, for example, in mole, about 20% to about 70%, about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about 40%.

[0473] In some embodiments, the ratio of cationic lipid to RNA is about 3 to about 15, such as about 5 to about 13 or about 7 to about 11.

[0474] Reduces Aggregated Lipids:

[0475] The term "lipids that reduce aggregation" refers to molecules comprising a lipid portion and a portion suitable for reducing or preventing aggregation of lipid-based carriers. Under storage conditions or during formulation, lipid-based carriers may undergo charge-induced aggregation, which may be undesirable for the stability of lipid-based carriers. Therefore, it is desirable to include lipid compounds that can reduce aggregation, such as lipid compounds that reduce aggregation by stabilizing the lipid-based carrier space. When the compound has a large but uncharged portion that shields or isolates the charged portion of the lipid-based carrier so that it does not approach other lipid-based carriers in the composition at close range, this spatial stabilization can occur. In the context of the present disclosure, the stabilization of the lipid-based carrier is achieved by including a lipid, which may include a lipid with a large sterically hindered group, which is, for example, located outside the lipid-based carrier after the lipid-based carrier is formed. Suitable groups that reduce aggregation include hydrophilic groups, such as monosialoganglioside GM1, polyamide oligomers (PAO) or certain polymers, such as poly (oxyalkylenes), such as poly (ethylene glycol) or poly (propylene glycol).

[0476] Lipids that contain a polymer as an aggregation-reducing group are referred to herein as "polymer-conjugated lipids."

[0477] The term "polymer-conjugated lipid" refers to a molecule comprising a lipid portion and a polymer portion, wherein the polymer is suitable for reducing or preventing aggregation of a lipid-based vector comprising RNA. A polymer must be understood as a substance or material consisting of very large molecules, or a macromolecule consisting of many repeating subunits. In the context of the present disclosure, a suitable polymer may be a hydrophilic polymer. An example of a polymer-conjugated lipid is a PEGylated or PEG-conjugated lipid.

[0478] In one embodiment, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid selected from polymer-conjugated lipids.

[0479] In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (or PEGylated lipid, PEG lipid).

[0480] The average molecular weight of the PEG moiety in the PEG conjugated lipid is, for example, about 500 Daltons to about 8000 Daltons (e.g., about 1000 Daltons to about 4000 Daltons). In one embodiment, the average molecular weight of the PEG moiety is about 2000 Daltons.

[0481] In some embodiments, the PEG conjugated lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is DMG-PEG 2000. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP comprises PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0482] In some embodiments, the polymer conjugated lipid, such as a PEG conjugated lipid, is selected from or derived from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG-PEG 2000). As used in the art, "DMG-PEG 2000" is generally considered to be a mixture of 1,2-DMG PEG 2000 and 1,3-DMG PEG 2000, wherein the ratio of the two is about 97:3.

[0483] In other embodiments, the polymer-conjugated lipid, such as a PEG-conjugated lipid, is selected from or derived from C10-PEG2K or Cer8-PEG2K.

[0484] In one embodiment, the polymer conjugated lipid, such as a PEG conjugated lipid, is selected from or derived from formula (IVa):

[0485] (IVa)

[0486] For example, wherein n has an average value of 30 to 60, such as about 30 ± 2, 32 ± 2, 34 ± 2, 36 ± 2, 38 ± 2, 40 ± 2, 42 ± 2, 44 ± 2, 46 ± 2, 48 ± 2, 50 ± 2, 52 ± 2, 54 ± 2, 56 ± 2, 58 ± 2 or 60 ± 2. In one embodiment, n is about 49. In another embodiment, n is 45. In other embodiments, the PEG lipid is of formula (IVa), wherein n is selected to be an integer such that the average molecular weight of the PEG lipid is from about 2000 g / mole to about 3000 g / mole or from about 2300 g / mole to about 2700 g / mole. In another embodiment, the PEG lipid is of formula (IVa), wherein n is selected to be an integer such that the average molecular weight of the PEG lipid is about 2000 g / mole.

[0487] The PEG conjugated lipid of formula IVa as used herein has the chemical structure 2[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide, which is also known as ALC-0159.

[0488] Thus, in some embodiments, the aggregation reducing lipid is a PEG conjugated lipid selected from or derived from DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K, or ALC-0159.

[0489] In one embodiment, the lipid-based carrier, such as LNP of the pharmaceutical composition, comprises an aggregation-reducing lipid selected from or derived from ALC-0159.

[0490] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid, wherein the aggregation-reducing lipid is not a PEG-conjugated lipid.

[0491] In some embodiments, based on the total mole of lipid in the lipid-based carrier, the lipid-based carrier comprises less than about 3 moles, 2 moles or 1 mole of the lipid that reduces aggregation. In other embodiments, based on moles (based on 100% total moles of lipid in the lipid-based carrier), the lipid-based carrier comprises about 0.1% to about 10% of the lipid that reduces aggregation, for example, based on moles, about 0.5% to about 10%, about 0.5% to about 5%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1%, about 0.5% or about 0.3%. In other embodiments, in mole (based on 100% total mole of lipid in lipid-based carrier), lipid-based carrier comprises about 1.0% to about 2.0% of the lipid that reduces aggregation, for example, about 1.2% to about 1.9%, about 1.2% to about 1.8%, about 1.3% to about 1.8%, about 1.4% to about 1.8%, about 1.5% to about 1.8%, about 1.6% to about 1.8%, particularly about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, for example 1.7%. In other embodiments, in mole (based on 100% total mole of lipid in lipid-based carrier), lipid-based carrier comprises about 2.5%, 3%, 3.5%, 4%, 4.5% or 5% such as 2.5% of the lipid that reduces aggregation. In various embodiments, the molar ratio of cationic lipid to the lipid that reduces aggregation is about 100:1 to about 25:1.

[0492] Neutral lipids:

[0493] In some embodiments, the lipid-based carrier (eg, LNP) comprises a neutral lipid or a phospholipid.

[0494] The term "neutral lipid" refers to any one of lipid species (lipid species) existing in the form of uncharged or neutral zwitterionic under physiological pH. Suitable neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. The selection of neutral lipids for particles described herein is usually guided by considering, for example, lipid particle size and the stability of lipid particles in the blood stream. For example, neutral lipids are lipids (such as diacylphosphatidylcholine and diacylphosphatidylethanolamine) with two acyl groups. In one embodiment, neutral lipids include saturated fatty acids of carbon chain lengths of C10 to C20. In another embodiment, monounsaturated fatty acids or diunsaturated fatty acids with carbon chain lengths of C10 to C20 are used. In addition, neutral lipids with a mixture of saturated fatty acid chains and unsaturated fatty acid chains can be used.

[0495] In some embodiments, the lipid-based carrier comprises one or more than one neutral lipid, wherein the neutral lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (transDOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (DPhyPE), or a mixture thereof.

[0496] In some embodiments, the neutral lipid (eg, LNP) of the lipid-based carrier of the pharmaceutical composition is selected from or derived from 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC).

[0497] In other embodiments, the neutral lipid (eg, LNP) of the lipid-based carrier of the pharmaceutical composition is selected from or derived from 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (DPhyPE).

[0498] Thus, in some embodiments, the lipid-based carrier (eg, LNP) of the pharmaceutical composition comprises a neutral lipid selected from or derived from DSPC, DHPC, or DPhyPE.

[0499] In some embodiments, the lipid-based carrier, such as the LNP of the pharmaceutical composition, comprises a neutral lipid selected from or derived from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0500] In various embodiments, the molar ratio of cationic lipid to neutral lipid in the lipid-based carrier is from about 2:1 to about 8:1.

[0501] Neutral lipid accounts for the total lipid present in the carrier based on lipid, for example about 5 % by mole to about 90 % by mole, about 5 % by mole to about 10 % by mole, about 5 % by mole, 10 % by mole, 15 % by mole, 20 % by mole, 25 % by mole, 30 % by mole, 35 % by mole, 40 % by mole, 45 % by mole, 50 % by mole, 55 % by mole, 60 % by mole, 65 % by mole, 70 % by mole, 75 % by mole, 80 % by mole, 85 % by mole or about 90 % by mole. In one embodiment, in mole, the carrier based on lipid comprises about 0 % to about 15 % or 45 % neutral lipid, for example about 3 % to about 12 % or about 5 % to about 10 %. For example, in mole (based on 100 % total mole of the lipid in the carrier based on lipid), the carrier based on lipid can comprise about 15 %, about 10 %, about 7.5 % or about 7.1 % neutral lipid.

[0502] Steroids, steroid analogs, or steroids:

[0503] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, a steroid analog, or a sterol.

[0504] Suitably, steroids, steroid analogs or sterols can be derived from or selected from cholesterol, cholesterol hemisuccinate (CHEMS) and derivatives thereof. In other embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, steroid analog or sterol derived from a plant sterol (e.g., sitosterol, such as β-sitosterol), for example, derived from a compound having a structure of formula I as disclosed in claim 1 of WO2020061332; The disclosure of WO2020061332, in particular the disclosure of formula I and plant sterols, is incorporated herein by reference. In other embodiments, steroids are imidazole cholesterol esters or "ICE" as disclosed in paragraphs

[0320] and paragraphs

[0339] to

[0340] of WO2019226925; WO2019226925 is incorporated herein by reference as a whole.

[0505] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, steroid analog, or sterol selected from or derived from cholesterol or comprising cholesterol.

[0506] The molar ratio of cationic lipid to cholesterol in the lipid-based carrier may be from about 2: 1 to about 1: 1. In some embodiments, the cholesterol may be PEGylated.

[0507] In some embodiments, the carrier based on lipid (based on 100% total mole of the lipid in the carrier based on lipid) comprises about 10 mol % to about 60 mol % or about 25 mol % to about 40 mol % sterol. In one embodiment, sterol accounts for about 10 mol, 15 mol, 20 mol, 25 mol, 30 mol, 35 mol, 40 mol, 45 mol, 50 mol, 55 mol or about 60 mol of the total lipid present in the carrier based on lipid. In another embodiment, in mole (based on 100% total mole of the lipid in the carrier based on lipid), the carrier based on lipid comprises about 5% to about 50% sterol, for example, in mole, about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 30%. In some embodiments, the lipid-based carrier (based on 100% total moles of lipids in the lipid-based carrier) comprises about 28%, about 29%, or about 30% sterol. In some embodiments, the lipid-based carrier (based on 100% total moles of lipids in the lipid-based carrier) comprises about 40.9% sterol.

[0508] References to other suitable cationic or ionizable, neutral, steroid / sterol or aggregation reducing lipids:

[0509] Other suitable cationic or ionizable, neutral, steroid / sterol or aggregation reducing lipids are described in WO2010053572, WO2011068810, WO2012170889, WO2012170930, WO2013052523, WO2013090648, WO2013149140, WO2013149141, WO2013151663, WO2013151664, WO2013151665, WO2013151666, WO2013151667, WO2013151668, WO2013151669, WO2013151670, WO2013151671 671. WO2013151672, WO2013151736, WO2013185069, WO2014081507, WO2014089486, WO2014093924, WO2014144196, WO2014152211, WO2014152774, WO2 014152940, WO2014159813, WO2014164253, WO2015061461, WO2015061467, WO2015061500, WO2015074085, WO2015105926, WO2015148247, WO20151646 74. WO2015184256, WO2015199952, WO2015200465, WO2016004318, WO2016022914, WO2016036902, WO2016081029, WO2016118724, WO2016118725, WO20 16176330, WO2017004143, WO2017019935, WO2017023817, WO2017031232, WO2017049074, WO2017049245, WO2017070601, WO2017070613, WO201707061 6. WO2017070618, WO2017070620, WO2017070622, WO2017070623, WO2017070624, WO2017070626, WO2017075038, WO2017075531, WO2017099823, WO201 7106799、WO2017112865、WO2017117528、WO2017117530、WO2017180917、WO 2017201325, WO2017201340, WO2017201350, WO2017201352, WO2017218704,WO2017223135、WO2018013525、WO2018081480、WO2018081638、WO2018089540、WO2018089790、WO2018089801、WO2018089851、WO2018107026、WO2018118102、WO2018119163、WO2018157009、WO2018165257、WO2018170245、WO2018170306、WO2018170322、WO2018170336、WO2018183901、WO2018187590、WO2018191657、WO2018191719、WO2018200943、WO2018231709、WO2018231990、WO2018232120、WO2018232357、WO2019036000、WO2019036008、WO2019036028、WO2019036030、WO2019040590、WO2019089818、WO2019089828、WO2019140102、WO2019152557、WO2019152802、WO2019191780、WO2019222277、WO2019222424、WO2019226650、WO2019226925、WO2019232095、WO2019232097、WO2019232103、WO2019232208、WO2020061284、WO2020061295、WO2020061332、WO2020061367、WO2020081938、WO2020097376、WO2020097379、WO2020097384、WO2020102172、WO2020106903、WO2020146805、WO2020214946、WO2020219427、WO2020227085、WO2020232276、WO2020243540、WO2020257611、WO2020257716、WO2021007278、WO2021016430、WO2021022173、WO2021026358、WO2021030701、WO2021046260、WO2021050986、WO2021055833、WO2021055835、WO2021055849、WO2021127394、WO2021127641、WO2021202694、WO2021231697、WO2021231901, WO2008103276, WO2009086558, WO2009127060, WO2010048536, WO20100 54406, WO2010080724, WO2010088537, WO2010129709, WO201021865, WO2011022460, WO2 011043913, WO2011090965, WO2011149733, WO2011153120, WO2011153493, WO201204018 4. WO2012044638, WO2012054365, WO2012061259, WO2013063468, WO2013086354, WO2013 086373, US7893302B2, US7404969B2, US8158601B2, US8283333B2, US8466122B2, US856 9256B2, US20100036115, US20110256175, US20120202871, US20120027803, US20120128 760, US20130064894, US20130129785, US20130150625, US20130178541, US20130225836 and US20140039032; the disclosures of the above documents specifically related to cationic or ionizable, neutral, sterol or aggregation-reducing lipids suitable for lipid-based carriers are incorporated herein by reference.

[0510] For example, suitable cationic lipids or cationizable or ionizable lipids include, but are not limited to, DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium chloride propane (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), ckk-E12 (WO2015200465), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-di ...DOTAP). Linolenoyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenoyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-dilinoleylaminocarbonyloxy-3-dimethylaminopropane (DLin-C-DAP), ICE (based on imidazole), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLincarbDAP, DLinCdAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]dioxolane) HGT4003, 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (MC3, US20100324120), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxolane-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N,N-1 ,6-diundecyl-4,7,10,13-tetraazahexadecane-l,16-diamine), (6Z,9Z,28Z,31Z)-triacontahedral-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-triacontahedral-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-triacontahedral-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN. ® (a cationic lipid commercially available from GIBCO / BRL Grand Island, NY, comprising DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE)); LIPOFECTAMINE ® (a cationic lipid commercially available from GIBCO / BRL comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminylcarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE); and TRANSFECTAM ®(commercially available from Promega Corp., Madison, Wis., a cationic lipid comprising di(octadecanoyl)aminoglycylcarboxyspermine (DOGS) dissolved in ethanol) or any combination of any of the foregoing. Other suitable cationic or ionizable lipids include 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazane-diyl)di(dodecan-2-ol) (C12-200) described in international patent document WO2010053572 (and particularly in paragraph

[00225] of WO2010053572) and that described in WO2012170930. WO2010053572 and WO2012170930 are incorporated herein by reference, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US2015140070), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane alkane (DLinDAP), 1,2-dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol alcohol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA, WO2010042877); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA).

[0511] Lipid-based carrier composition:

[0512] In some embodiments, the lipid-based carrier of the pharmaceutical composition, such as LNP, comprises the encoding RNA as defined in the first aspect, a cationic lipid as defined herein, an aggregation-reducing lipid as defined herein, optionally comprises a neutral lipid as defined herein, and optionally comprises a steroid or a steroid analog as defined herein.

[0513] In some embodiments, the lipid-based vector comprising the RNA encoding the first aspect comprises

[0514] (i) at least one cationic lipid or ionizable lipid, such as defined herein;

[0515] (ii) at least one neutral lipid or phospholipid, such as defined herein;

[0516] (iii) at least one steroid or steroid analogue, such as defined herein; and

[0517] (iv) at least one aggregation reducing lipid, such as defined herein.

[0518] In some embodiments, the lipid-based vector comprising the RNA encoding the first aspect comprises

[0519] (i) at least one cationic lipid selected from or derived from ALC-0315, SM-102, SS-33 / 4PE-15, HEXA-C5DE-PipSS or compound C26 (see C26 in Table 1 of WO2021123332);

[0520] (ii) at least one neutral lipid selected from or derived from DSPC, DHPC or DPhyPE;

[0521] (iii) at least one steroid or steroid analogue selected from or derived from cholesterol; and

[0522] (iv) at least one aggregation-reducing lipid selected from or derived from DMG-PEG 2000, C10-PEG2K, Cer8-PEG2K, or ALC-0159; and

[0523] The lipid-based vector encapsulates RNA.

[0524] In some embodiments, the cationic lipid (as defined herein), the neutral lipid (as defined herein), the steroid or steroid analog (as defined herein), and / or the aggregation reducing lipid (as defined herein) may be combined in various relative proportions.

[0525] In some embodiments, the lipid-based vector comprises (i) to (iv) in a molar ratio of about 20% to 60% of a cationic lipid or ionizable lipid, about 5% to 25% of a neutral lipid, about 25% to 55% of a steroid or steroid analog, and about 0.5% to 15% of an aggregation-reducing lipid, such as a polymer-conjugated lipid, e.g., wherein the lipid-based vector encapsulates RNA.

[0526] For example, the ratio between the cationic or ionizable lipid, to the neutral lipid, to the steroid or steroid analog, to the aggregation-reducing lipid can be about 30 to 60:20 to 35:20 to 30:1 to 15, or about 40:30:25:5, 50:25:20:5, 50:20:25:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:32:25:3 or 40:33:25:2, respectively.

[0527] In some embodiments, a lipid-based carrier, such as an LNP encoding the RNA of the first aspect, comprises

[0528] (i) at least one cationic lipid selected from SM-102;

[0529] (ii) at least one neutral lipid selected from DSPC;

[0530] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0531] (iv) at least one aggregation-reducing lipid selected from DMG-PEG 2000; and

[0532] wherein the lipid-based carrier encapsulates RNA, e.g., wherein i) to iv) are about 50% cationic lipid, about 10% neutral lipid, about 38.5% steroid or steroid analog, and about 1.5% aggregation-reducing lipid in a weight ratio,

[0533] For example, wherein a lipid-based vector encapsulates RNA.

[0534] In some embodiments, a lipid-based carrier, such as an LNP encoding the RNA of the first aspect, comprises

[0535] (i) at least one cationic lipid selected from SM-102;

[0536] (ii) at least one neutral lipid selected from DSPC;

[0537] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0538] (iv) at least one aggregation-reducing lipid selected from DMG-PEG 2000; and

[0539] wherein the lipid-based carrier encapsulates RNA, e.g., wherein i) to iv) are in a weight ratio of about 48.5% cationic lipid, about 11.1% neutral lipid, about 38.9% steroid or steroid analog, and about 1.5% aggregation-reducing lipid,

[0540] For example, wherein the lipid-based carrier encapsulates RNA. A suitable N / P ratio for the formulation is about 4.85 (molar ratio of lipid to RNA).

[0541] In some embodiments, a lipid-based carrier, such as an LNP encoding the RNA of the first aspect, comprises

[0542] (i) at least one cationic lipid selected from SS-33 / 4PE-15, HEXA-C5DE-PipSS or compound C26 (see C26 in Table 1 of WO2021123332);

[0543] (ii) at least one neutral lipid selected from DPhyPE;

[0544] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0545] (iv) at least one aggregation-reducing lipid selected from DMG-PEG 2000; and

[0546] Wherein the lipid-based carrier encapsulates RNA.This LNP is referred to herein as GN-LNP.

[0547] In one embodiment, a lipid-based carrier, such as an LNP encoding the RNA of the first aspect, comprises

[0548] (i) at least one cationic lipid selected from ALC-0315;

[0549] (ii) at least one neutral lipid selected from DSPC;

[0550] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0551] (iv) at least one aggregation-reducing lipid selected from ALC-0159.

[0552] In one embodiment, a lipid-based carrier, preferably comprising the RNA-encoding LNP of the first aspect, comprises

[0553] (i) at least one cationic lipid selected from ALC-0315;

[0554] (ii) at least one neutral lipid selected from DSPC;

[0555] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0556] (iv) at least one aggregation-reducing lipid selected from ALC-0159; and

[0557] Wherein the lipid-based carrier encapsulates RNA, for example wherein i) to iv) is about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% molar ratio of the lipid that reduces aggregation, for example wherein the lipid-based carrier encapsulates RNA. Such LNP is referred to herein as 315-LNP.

[0558] In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNP) having a molar ratio of about 50:10:38.5:1.5, for example, a molar ratio of 47.5:10:40.8:1.7, or for example, a molar ratio of 47.4:10:40.9:1.7 (i.e., the ratio (mol %) of cationic lipid (e.g., lipid III-3 (ALC-0315) described above), DSPC, cholesterol, and PEG-lipid (e.g., PEG-lipid of formula (IVa) with n=49 described above, for example, PEG-lipid of formula (IVa) with n=45 described above (ALC-0159)); dissolved in ethanol).

[0559] In one embodiment, a lipid-based carrier, such as an LNP encoding the RNA of the first aspect, comprises

[0560] (i) at least one cationic lipid selected from ALC-0315;

[0561] (ii) at least one neutral lipid selected from DSPC;

[0562] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0563] (iv) at least one aggregation-reducing lipid selected from ALC-0159; and

[0564] wherein the lipid-based carrier encapsulates RNA, e.g., wherein i) to iv) is a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, e.g., wherein the lipid-based carrier encapsulates RNA, wherein the RNA comprises or consists of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 8 NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: 995, SEQ ID NO: 998 to SEQ ID NO: 1020, SEQ ID NO: 1023 to SEQ ID NO: 1045, SEQ ID NO: 1070, SEQ ID NO: 1073 to SEQ ID NO: 1095, SEQ ID NO: 1098 to SEQ ID NO: 1120, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, or an RNA sequence or fragment or variant thereof that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270. In such an embodiment, the RNA is, for example, an mRNA comprising a cap 1 structure and an RNA sequence that is not chemically modified (e.g., composed of unmodified ribonucleotides).

[0565] The mRNA sequence in this context is, for example, SEQ ID NO: 829, SEQ ID NO: 679 or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 834, SEQ ID NO: 684 or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 833, SEQ ID NO: 683 or a fragment or variant of any of these sequences.

[0566] In some embodiments, a lipid-based carrier, such as an LNP encoding the RNA of the first aspect, comprises

[0567] (i) at least one cationic lipid selected from ALC-0315;

[0568] (ii) at least one neutral lipid selected from DSPC;

[0569] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0570] (iv) at least one aggregation-reducing lipid selected from ALC-0159; and

[0571] wherein the lipid-based carrier encapsulates RNA, e.g., wherein i) to iv) is a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, e.g., wherein the lipid-based carrier encapsulates RNA, wherein the RNA comprises or consists of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 8 NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: 995, SEQ ID NO: 998 to SEQ ID NO: 1020, SEQ ID NO: 1023 to SEQ ID NO: 1045, SEQ ID NO: 1070, SEQ ID NO: 1073 to SEQ ID NO: 1095, SEQ ID NO: 1098 to SEQ ID NO: 1120, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: : The invention relates to an RNA sequence or a fragment or variant thereof that is identical to or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, SEQ ID NO: 1271 to SEQ ID NO: 1276.In such an embodiment, the RNA is, for example, an mRNA comprising a cap 1 structure and an RNA sequence in which all uracils are replaced by pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0572] The mRNA sequence in this context is, for example, SEQ ID NO: 829, SEQ ID NO: 679, SEQ ID NO: 1271, SEQ ID NO: 1274, or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 834, SEQ ID NO: 684, SEQ ID NO: 1273, SEQ ID NO: 1276, or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 833, SEQ ID NO: 683, SEQ ID NO: 1272, SEQ ID NO: 1275, or a fragment or variant thereof.

[0573] In some embodiments, the weight ratio of lipid to RNA in the lipid-based carrier is about 10: 1 to about 60: 1, such as about 40: 1. In some embodiments, the weight ratio of lipid to RNA is about 20: 1 to about 30: 1, such as about 25: 1. In other embodiments, the weight ratio of lipid to RNA is 20 to 60, such as about 3 to about 15, about 5 to about 13, about 4 to about 8, or about 7 to about 11.

[0574] The amount of lipid contained in the lipid-based carrier can be selected taking into account the amount of RNA cargo. In one embodiment, these amounts are selected so that the N / P ratio of the lipid-based carrier of the encapsulated RNA is about 0.1 to about 20. The N / P ratio is defined as the molar ratio of the nitrogen atom ("N") of the basic nitrogen-containing group of the lipid to the phosphate group ("P") of the RNA used as cargo. The N / P ratio can be calculated based on, for example, 1 μg RNA usually contains about 3 nanomoles of phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The "N" value of a lipid or lipidoid can be calculated based on its molecular weight and the relative content of the group of permanent cations and - if present - the group of cations.

[0575] In embodiments, the N / P ratio can be from about 1 to about 50. In other embodiments, the N / P ratio is from about 1 to about 20, such as from about 1 to about 15, from about 1 to about 10, or from about 5 to about 7. For "GN-LNP", a suitable N / P (molar ratio of lipid to RNA) is about 14 or about 17. For "315-LNP", a suitable N / P (molar ratio of lipid to RNA) is about 6. Another suitable N / P ratio is about 4.85 or 5 (molar ratio of lipid to RNA).

[0576] In various embodiments, the pharmaceutical composition comprises a lipid-based carrier (encapsulating RNA) of a well-defined size (particle size, uniform size distribution).

[0577] The size of the lipid-based carrier of pharmaceutical composition is generally described as Z-average particle diameter in this article. The term "mean diameter", "diameter" or "size" of particle (e.g., lipid-based carrier) is used synonymously with the value of Z-average. The term "Z-average particle diameter" refers to the average diameter of particles measured by dynamic light scattering (DLS), wherein a so-called cumulant algorithm is used for data analysis, and the algorithm provides (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO13321) as a result of the so-called Z-average and dimensionless polydispersity index (PI) of the unit length.

[0578] The term "dynamic light scattering" or "DLS" refers to a method for analyzing particles in a liquid, wherein the liquid is typically illuminated with a monochromatic light source, and wherein light scattered by the particles in the liquid is detected. Thus, DLS can be used to measure particle size in a liquid. Suitable DLS procedures are known in the art. Commercially available DLS instruments (such as the Zetasizer Nano series, Malvern Instruments, Worcestershire, UK) are available. DLS instruments use a 90° detector (e.g., the DynaPro ® NanoStar ® or Zetasizer Nano S90 from Malvern Instruments ® ), or a backscatter detection system with 173° for incident light at nearly 180° (e.g., the Zetasizer NanoS from Malvern Instruments). ® ) and 158° backscatter detection system (DynaPro Plate Reader from Malvern Instruments ® ). Typically, DLS measurements are performed at a temperature of about 25° C. In the context of the present disclosure, the DLS method is also used to determine the polydispersity index (PDI) and / or the main peak diameter of the lipid-based carriers incorporating RNA.

[0579] In various embodiments, the lipid-based carrier of the pharmaceutical composition encapsulating RNA has a diameter of about 50 nm to about 200 nm, about 50 nm to about 190 nm, about 50 nm to about 180 nm, about 50 nm to about 170 nm, about 50 nm to about 160 nm, 50 nm to about 150 nm, 50 nm to about 140 nm, 50 nm to about 130 nm, 50 nm to about 120 nm, 50 nm to about 110 nm, 50 nm to about 100 nm, 50 nm to about 90 nm, 50 nm to about 80 nm, 50 nm to about 70 nm, 50 nm to about 60 nm, 60 nm to about 200 nm, about 60 nm to about 190 nm, about 60 nm to about 180 nm, about 60 nm to about 170 nm, about 60 nm to about 160 nm, In some embodiments, the invention relates to a Z-average particle size of about 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm.

[0580] In one embodiment, the lipid-based carrier of the RNA encapsulated pharmaceutical composition has a Z-average particle size of about 50 nm to about 200 nm, such as about 50 nm to about 150 nm, such as about 50 nm to about 120 nm.

[0581] Suitably, the pharmaceutical composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of a lipid-based carrier having a particle size greater than about 500 nm.

[0582] Suitably, the pharmaceutical composition comprises less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% LNPs having a particle size of less than about 20 nm.

[0583] Suitably, at least about 80%, 85%, 90%, 95% of the lipid-based carriers of the composition have a spherical morphology.

[0584] In embodiments, the polydispersity index (PDI) of the lipid-based carrier is typically between 0.1 and 0.5. In particular embodiments, the PDI is below 0.2. Typically, the PDI is determined by dynamic light scattering.

[0585] In some embodiments, 80% RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier, for example, 85% RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier, for example, 90% RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier, for example, 95% RNA contained in the pharmaceutical composition is encapsulated in a lipid-based carrier. The percentage of encapsulation can be determined by RiboGreen assay known in the art.

[0586] According to some embodiments, the lipid-based vector, e.g., a lipid-based vector encapsulating or comprising RNA, has been purified by at least one purification step, e.g., by at least one TFF step and / or at least one clarification step and / or at least one filtration step.

[0587] Antagonists of RNA sensing pattern recognition receptors:

[0588] In some embodiments, the pharmaceutical composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor.Such an antagonist may, for example, be co-formulated into a lipid-based carrier as defined herein.

[0589] Suitable antagonists of at least one RNA sensing pattern recognition receptor are disclosed in published PCT patent application WO2021028439, the entire disclosure of which is incorporated herein by reference. In particular, the disclosure of suitable antagonists of at least one RNA sensing pattern recognition receptor as defined in any one of claims 1 to 94 of WO2021028439 is incorporated herein by reference.

[0590] In some embodiments in this context, the pharmaceutical composition comprises at least one antagonist of at least one RNA sensing pattern recognition receptor selected from Toll-like receptors, such as TLR7 and / or TLR8.

[0591] In embodiments in this context, the at least one antagonist of at least one RNA sensing pattern recognition receptor is selected from nucleotides, nucleotide analogs, nucleic acids, peptides, proteins, small molecules, lipids or fragments, variants or derivatives of any of these.

[0592] In some embodiments within this context, the at least one antagonist of at least one RNA sensing pattern recognition receptor is a single stranded oligonucleotide, such as a single stranded RNA oligonucleotide.

[0593] In an embodiment of this context, at least one antagonist of at least one RNA sensing pattern recognition receptor is a single-stranded oligonucleotide comprising or consisting of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a nucleic acid sequence selected from SEQ ID NO: 85 to SEQ ID NO: 212 of WO2021028439, or a fragment of any of these sequences.

[0594] In some embodiments in this context, the antagonist of at least one RNA sensing pattern recognition receptor is a single-stranded oligonucleotide comprising or consisting of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a nucleic acid sequence selected from SEQ ID NO: 85 to SEQ ID NO: 87, SEQ ID NO: 149 to SEQ ID NO: 212 of WO2021028439, or a fragment of any of these sequences.

[0595] A suitable antagonist of at least one RNA sensing pattern recognition receptor in the context of the present disclosure is 5'-GAGCGmG CCA-3' (SEQ ID NO: 85 of WO2021028439) or a fragment thereof.

[0596] In some embodiments of this context, the molar ratio of at least one antagonist of at least one RNA sensing pattern recognition receptor as defined herein to at least one RNA encoding an antigenic peptide or protein as defined herein is suitably from about 1:1 to about 100:1, or from about 20:1 to about 80:1.

[0597] In some embodiments of this context, the weight ratio of at least one antagonist of at least one RNA sensing pattern recognition receptor as defined herein to at least one RNA encoding an antigenic peptide or protein as defined herein is suitably about 1:1 to about 1:30, or about 1:2 to about 1:10.

[0598] In an embodiment of this context, at least one antagonist of at least one RNA sensing pattern recognition receptor as defined herein and at least one RNA encoding an antigenic peptide or protein as defined herein are formulated separately, for example separately into a lipid-based carrier as defined herein.

[0599] In some embodiments in this context, at least one antagonist of at least one RNA sensing pattern recognition receptor as defined herein and at least one RNA encoding an antigenic peptide or protein as defined herein are co-formulated, for example co-formulated into a lipid-based carrier as defined herein.

[0600] In an embodiment of the composition comprising at least one antagonist of at least one RNA sensing pattern recognition receptor, the at least one RNA encoding an antigenic peptide or protein as defined herein, for example, does not comprise chemically modified nucleotides as defined herein (e.g., pseudouridine (ψ) or N1-methylpseudouridine (m1ψ)).

[0601] Submission:

[0602] In some embodiments, the pharmaceutical composition is lyophilized, spray dried, or spray freeze dried.

[0603] Therefore, the pharmaceutical composition is lyophilized (e.g. according to WO2016165831 or WO2011069586) to obtain a temperature-stable composition. Spray drying or spray freeze drying (e.g. according to WO2016184575 or WO2016184576) can also be used to dry the pharmaceutical composition to obtain a temperature-stable composition (powder) as defined herein.

[0604] The freeze-dried and / or spray-dried lyoprotectants may be selected from trehalose, sucrose, mannose, dextran and inulin. Suitable lyoprotectants are sucrose, optionally containing other lyoprotectants. Other suitable lyoprotectants are trehalose, optionally containing other lyoprotectants. Therefore, the pharmaceutical composition may contain at least one lyoprotectant.

[0605] In some embodiments, the pharmaceutical composition is a liquid composition or a dried composition, such as a lyophilized / spray-dried composition that can be reconstituted in a liquid carrier.

[0606] In some embodiments, the pharmaceutical composition (or liquid carrier) comprises a sugar at a concentration of about 50 mM to about 300 mM, such as sucrose at a concentration of about 150 mM.

[0607] In some embodiments, the pharmaceutical composition (or liquid carrier) comprises a salt at a concentration of about 10 mM to about 200 mM, such as NaCl at a concentration of about 75 mM.

[0608] In some embodiments, the pharmaceutical composition (or liquid carrier) comprises a buffer at a concentration of 1 mM to about 100 mM, such as Na2HPO4, Na3PO4 or Tris (tromethamine). In other embodiments, the pharmaceutical composition (or liquid carrier) comprises about 2.4 mM Tris (tromethamine), about 1.4 mM glacial acetic acid, about 3.9 mM acetic acid and about 254 mM sugar.

[0609] In some embodiments, the pharmaceutical composition (or liquid carrier) has a pH of about pH 7.0 to about pH 8.0, such as a pH of about pH 7.4.

[0610] 3: Vaccines comprising RNA encoding E. coli FimH or antigenic polypeptides derived from E. coli FimH

[0611] In a third aspect, a vaccine against E. coli is provided.

[0612] It is noteworthy that the embodiment relating to the composition of the second aspect can similarly continue to be read and understood as a suitable embodiment of the vaccine of the third aspect. The embodiment relating to the vaccine of the third aspect can also similarly continue to be read and understood as a suitable embodiment of the composition of the second aspect. In addition, the features and embodiments (coding RNA of the present disclosure) described in the context of the first aspect must continue to be read and must be understood as a suitable embodiment of the vaccine of the third aspect.

[0613] In some embodiments, the vaccine comprises an RNA encoding the first aspect, or at least one composition of the second aspect.

[0614] The term "vaccine" will be recognized and understood by those of ordinary skill in the art, and is intended, for example, to be a preventive or therapeutic material that provides at least one epitope or antigen, such as an immunogen. In the context of the present disclosure, the antigen or antigenic function is suitably provided by the RNA of the first aspect (the encoding RNA comprising a coding sequence encoding an antigenic polypeptide selected from or derived from E. coli FimH) or the composition of the second aspect (which comprises the encoding RNA of the first aspect).

[0615] In some embodiments, the vaccine induces an adaptive immune response, such as a protective adaptive immune response against E. coli. In particular, the E. coli is selected from the group consisting of: E. coli J96, E. coli 536, E. coli CFT073, E. coli UMN026, E. coli CLONE D i14, E. coli CLONE D i2, E. coli IA139, E. coli NA114, E. coli IHE3034, E. coli 789, E. coli F11, and E. coli UTI89.

[0616] In some embodiments, the vaccine, e.g., administering the vaccine to a subject, induces a humoral immune response against E. coli. In one embodiment, the humoral immune response is against E. coli FimH. In one embodiment, the vaccine, e.g., administering the vaccine to a subject, induces a humoral immune response against E. coli, e.g., against E. coli FimH, in the urine of the subject after administration of the vaccine.

[0617] In one embodiment, the vaccine, e.g., administering the vaccine to a subject, induces neutralizing antibody titers against E. coli. In one embodiment, the antibody is an IgG antibody. In one embodiment, the antibody is against E. coli FimH. In one embodiment, the vaccine, e.g., administering the vaccine to a subject, induces neutralizing antibody titers against E. coli, e.g., against E. coli FimH, in the urine of the subject after administration of the vaccine.

[0618] In one embodiment, the vaccines of the present disclosure induce antibodies capable of inhibiting bacterial adhesion to urothelial cells. Suitable methods for detecting inhibition of bacterial adhesion are described herein and in the Examples.

[0619] Methods for detecting bacterial adhesion are known in the art. Suitably, the method described in Thomas WE et al., Cell. 2002 Jun 28; 109(7): 913-23; 109(7): 913-23, which is incorporated herein by reference; or Hartmann M et al., FEBS Lett. 2012 May 21; 586(10): 1459-65, which is incorporated herein by reference; or Falk P et al., Methods Cell Biol. 1994; 45: 165-92, which is incorporated herein by reference; or García Méndez KB et al., Int J Exp Pathol. 2016 Apr; 97(2): 194-201, which is incorporated herein by reference. In one embodiment, bacterial adhesion is (briefly) detected using the BAI assay as described below and in the Examples: UPEC strains engineered to express the mCherry fluorescent marker are cultured with a monolayer of SV-HUC-1 (ATTCC) in a 96-well plate for 30 minutes in the presence of specific sera against FimH derivatives or positive / negative controls. After adhesion, the cells are washed extensively to remove unbound bacteria and fixed with formaldehyde. Finally, specific fluorescent signals associated with adherent bacteria are recorded using an automated high-content screening microscope (Opera Phenix) and quantified using Harmony software.

[0620] In some embodiments, the immune response is effective to prevent or treat one or more than one symptom associated with UTI in a subject in need thereof. In certain embodiments, the immune response is effective to prevent or reduce UTI symptoms, such as in at least 30%, such as in at least 40%, such as in at least 50% of subjects administered the vaccine. Symptoms of UTI can vary depending on the nature of the infection and include, but are not limited to, difficulty urinating, frequent or urgent urination, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea.

[0621] In certain embodiments, the immune response is effective to prevent or reduce organ failure caused by UTI. In certain embodiments, the immune response is effective to reduce the likelihood of hospitalization of a subject with UTI. In some embodiments, the immune response is effective to reduce the length of hospital stay of a subject with UTI.

[0622] According to the present disclosure, vaccines may be used for human medical purposes, as well as for veterinary medical purposes (mammalian, vertebrate or avian species).

[0623] Suitable routes of administration of the vaccine include intranasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal or subcutaneous. Thus, in some embodiments, the vaccine is suitable for intranasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal or subcutaneous administration.

[0624] In one embodiment, the vaccine is suitable for intramuscular administration.

[0625] In one embodiment, the vaccine comprises a lipid-based carrier comprising

[0626] (i) at least one cationic lipid selected from ALC-0315;

[0627] (ii) at least one neutral lipid selected from DSPC;

[0628] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0629] (iv) at least one aggregation-reducing lipid selected from ALC-0159; and

[0630] wherein the lipid-based carrier encapsulates the RNA of the present disclosure, e.g., wherein i) to iv) is a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, wherein the RNA comprises or consists of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: SEQ ID NO: 1095, SEQ ID NO: 1098 to SEQ ID NO: 1120, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145 NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, or an RNA sequence or fragment or variant thereof that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270. In such embodiments, the RNA is, for example, an mRNA comprising a cap 1 structure, and the RNA sequence is not chemically modified (e.g., composed of unmodified ribonucleotides).The mRNA sequence in this context is, for example, SEQ ID NO: 829, SEQ ID NO: 679 or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 834, SEQ ID NO: 684 or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 833, SEQ ID NO: 683 or a fragment or variant of any of these sequences.

[0631] In one embodiment, the vaccine comprises a lipid-based carrier comprising

[0632] (i) at least one cationic lipid selected from ALC-0315;

[0633] (ii) at least one neutral lipid selected from DSPC;

[0634] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0635] (iv) at least one aggregation-reducing lipid selected from ALC-0159; and

[0636] wherein the lipid-based carrier encapsulates the RNA of the present disclosure, e.g., wherein i) to iv) is a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, wherein the RNA comprises or consists of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: SEQ ID NO: 1095, SEQ ID NO: 1098 to SEQ ID NO: 1120, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145 NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, SEQ ID NO: 1274 to SEQ ID NO: 1276, or an RNA sequence or fragment or variant thereof that is identical or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In such an embodiment, the RNA is, for example, an mRNA comprising a cap 1 structure, and the uracil of the RNA sequence is replaced by pseudouridine (ψ).The mRNA sequence in this context is, for example, SEQ ID NO: 829, SEQ ID NO: 679, SEQ ID NO: 1274 or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 834, SEQ ID NO: 684, SEQ ID NO: 1276 or a fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 833, SEQ ID NO: 683, SEQ ID NO: 1275 or a fragment or variant of any of these sequences.

[0637] In one embodiment, the vaccine comprises a lipid-based carrier comprising

[0638] (i) at least one cationic lipid selected from ALC-0315;

[0639] (ii) at least one neutral lipid selected from DSPC;

[0640] (iii) at least one steroid or steroid analogue selected from cholesterol; and

[0641] (iv) at least one aggregation-reducing lipid selected from ALC-0159; and

[0642] wherein the lipid-based carrier encapsulates the RNA of the present disclosure, e.g., wherein i) to iv) is a molar ratio of about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, wherein the RNA comprises or consists of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: SEQ ID NO: 1095, SEQ ID NO: 1098 to SEQ ID NO: 1120, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145, SEQ ID NO: 1148 to SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1123 to SEQ ID NO: 1145 NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, SEQ ID NO: 1271 to SEQ ID NO: 1273, or an RNA sequence or fragment thereof that is identical or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.In such an embodiment, the RNA is, for example, an mRNA comprising a cap 1 structure, and the uracil of the RNA sequence is replaced by N1-methyl pseudouridine (m1ψ). The mRNA sequence in this context is, for example, SEQ ID NO: 829, SEQ ID NO: 679, SEQ ID NO: 1271, or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 834, SEQ ID NO: 684, SEQ ID NO: 1273, or a fragment or variant of any of these sequences. Other mRNA sequences in this context are SEQ ID NO: 833, SEQ ID NO: 683, SEQ ID NO: 1272, or a fragment or variant of any of these sequences.

[0643] 4: Test kit or kit set

[0644] In a fourth aspect, a kit or kit set for treating or preventing an infection caused by E. coli is provided. It is noteworthy that the embodiments of the RNA of the first aspect can be similarly continued to be read and understood as suitable embodiments of the kit or kit set of the fourth aspect. The embodiments of the pharmaceutical composition of the second aspect or the vaccine of the third aspect can also be similarly continued to be read and understood as suitable embodiments of the kit or kit set of the fourth aspect,

[0645] In some embodiments, a kit or kit-of-parts comprises at least one RNA encoding the first aspect, at least one composition of the second aspect, and / or at least one vaccine of the third aspect.

[0646] In addition, the kit or kit set may include a liquid carrier for dissolution, and / or technical instructions providing information on administration and dosage of the components.

[0647] The kit may further comprise other components as described in the context of the composition of the second aspect and / or the vaccine of the third aspect.

[0648] The technical instructions of the kit may contain information about administration and dosage and patient populations. Such a kit, such as a kit set, can be applied to any application or use described herein, such as the use of the RNA of the first aspect, the composition of the second aspect, the vaccine of the third aspect, and the treatment or prevention of infection or disease caused by Escherichia coli, or a condition related thereto.

[0649] Suitably, the encoding RNA, composition or vaccine is provided in separate parts of the kit.

[0650] In some embodiments, the encoding RNA, composition, or vaccine is lyophilized or spray (freeze) dried.

[0651] In embodiments where the pharmaceutical composition is provided as a lyophilized or spray-freeze-dried or spray-dried composition, the kit or kit set may suitably comprise a buffer for reconstitution of the lyophilized or spray-freeze-dried or spray-dried composition.

[0652] Thus, the kit or kit set may additionally comprise a buffer for reconstitution and / or dilution of the RNA, composition or vaccine.

[0653] In some embodiments, the buffer used for recombinant and / or dilution is a sterile buffer. In some embodiments, the buffer comprises a salt, such as NaCl, optionally at a concentration of about 0.9%. Such a buffer may optionally comprise a preservative.

[0654] In some embodiments, a kit or kit-set as defined herein comprises at least one syringe.

[0655] In some embodiments, the kit or kit set comprises the following components:

[0656] a) at least one container or vial comprising a composition or vaccine as defined herein.

[0657] b) optionally, at least one dilution container or vial comprising a sterile dilution buffer, suitably a buffer comprising NaCl, optionally comprising a preservative;

[0658] c) optionally, at least one means for transferring the composition or vaccine from the container to a dilution container; and

[0659] d) at least one syringe for administering the composition or vaccine to a subject, such as a syringe configured for intramuscular administration to a human subject.

[0660] 5: Medical use

[0661] In other aspects, there is provided the medical use of an encoding RNA as defined herein, a composition as defined herein, a vaccine as defined herein, or a kit or a kit-of-parts as defined herein.

[0662] It is noteworthy that the embodiments relating to the aforementioned aspects may similarly continue to be read and understood as suitable embodiments for medical use of the present invention (and vice versa).

[0663] Thus, provided are the encoding RNA of the disclosure, and / or the composition of the disclosure, and / or the vaccine of the disclosure, and / or the kit or kit-of-kits of the disclosure for use as a medicament.

[0664] In other aspects, a second medical use of an encoding RNA as defined herein, a composition as defined herein, a vaccine as defined herein, or a kit or a kit-of-parts as defined herein is provided.

[0665] Therefore, the disclosed RNA encoding, and / or the disclosed composition, and / or the disclosed vaccine, and / or the disclosed kit or kit set for treating or preventing a disease caused by Escherichia coli are provided. In particular, the Escherichia coli is selected from: Escherichia coli J96, Escherichia coli 536, Escherichia coli CFT073, Escherichia coli UMN026, Escherichia coli CLONED i14, Escherichia coli CLONE D i2, Escherichia coli IA139, Escherichia coli NA114, Escherichia coli IHE3034, Escherichia coli 789, Escherichia coli F11 and Escherichia coli UTI89.

[0666] In some embodiments, the disclosed RNA encoding, and / or the disclosed composition, and / or the disclosed vaccine, and / or the disclosed kit or kit set is provided for treating or preventing one or more symptoms associated with UTI in a subject in need thereof. In certain embodiments, the use is for treating or preventing UTI symptoms, such as treating or preventing UTI symptoms in at least 30%, such as at least 40%, such as at least 50% of the subjects to whom the vaccine is administered. The symptoms of UTI may vary depending on the nature of the infection and include, but are not limited to, dysuria, frequent or urgent urination, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea.

[0667] In certain embodiments, the use is for preventing or reducing organ failure caused by UTI. In certain embodiments, the use is for reducing the likelihood of hospitalization of a subject with UTI. In some embodiments, the use is for reducing the duration of hospitalization of a subject with UTI.

[0668] In the context of medical use, the encoding RNA of the present disclosure, and / or the composition of the present disclosure, and / or the vaccine of the present disclosure, and / or the kit or kit-of-kits of the present disclosure can be administered, for example, topically.

[0669] In this context, administration may be intranasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal or subcutaneous, e.g. intramuscular.

[0670] In one embodiment, administration may be by conventional needle injection, such as intramuscular injection.

[0671] In some embodiments, the use can be used for human medical purposes, and can also be used for veterinary medical purposes. In one embodiment, the use can be used for human medical purposes.

[0672] In some embodiments, the use is for naive subjects, i.e., subjects who do not have an E. coli infection or have never had a UTI. In one embodiment, the use is for subjects at risk of acquiring or developing a UTI, such as immunocompromised or immunodeficient individuals, before symptoms develop or symptoms become severe. In certain embodiments, the use is for subjects who have been diagnosed or have been diagnosed with a UTI.

[0673] As used herein, the term "high-risk subject" refers to a person who is more susceptible to disease than the general adult population. Examples of "high-risk subjects" include people with one or more than one risk factor for UTI, which may include, but are not limited to, the elderly, immunocompromised people, people with diabetes, people with a known history of rUTI, people with urinary tract obstruction such as kidney stones, sexually active women, postmenopausal women, people who use catheters, people who are incontinent, people who have recently undergone urological surgery such as urinary tract surgery, etc.

[0674] In certain embodiments, the use is for subjects who have been diagnosed or have been diagnosed with UPEC infection. In some embodiments, the use is for subjects with recurrent UTI. In some embodiments, the use is for subjects with recurrent UTI, but the subject is healthy at the time of treatment. In some embodiments, the use is for subjects with bacteremia or sepsis of Escherichia coli or subjects at risk of acquiring bacteremia or sepsis of Escherichia coli. In some embodiments, the subject to be administered or applied the composition or method of the present disclosure suffers from a condition that requires them to use a catheter such as a urinary catheter (which causes the risk of CAUTI, i.e., catheter-related UTI). In some embodiments, the use is for subjects undergoing scheduled surgery.

[0675] In certain embodiments, the use is for adults older than 50 years. In certain embodiments, the use is for adults older than 55 years, older than 60 years, or older than 65 years. In certain embodiments, the use is for women aged about 16 to 50 years, such as women aged about 16 to 35 years. In certain embodiments, the use is for subjects with diabetes.

[0676] 6: Treatment methods

[0677] In other aspects, methods of treating or preventing a disease caused by E. coli are provided. The method comprises administering to a subject in need thereof an effective amount of an encoding RNA according to the first aspect, a pharmaceutical composition according to the second aspect, a vaccine according to the third aspect, or a kit or kit set according to the fourth aspect. Methods for inducing an immune response in a subject in need thereof are also provided. Suitably, the immune response is effective to prevent or treat one or more symptoms associated with UTI in a subject in need thereof.

[0678] Also provided is the use of the encoding RNA according to the first aspect, the pharmaceutical composition according to the second aspect, the vaccine according to the third aspect, or the kit or kit set according to the fourth aspect for eliciting an immune response in a mammal, for example, to treat and / or prevent a disease. Also provided is the use of the encoding RNA according to the first aspect, the pharmaceutical composition according to the second aspect, the vaccine according to the third aspect, or the kit or kit set according to the fourth aspect for the preparation of a medicament for eliciting an immune response in a mammal, for example, to treat and / or prevent a disease, such as an E. coli infection.

[0679] It is noteworthy that the embodiments related to the aforementioned aspects can similarly continue to be read and understood as suitable embodiments for medical use of the present disclosure.

[0680] Furthermore, specific features and embodiments relating to methods of treatment as provided herein may also be applicable to the medical uses of the present disclosure, and vice versa.

[0681] Prevention (inhibition) or treatment of disease, particularly infection caused by E. coli, involves inhibiting the full development of the disease or condition, for example, inhibiting the full development of the disease or condition in a subject at risk of suffering from a disease such as infection. "Treatment" refers to a therapeutic intervention that improves its signs or symptoms after the disease or pathological condition begins to develop. With respect to a disease or pathological state, the term "improvement" refers to any observable beneficial effect of treatment. Inhibiting a disease can include preventing or reducing the risk of a disease, such as preventing or reducing the risk of E. coli infection. The beneficial effect can be demonstrated, for example, by delayed onset of clinical symptoms of the disease in susceptible subjects, reduced severity of some or all clinical symptoms of the disease, slowed disease progression, reduction in viral load, improvement of the overall health or health of the subject, or by other parameters specific to a particular disease. "Preventive" treatment is a treatment administered to a subject that does not show signs of the disease or only shows early signs, with the purpose of reducing the risk of a pathological state.

[0682] In some embodiments, a method for treating or preventing a disease, disorder, or pathological condition is provided, wherein the method comprises administering or applying the encoding RNA of the present disclosure, and / or the composition of the present disclosure, and / or the vaccine of the present disclosure, and / or the kit or kit set of the present disclosure to a subject in need thereof.

[0683] In some embodiments, the disease, disorder, or pathological condition is an infectious disease caused by E. coli or a condition associated with such an infectious disease.

[0684] In specific embodiments, the methods of inducing an immune response in a subject of the present disclosure result in vaccination of the subject to induce protective immunity against infection with an E. coli strain expressing FimH.

[0685] In one embodiment, the method induces a humoral immune response against E. coli.

[0686] In one embodiment, the humoral immune response is against E. coli FimH. In one embodiment, the method induces a humoral immune response against E. coli, such as against E. coli FimH, in the urine of the subject.

[0687] In one embodiment, the method induces neutralizing antibody titers against E. coli. In one embodiment, the antibody is an IgG antibody. In one embodiment, the antibody is against E. coli FimH. In one embodiment, the method induces neutralizing antibody titers against E. coli, such as against E. coli FimH, in the urine of the subject.

[0688] In one embodiment, the methods of the present disclosure induce antibodies capable of inhibiting bacterial adhesion to urothelial cells. Suitable methods for detecting inhibition of bacterial adhesion are described herein and in the Examples.

[0689] In certain embodiments, following administration of an encoding RNA, pharmaceutical composition, or vaccine according to the present disclosure, the immune response induced in a subject is effective to eliminate the UTI.

[0690] In certain embodiments, following administration of an RNA encoding, pharmaceutical composition or vaccine according to the present disclosure, following an immune response induced in a subject, the immune response induced in the subject is effective in preventing or alleviating UTI symptoms, such as in at least 30%, such as in at least 40%, such as in at least 50% of the subjects to whom the composition was administered. The symptoms of UTI may vary depending on the nature of the infection, including but not limited to: dysuria, frequent or urgent urination, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills and / or nausea.

[0691] In certain embodiments, following the administration of the coding RNA, pharmaceutical composition or vaccine according to the present disclosure, the immune response induced in the subject is effective to prevent or reduce organ failure caused by UTI. In certain embodiments, following the immune response induced in the subject after the administration of the coding RNA, pharmaceutical composition or vaccine according to the present disclosure, the immune response induced in the subject is effective to reduce the likelihood of hospitalization of the subject with UTI. In some embodiments, following the administration of the composition of the present disclosure, the immune response induced in the subject is effective to reduce the duration of hospitalization of the subject with UTI.

[0692] In some embodiments, the application or administration is by intranasal administration, oral administration, sublingual administration, intramuscular injection, intravenous injection, percutaneous injection, or intradermal injection. In one embodiment, the application or administration is carried out by intramuscular injection.

[0693] As used herein in the context of the present disclosure, the term "effective amount" refers to an amount sufficient to induce a desired immune effect or immune response in a subject. In certain embodiments, an "effective amount" refers to an amount sufficient to produce immunity in a subject to achieve one or more of the following effects in a subject: (i) preventing the development or occurrence of UTI or its associated symptoms; (ii) preventing or reducing the recurrence of UTI or its associated symptoms; (iii) preventing, alleviating or improving the severity of UTI or its associated symptoms; (iv) shortening the duration of infection with UTI or its associated symptoms; (v) preventing clinical progression of UTI or its associated symptoms; (vi) causing regression of UTI or its associated symptoms; (vii) preventing or alleviating organ failure caused by UTI; (viii) reducing the likelihood or frequency of hospitalization of subjects with UTI; (ix) reducing the length of hospitalization of subjects with UTI; (x) eliminating UTI; and / or (xi) enhancing or improving the preventive or therapeutic effect of another therapy.

[0694] The selection of a specific effective dose can be determined by those skilled in the art based on consideration of several factors (e.g., through clinical trials), including the disease to be treated or prevented, the symptoms involved, the subject's medical history, the subject's physical condition, such as the subject's age, weight and / or immune status, the composition administered, and other factors known to those skilled in the art. The precise dose used in the formulation also depends on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and the circumstances of each patient. The effective dose can be inferred from the dose-response curve of a test system derived from an in vitro model or an animal model.

[0695] In one embodiment, the subject in need thereof is a mammalian subject, such as a human subject.

[0696] In certain embodiments, the methods of the present disclosure are administered or applied to naive subjects, i.e., subjects who do not have an E. coli infection or have never had a UTI. In one embodiment, the compositions or methods of the present disclosure are administered or applied to subjects at risk of acquiring or developing a UTI, such as immunocompromised or immunodeficient individuals, before symptoms develop or symptoms become severe. In certain embodiments, the methods of the present disclosure are administered or applied to subjects who have been diagnosed or have been diagnosed with a UTI.

[0697] In certain embodiments, the methods of the present disclosure are administered or applied to subjects who have been diagnosed or have been diagnosed with UPEC infection. In some embodiments, the compositions or methods of the present disclosure are administered or applied to subjects with recurrent UTI. In some embodiments, the methods of the present invention are administered or applied to subjects with recurrent UTI, but the subjects are healthy at the time of treatment. In some embodiments, the methods of the present disclosure are administered or applied to subjects with bacteremia or sepsis of Escherichia coli or subjects at risk of acquiring bacteremia or sepsis of Escherichia coli. In some embodiments, the subjects to be applied the methods of the present disclosure suffer from conditions that require them to use catheters such as urinary catheters (which lead to the risk of CAUTI, i.e., catheter-related UTI). In some embodiments, the methods of the present disclosure are applied to subjects undergoing scheduled surgery.

[0698] In certain embodiments, the subject to which the methods of the present disclosure are applied is a human subject, e.g., a human subject at risk for UTI disease. In certain embodiments, the subject to which the compositions or methods of the present disclosure are applied is an adult greater than 50 years of age. In certain embodiments, the subject to which the methods of the present disclosure are applied is an adult greater than 55 years of age, greater than 60 years of age, or greater than 65 years of age.

[0699] In certain embodiments, the subject to whom the methods of the present disclosure are applied is a woman between the ages of about 16 and 50, such as a woman between the ages of about 16 and 35. In certain embodiments, the subject to whom the methods of the present disclosure are applied has diabetes. Table Description

[0700] Table 1: Sequences (amino acid sequence and coding sequence)

[0701] Table 2: RNA constructs

[0702] Table 3: RNA constructs encoding antigen designs used in the examples

[0703] Table 4: Lipid-based carrier compositions of the examples

[0704] Table 5: RNA constructs used for Western blot analysis (Example 2.1)

[0705] Table 6: Vaccination schedule (Example 2.2)

[0706] Table 7: BAI titers of serum antibody responses designed against E. coli FimH antigen (Example 2.3)

[0707] Table 8: Vaccination schedule (Example 3.1)

[0708] Table 9: BAI titers of serum antibody responses designed against E. coli FimH antigen (Example 3.2)

[0709] Table 10: mRNA constructs used for Western blot analysis (Example 4.1)

[0710] Table 11: Vaccination schedule (Example 4.2)

[0711] Table 12: BAI titers of serum antibody responses designed against E. coli FimH antigen (Example 4.3)

[0712] Table 13: Other sequences shorter than 10 specifically defined nucleotides or shorter than 4 specifically defined amino acids. Numbered implementation plan

[0713] Hereinafter, embodiments of the present invention are provided as a numbered list of embodiments (Embodiment 1 to Embodiment 110).

[0714] Implementation 1 . Encoding RNA comprising at least one untranslated region (UTR) and at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli type 1 fimbriae D-mannose-specific adhesin (FimH).

[0715] Implementation 2 . The encoding RNA according to embodiment 1, wherein the Escherichia coli FimH comprises an amino acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, or an immunogenic fragment or immunogenic variant thereof.

[0716] Implementation 3 . The coding RNA according to embodiment 1 or 2, wherein the coding sequence additionally encodes one or more other peptides or protein elements, wherein the one or more other peptides or protein elements are selected from: donor chain peptides, signal peptides, antigen aggregation domains or transmembrane domains.

[0717] Implementation 4 . The coding RNA according to embodiment 3, wherein one or more than one other peptide or protein element is a donor chain peptide, optionally wherein the coding sequence encodes the following elements in the N-terminal to C-terminal direction: an antigenic polypeptide selected from or derived from Escherichia coli FimH; and a donor chain peptide.

[0718] Implementation 5 . An encoding RNA according to embodiment 4, wherein the donor chain peptide comprises or consists of an amino acid sequence of SEQ ID NO: 338 or SEQ ID NO: 339 or a variant thereof, optionally wherein the variant of SEQ ID NO: 338 or SEQ ID NO: 339 has 1 to 5 single amino acid mutations compared to SEQ ID NO: 338 or SEQ ID NO: 339, such as 1, 2, 3 or 4 single amino acid mutations.

[0719] Implementation 6 . The encoding RNA according to embodiment 4, wherein the donor chain peptide comprises or consists of an amino acid sequence of SEQ IDNO: 338.

[0720] Implementation Plan 7 . The encoding RNA according to any one of embodiments 1 to 6, wherein the coding sequence additionally encodes a peptide linker.

[0721] Implementation 8 . The encoding RNA according to embodiments 1 to 7, wherein the coding sequence encodes the following elements in the direction from N-terminus to C-terminus: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker element; and a donor chain peptide.

[0722] Implementation Plan 9 . The encoding RNA according to embodiment 7 or 8, wherein the peptide linker comprises or consists of any one of SEQ ID NO: 352 to SEQ ID NO: 358.

[0723] Implementation 10 . The encoding RNA according to embodiments 7 to 9, wherein the peptide linker comprises or consists of SEQID NO: 352.

[0724] Implementation Plan 11 . The encoding RNA according to any of the preceding embodiments, wherein the antigenic polypeptide is in a low mannose binding affinity conformation.

[0725] Implementation 12 . An encoding RNA according to any one of embodiments 1 to 11, wherein the coding sequence additionally encodes an antigen aggregating domain.

[0726] Implementation 13. The encoding RNA according to embodiment 12, wherein the antigen aggregation domain is selected from or derived from ferritin or dioxotetrahydropterin synthase.

[0727] Implementation Plan 14 . An encoding RNA according to any one of embodiments 12 or 13, wherein the amino acid sequence of the antigen aggregating domain is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of the amino acid sequences of SEQ ID NO: 457 to SEQ ID NO: 459, SEQ ID NO: 443, SEQID NO: 444, or fragments or variants thereof.

[0728] Implementation Plan 15 . The coding RNA according to any one of embodiments 1 to 11, wherein the coding sequence additionally encodes a transmembrane domain.

[0729] Implementation Plan 16 . The encoding RNA according to claim 15, wherein the transmembrane domain is heterologous and is optionally selected from or derived from an influenza HA transmembrane domain, for example selected from or derived from SEQ ID NO: 478.

[0730] Implementation Plan 17 . The encoding RNA according to any one of embodiments 1 to 16, wherein the coding sequence additionally encodes a signal peptide.

[0731] Implementation Plan 18 . The encoding RNA according to embodiment 17, wherein the signal peptide is selected from or derived from FimH, FimC, immunoglobulin kappa IgK (IgK), immunoglobulin IgE (IgE), tissue plasminogen activator (TPA or HsPLAT), human serum albumin (HSA or HsALB), or MHC class I lymphocyte antigen (HLA-A2), optionally wherein the amino acid sequence of the signal peptide is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of the amino acid sequences SEQ ID NO: 394 to SEQ ID NO: 400, or is a fragment or variant thereof.

[0732] Implementation Plan 19. The encoding RNA according to embodiment 17 or 18, wherein the signal peptide is selected from or derived from IgE or IgK, optionally wherein the amino acid sequence of the signal peptide is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences SEQ ID NO: 394, SEQ ID NO: 395, or is a fragment or variant thereof.

[0733] Implementation 20(a) . The coding RNA according to any one of embodiments 1 to 19, wherein the coding sequence encodes the following elements, for example, in the direction from N-terminus to C-terminus:

[0734] a) Signal peptide, antigenic peptide;

[0735] b) signal peptide, antigenic peptide, peptide linker, donor chain peptide;

[0736] c) antigen aggregation domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide;

[0737] d) signal peptide, antigen aggregation domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide;

[0738] e) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, antigen aggregation domain; or

[0739] f) Signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, transmembrane domain.

[0740] Implementation 20(b) . An encoding RNA according to any one of embodiments 1 to 19 and 20(a), wherein the coding sequence encodes the following elements, for example, in the direction from N-terminus to C-terminus: a signal peptide, an antigenic polypeptide, a peptide linker and a donor chain peptide; optionally wherein the signal peptide is selected from SEQ ID NO: 394 to SEQ ID NO: 400, optionally wherein the signal peptide is SEQ ID NO: 395; the antigenic polypeptide is selected from SEQ ID NO: 247 to SEQ ID NO: 256, optionally wherein the antigenic polypeptide is SEQ ID NO: 247; the peptide linker is selected from SEQ ID NO: 352 to SEQ ID NO: 354, optionally wherein the peptide linker is SEQ ID NO: 352; the donor chain peptide is selected from SEQ ID NO: 338, SEQ ID NO: 339, optionally wherein the donor chain peptide is SEQ ID NO: 338.

[0741] Implementation Plan 21 . An encoding RNA according to any one of embodiments 1 to 19, wherein the coding sequence encodes the following elements, for example, in the direction from N-terminus to C-terminus: a signal peptide, an antigenic polypeptide as defined herein, a (first) peptide linker, a donor strand peptide, a (second) peptide linker; and an antigen aggregation domain; optionally wherein the signal peptide is selected from SEQ ID NO: 394 to SEQ ID NO: 400, optionally wherein the signal peptide is SEQ ID NO: 394; the antigenic polypeptide is selected from SEQ ID NO: 247 to SEQ ID NO: 256, optionally wherein the antigenic polypeptide is SEQ ID NO: 247; the (first) peptide linker is selected from SEQ ID NO: 352 to SEQ ID NO: 354, optionally wherein the (first) peptide linker is SEQ ID NO: 352; the donor strand peptide is selected from SEQ ID NO: 338, SEQ ID NO: 339, optionally wherein the donor strand peptide is SEQ ID NO: 338; the (second) peptide linker is selected from SEQ ID NO: 355 to SEQ ID NO:358, optionally wherein the (second) peptide linker is SEQ ID NO: 355; the antigen aggregation domain is selected from SEQ ID NO: 443, SEQ ID NO: 444, SEQ ID NO: 457 to SEQ ID NO: 459, optionally wherein the peptide linker is SEQ ID NO: 444 or SEQ ID NO: 459.

[0742] Implementation Plan 22 . An encoding RNA according to any one of the preceding embodiments, wherein the coding sequence encodes an amino acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, SEQ ID NO: 498 to SEQ ID NO: 520, SEQ ID NO: 1277, or an immunogenic fragment or immunogenic variant thereof.

[0743] Implementation 23(a). An encoding RNA according to any of the preceding embodiments, wherein the coding sequence encodes an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 504, SEQ ID NO: 508 and SEQ ID NO: 509, or an immunogenic fragment or immunogenic variant thereof.

[0744] Implementation 23(b) . An encoding RNA according to any of the preceding embodiments, wherein the coding sequence encodes an amino acid sequence that is identical to any one of SEQ ID NO: 504 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or an immunogenic fragment or immunogenic variant thereof.

[0745] Implementation 23(c) . An encoding RNA according to any of the preceding embodiments, wherein the coding sequence encodes an amino acid sequence that is identical to any one of SEQ ID NO: 508 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or an immunogenic fragment or immunogenic variant thereof.

[0746] Implementation 23(d) . An encoding RNA according to any of the preceding embodiments, wherein the coding sequence encodes an amino acid sequence that is identical to any one of SEQ ID NO: 509 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or an immunogenic fragment or immunogenic variant thereof.

[0747] Implementation Plan 24. An encoding RNA according to any one of embodiments 1 to 22, wherein the coding sequence comprises a nucleic acid sequence or a fragment or variant thereof that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence according to any one of SEQ ID NO: 187 to SEQ ID NO: 246, SEQ ID NO: 257 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO: 670.

[0748] Implementation Plan 25 . The coding RNA according to any of the preceding embodiments, wherein the coding sequence is a codon-modified coding sequence, wherein the amino acid sequence encoded by the coding sequence modified by at least one codon is optionally unmodified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence, optionally wherein the coding sequence modified by at least one codon is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage-adaptive coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof.

[0749] Implementation 26(a) . An encoding RNA according to embodiment 25, wherein the coding sequence comprises at least one of a nucleic acid sequence or a fragment or variant thereof that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQID NO: 648 to SEQ ID NO: 670.

[0750] Implementation 26(b). An encoding RNA according to any of the preceding embodiments, wherein the coding sequence comprises a nucleic acid sequence or at least one of a fragment or variant thereof that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 529, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 554, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 579, SEQ ID NO: 583, SEQ ID NO: 584, SEQ ID NO: 604, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 629, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 654, SEQ ID NO: 658, SEQ ID NO: 659.

[0751] Implementation Plan 27 . The coding RNA according to any one of the preceding embodiments, wherein the coding sequence is a G / C optimized coding sequence.

[0752] Implementation Plan 28 . An encoding RNA according to embodiment 27, wherein the coding sequence comprises at least one of a nucleic acid sequence or a fragment or variant thereof that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 648 to SEQ ID NO: 670.

[0753] Implementation Plan 29. The coding RNA according to embodiment 27, wherein the coding sequence comprises a nucleic acid sequence or at least one of its fragments or variants that is identical to any one of SEQ ID NO: 529, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 554, SEQ ID NO: 558, SEQ ID NO: 559, SEQ ID NO: 654, SEQ ID NO: 658, SEQ ID NO: 659, or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0754] Implementation Plan 30 . The encoding RNA according to any of the preceding embodiments, wherein at least one UTR is selected from at least one 5'-UTR and / or at least one 3'-UTR, optionally wherein the at least one UTR is selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR.

[0755] Implementation Plan 31 . The coding RNA according to embodiment 30, wherein the coding RNA comprises at least one 3'-UTR, wherein the at least one 3'-UTR comprises or consists of a nucleic acid sequence derived from the 3'-UTR of a gene selected from the group consisting of PSMB3, ALB7, α-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a nucleic acid sequence derived from a homolog, fragment or variant of any one of these genes.

[0756] Implementation Plan 32 . An encoding RNA according to embodiment 31, wherein at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence or a fragment or variant thereof that is identical to SEQ ID NO: 67 to SEQ ID NO: 90, SEQ ID NO: 109 to SEQ ID NO: 120 or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0757] Implementation Plan 33 . The coding RNA according to embodiment 31, wherein the coding RNA comprises a 3'-UTR derived from or selected from the PSMB3 gene.

[0758] Implementation Plan 34. The encoding RNA according to embodiment 33, wherein the 3'-UTR derived from or selected from the PSMB3 gene comprises or consists of a nucleic acid sequence or a fragment or variant thereof that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 109 to SEQ ID NO: 120.

[0759] Implementation Plan 35 . An encoding RNA according to any one of embodiments 30 to 34, wherein the encoding RNA comprises at least one 5'-UTR, wherein the at least one (heterologous) 5'-UTR comprises or consists of a nucleic acid sequence derived from the 5'-UTR of a gene selected from the group consisting of HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a nucleic acid sequence derived from a homologue, fragment or variant thereof.

[0760] Implementation Plan 36 . The encoding RNA according to embodiment 35, wherein at least one (heterologous) 5'-UTR derived from or selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2 comprises or consists of a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1 to SEQ ID NO: 32, SEQ ID NO: 65, SEQ ID NO: 66, or a fragment or variant thereof.

[0761] Implementation Plan 37 . The encoding RNA according to embodiment 35 or 36, wherein at least one (heterologous) 5'-UTR is selected from HSD17B4, optionally wherein the 5'-UTR derived from or selected from HSD17B4 comprises or consists of a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 65, SEQ ID NO: 66, or a fragment or variant thereof.

[0762] Implementation Plan 38 . The encoding RNA according to any one of embodiments 30 to 37, wherein at least one (heterologous) 5'-UTR is selected from HSD17B4 and at least one (heterologous) 3'-UTR is selected from PSMB3.

[0763] Implementation Plan 39 . The coding RNA according to any one of the preceding embodiments, wherein the coding RNA of the present invention is a monocistronic.

[0764] Implementation Plan 40 . The encoding RNA according to any one of the preceding embodiments, comprising at least one poly(A) sequence, optionally wherein the at least one poly(A) sequence comprises about 40 to about 500 adenosine nucleotides, such as about 60 to about 250 adenosine nucleotides, such as about 60 to about 150 adenosine nucleotides.

[0765] Implementation Plan 41 . The encoding RNA according to embodiment 40, wherein at least one poly (A) sequence comprises about 100 adenosine nucleotides.

[0766] Implementation Plan 42 . The encoding RNA according to embodiment 40 or 41, wherein at least one poly(A) sequence is located directly at the 3' end, optionally wherein the 3' terminal nucleotide is adenosine.

[0767] Implementation Plan 43 . The coding RNA according to any of the preceding embodiments, comprising at least one poly(C) sequence and / or at least one miRNA binding site and / or histone stem-loop sequence.

[0768] Implementation Plan 44 . The encoding RNA according to embodiment 43 comprises at least one histone stem-loop.

[0769] Implementation Plan 45 . The coding RNA according to embodiment 44, wherein the histone stem-loop sequence comprises or consists of a nucleic acid sequence that is identical to any one of SEQ ID NO: 136, SEQ ID NO: 137, or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof.

[0770] Implementation Plan 46. The coding RNA according to any one of the preceding embodiments, comprising at least one 3' terminal sequence element, optionally wherein the 3' terminal sequence element comprises or consists of an RNA sequence or a fragment or variant thereof that is identical to any one of SEQ ID NO: 138 to SEQ ID NO: 172 or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0771] Implementation Plan 47 . The coding RNA according to embodiment 47 comprises a 3' terminal sequence element, which comprises or consists of an RNA sequence or a fragment or variant thereof that is identical to SEQ ID NO: 144 or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0772] Implementation Plan 48 . The coding RNA according to any one of the preceding embodiments, comprising a 5' terminal sequence element, wherein the 5' terminal sequence element comprises or consists of an RNA sequence that is identical to or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 121 to SEQ ID NO: 127, or a fragment or variant of these sequences.

[0773] Implementation Plan 49 . The coding RNA according to embodiment 48 comprises a 5' terminal sequence element, wherein the 5' terminal sequence element comprises or consists of an RNA sequence or a fragment or variant thereof that is identical to SEQ ID NO: 122 or has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0774] Implementation Plan 50 . The encoding RNA according to any one of the preceding embodiments, comprising a 5' cap structure.

[0775] Implementation Plan 51 . The encoding RNA according to embodiment 50, wherein the 5' cap structure is selected from the cap 1 structure or the modified cap 1 structure.

[0776] Implementation Plan 52 . An encoding RNA according to embodiment 50 or 51, wherein the 5' cap structure is co-transcriptionally added using a trinucleotide cap analog, particularly during RNA in vitro transcription.

[0777] Implementation Plan 53. An encoding RNA according to any one of embodiments 50 to 52, comprising a cap 1 structure.

[0778] Implementation Plan 54 . An encoding RNA according to embodiment 53, wherein the cap 1 structure is formed by co-transcriptional capping using the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG.

[0779] Implementation Plan 55 . The encoding RNA according to embodiment 54, wherein the cap 1 analog is m7G(5')ppp(5')(2'OMeA)pG.

[0780] Implementation Plan 56 . The encoding RNA according to any one of the preceding embodiments, comprising at least one modified nucleotide, the modified nucleotide being optionally selected from pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0781] Implementation Plan 57 . The coding RNA according to embodiment 56, wherein the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), optionally wherein substantially all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0782] Implementation Plan 58 . The encoding RNA according to any of the preceding embodiments, wherein the nucleic acid comprises at least one modified nucleotide, the modified nucleotide being N1-methylpseudouridine (m1ψ).

[0783] Implementation Plan 59 . The encoding RNA according to embodiment 57 or 58, wherein substantially all uracil nucleotides are replaced by N1-methylpseudouridine (m1ψ) nucleotides.

[0784] Implementation Plan 60 . The coding RNA according to any one of the preceding embodiments, wherein the coding RNA is selected from mRNA, coding self-replicating RNA, coding circular RNA, coding viral RNA or coding replicon RNA.

[0785] Implementation Plan 61 . The coding RNA according to embodiment 60, wherein the coding RNA is mRNA.

[0786] Implementation Plan 62. The coding RNA according to any of the preceding embodiments, wherein the coding RNA is an in vitro transcribed RNA, optionally wherein the RNA in vitro transcription has been performed in the presence of a sequence-optimized nucleotide mixture.

[0787] Implementation Plan 63 . The encoding RNA according to any of the preceding embodiments, wherein the encoding RNA is a purified RNA, optionally wherein the RNA has been purified by RP-HPLC, AEX, SEC, hydroxyapatite chromatography, TFF, filtration, precipitation, core-bead flow-through chromatography, oligo(dT) purification, cellulose-based purification, or any combination thereof.

[0788] Implementation Plan 64 . The encoding RNA according to embodiment 63, wherein the RNA has been purified by RP-HPLC and / or TFF.

[0789] Implementation Plan 65 . The encoding RNA according to any of the preceding embodiments, wherein the encoding RNA has an integrity of at least about 50%, such as at least about 60%, more such as at least about 70%, such as at least about 80%.

[0790] Implementation Plan 66 . The coding RNA according to any one of the preceding embodiments, wherein it comprises the following elements, for example, in the 5' to 3' direction:

[0791] A) 5' cap structure;

[0792] B) a 5'-UTR, for example, a 5'-UTR selected from or derived from the HSD17B4 gene;

[0793] C) at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH;

[0794] D) a 3'-UTR, for example, selected from or derived from a 3'-UTR of a PSMB3 gene;

[0795] E) optionally, a histone stem-loop; and

[0796] F) A poly(A) sequence, for example comprising about 100 A nucleotides.

[0797] Implementation Plan 67. The coding RNA according to any one of the preceding embodiments, which comprises or consists of a sequence according to SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: 973 to SEQ ID NO: 995, SEQ ID NO: 998 to SEQ ID NO: SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1259, SEQ ID NO: 1261 to SEQ ID NO: 1262, SEQ ID NO: 1263 to SEQ ID NO: 1264, SEQ ID NO: 1265 to SEQ ID NO: 1266, SEQ ID NO: 1267 to SEQ ID NO: 1268, SEQ ID NO: 1269 to SEQ ID NO: 1300, SEQ ID NO: 1311 to SEQ ID NO: 1320 1270, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of 1270, or a fragment or variant thereof.

[0798] Implementation Plan 68 . The coding RNA according to embodiment 67, wherein at least one uracil nucleotide in the RNA sequence, such as all uracil nucleotides, is replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0799] Implementation Plan 69 . The coding RNA according to any one of the preceding embodiments, wherein the coding RNA comprises or consists of a polypeptide according to SEQ ID NO: 679, SEQ ID NO: 704, SEQ ID NO: 729, SEQ ID NO: 754, SEQ ID NO: 779, SEQ ID NO: 804, SEQ ID NO: 829, SEQ ID NO: 854, SEQ ID NO: 879, SEQ ID NO: 904, SEQ ID NO: 929, SEQ ID NO: 954, SEQ ID NO: 979, SEQ ID NO: 1004, SEQ ID NO: 1029, SEQ ID NO: 1054, SEQ ID NO: 1079, SEQ ID NO: 1104, SEQ ID NO: 1129, SEQ ID NO: 1154, SEQ ID NO: 1179, SEQ ID NO: 1204, SEQ ID NO: 1229, SEQ ID NO: 1254, or a nucleic acid sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of 1254, or a fragment or variant thereof, optionally encoding an RNA comprising a 5' end cap 1 structure.

[0800] Implementation Plan 70. The coding RNA according to any one of the preceding embodiments, wherein the coding RNA comprises or consists of a sequence according to SEQ ID NO: 683, SEQ ID NO: 708, SEQ ID NO: 733, SEQ ID NO: 758, SEQ ID NO: 783, SEQ ID NO: 808, SEQ ID NO: 833, SEQ ID NO: 858, SEQ ID NO: 883, SEQ ID NO: 908, SEQ ID NO: 933, SEQ ID NO: 958, SEQ ID NO: 983, SEQ ID NO: 1008, SEQ ID NO: 1033, SEQ ID NO: 1058, SEQ ID NO: 1083, SEQ ID NO: 1108, SEQ ID NO: 1133, SEQ ID NO: 1158, SEQ ID NO: 1183, SEQ ID NO: 1208, SEQ ID NO: 1233, SEQ ID NO: 1258, or a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof, optionally encoding an RNA comprising a 5' end cap 1 structure.

[0801] Implementation Plan 71. The coding RNA according to any one of the preceding embodiments, wherein in one embodiment, the coding RNA comprises or consists of a polypeptide according to SEQ ID NO: 684, SEQ ID NO: 709, SEQ ID NO: 734, SEQ ID NO: 759, SEQ ID NO: 784, SEQ ID NO: 809, SEQ ID NO: 834, SEQ ID NO: 859, SEQ ID NO: 884, SEQ ID NO: 909, SEQ ID NO: 934, SEQ ID NO: 959, SEQ ID NO: 984, SEQ ID NO: 1009, SEQ ID NO: 1034, SEQ ID NO: 1059, SEQ ID NO: 1084, SEQ ID NO: 1109, SEQ ID NO: 1134, SEQ ID NO: 1159, SEQ ID NO: 1184, SEQ ID NO: 1209, SEQ ID NO: 1234, SEQ ID NO: 1259, or a nucleic acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof, optionally encoding an RNA comprising a 5' end cap 1 structure.

[0802] Implementation 72(a) . The coding RNA according to embodiment 69, wherein the coding RNA is a 5'-capped (cap 1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, or unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides;

[0803] It is identical to the RNA sequence according to any one of SEQ ID NO: 679, SEQ ID NO: 829, SEQ ID NO: 979, SEQ ID NO: 1129, SEQ ID NO: 1271, SEQ ID NO: 1274, or a fragment or variant thereof.

[0804] Implementation 72(b). The coding RNA according to embodiment 69, wherein the coding RNA is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides;

[0805] It is identical to the RNA sequence according to SEQ ID NO: 1271 or a fragment or variant thereof.

[0806] Implementation 72(c) . The coding RNA according to embodiment 69, wherein the coding RNA is a 5'-capped (cap 1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides;

[0807] It is identical to the RNA sequence according to SEQ ID NO: 1274 or a fragment or variant thereof.

[0808] Implementation 73(a) . The coding RNA according to embodiment 70, wherein the coding RNA is a 5'-capped (cap 1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, or unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides;

[0809] It is identical to the RNA sequence according to any one of SEQ ID NO: 683, SEQ ID NO: 833, SEQ ID NO: 983, SEQ ID NO: 1133, SEQ ID NO: 1272, SEQ ID NO: 1275, or a fragment or variant thereof.

[0810] Implementation 73(b) . The coding RNA according to embodiment 70, wherein the coding RNA is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides;

[0811] It is identical to the RNA sequence according to SEQ ID NO: 1272 or a fragment or variant thereof.

[0812] Implementation 73(c). The coding RNA according to embodiment 70, wherein the coding RNA is a 5'-capped (cap 1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides;

[0813] It is identical to the RNA sequence according to SEQ ID NO: 1275 or a fragment or variant thereof.

[0814] Implementation 74(a) . The coding RNA according to embodiment 71, wherein the coding RNA is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, or unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides;

[0815] It is identical to the RNA sequence according to any one of SEQ ID NO: 684, SEQ ID NO: 834, SEQ ID NO: 984, SEQ ID NO: 1134, SEQ ID NO: 1273, SEQ ID NO: 1276, or a fragment or variant thereof.

[0816] Implementation 74(b) . The coding RNA according to embodiment 69, wherein the coding RNA is a 5'-capped (cap 1) mRNA, which comprises or consists of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides;

[0817] It is identical to the RNA sequence according to SEQ ID NO: 1273 or a fragment or variant thereof.

[0818] Implementation 74(c) . The coding RNA according to embodiment 69, wherein the coding RNA is a 5'-capped (cap 1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides;

[0819] It is identical to the RNA sequence according to SEQ ID NO: 1276 or a fragment or variant thereof.

[0820] Implementation Plan 75 . A pharmaceutical composition comprising the encoding RNA according to any one of the preceding embodiments.

[0821] Implementation Plan 76. According to embodiment 75, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier or excipient.

[0822] Implementation Plan 77 . A pharmaceutical composition according to embodiment 75 or 76, comprising a lipid-based carrier, optionally wherein the encoding RNA is formulated in the lipid-based carrier.

[0823] Implementation Plan 78 . A pharmaceutical composition according to embodiment 77, wherein the lipid-based carrier is selected from liposomes, lipid nanoparticles, liposome complexes, solid lipid nanoparticles, liposome complexes and / or nanoliposomes.

[0824] Implementation Plan 79 . A pharmaceutical composition according to embodiment 78, wherein the lipid-based carrier is a lipid nanoparticle, optionally wherein the lipid nanoparticle encapsulates the encoding RNA.

[0825] Implementation Plan 80 . A pharmaceutical composition according to any one of embodiments 75 to 79, wherein the encoding RNA is formulated in at least one cationic or polycationic compound.

[0826] Implementation Plan 81 . A pharmaceutical composition according to embodiment 80, wherein at least one cationic or polycationic compound is selected from a cationic or polycationic polymer, a cationic or polycationic polysaccharide, a cationic or polycationic lipid, a cationic or polycationic protein, a cationic or polycationic peptide, or any combination thereof.

[0827] Implementation Plan 82 . A pharmaceutical composition according to any one of embodiments 77 to 81, wherein the lipid-based carrier comprises at least one lipid selected from aggregation-reducing lipids, cationic lipids or ionizable lipids, neutral lipids or phospholipids, or steroids, steroid analogs or sterols, or any combination thereof.

[0828] Implementation Plan 83 . According to the pharmaceutical composition of embodiment 82, the lipid-based carrier comprises an aggregation-reducing lipid, a cationic lipid or an ionizable lipid, a neutral lipid or a phospholipid, and a steroid, a steroid analog or a sterol.

[0829] Implementation Plan 84 . A pharmaceutical composition according to any one of embodiments 77 to 83, wherein the lipid-based carrier comprises a cationic lipid selected from or derived from Formula III, such as Formula III-3.

[0830] Implementation Plan 85. A pharmaceutical composition according to any one of embodiments 77 to 84, wherein the lipid-based carrier comprises a cationic lipid selected from or derived from ALC-0315.

[0831] Implementation Plan 86 . A pharmaceutical composition according to any one of embodiments 77 to 85, wherein the lipid-based carrier comprises an aggregation-reducing lipid selected from polymer-conjugated lipids, optionally wherein the polymer-conjugated lipid is a PEG-conjugated lipid selected from or derived from formula IVa, such as a PEG-conjugated lipid se...

Claims

1. A coding RNA comprising at least one untranslated region (UTR) and at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli type 1 fimbriae D-mannose-specific adhesin (FimH).

2. The encoding RNA according to claim 1, wherein the E. coli FimH comprises an amino acid sequence that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, or an immunogenic fragment or immunogenic variant thereof.

3. The coding RNA according to claim 1 or 2, wherein the coding sequence additionally encodes one or more than one other peptide or protein element, wherein the one or more than one other peptide or protein element is selected from: a donor chain peptide, a signal peptide, an antigen aggregation domain or a transmembrane domain.

4. The coding RNA according to claim 3, wherein one or more than one other peptide or protein element is a donor strand peptide, optionally wherein the coding sequence encodes the following elements in the N-terminal to C-terminal direction: an antigenic polypeptide selected from or derived from Escherichia coli FimH; and a donor strand peptide.

5. The coding RNA according to claim 4, wherein the donor chain peptide comprises or consists of an amino acid sequence of SEQ ID NO: 338 or a variant thereof, optionally wherein the variant of SEQ ID NO: 338 has 1 to 5 single amino acid mutations compared to SEQ ID NO: 338, such as 1, 2, 3 or 4 single amino acid mutations.

6. The coding RNA according to any one of claims 4 or 5, wherein the coding sequence additionally encodes a peptide linker, optionally wherein the coding sequence encodes the following elements in the direction from the N-terminus to the C-terminus: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker element; and a donor strand peptide.

7. The coding RNA according to claim 6, wherein the peptide linker comprises or consists of SEQ ID NO:

352.

8. The coding RNA according to any one of claims 3 to 7, wherein the coding sequence additionally encodes an antigen aggregation domain, optionally wherein the antigen aggregation domain is selected from or derived from ferritin or dioxotetrahydropterin synthase.

9. The encoding RNA according to claim 8, wherein the amino acid sequence of the antigen aggregation domain is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NO: 457 to SEQ ID NO: 459, SEQ ID NO: 443, SEQ ID NO: 444, or is a fragment or variant thereof.

10. The coding RNA according to any one of claims 3 to 9, wherein the coding sequence additionally encodes a signal peptide, optionally wherein the signal peptide is selected from or derived from IgE or IgK, wherein the amino acid sequence of the signal peptide is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences of SEQ ID NO: 394, SEQ ID NO: 395, or is a fragment or variant thereof.

11. The coding RNA according to any one of claims 3 to 10, wherein the coding sequence optionally encodes the following elements in the direction from N-terminus to C-terminus: a) Signal peptide, antigenic peptide; b) signal peptide, antigenic peptide, peptide linker, donor chain peptide; c) antigen aggregation domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide; d) signal peptide, antigen aggregation domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide; e) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, antigen aggregation domain; or f) Signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, transmembrane domain.

12. The coding RNA according to any one of the preceding claims, wherein the coding sequence encodes an amino acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 177 to SEQ ID NO: 186, SEQ ID NO: 247 to SEQ ID NO: 256, SEQ ID NO: 498 to SEQ ID NO: 520, SEQ ID NO: 1277, or an immunogenic fragment or immunogenic variant thereof.

13. The coding RNA according to any one of the preceding claims, wherein the coding sequence comprises a nucleic acid sequence or a fragment or variant thereof that is identical to or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NO: 187 to SEQ ID NO: 246, SEQ ID NO: 257 to SEQ ID NO: 316, SEQ ID NO: 523 to SEQ ID NO: 545, SEQ ID NO: 548 to SEQ ID NO: 570, SEQ ID NO: 573 to SEQ ID NO: 595, SEQ ID NO: 598 to SEQ ID NO: 620, SEQ ID NO: 623 to SEQ ID NO: 645, SEQ ID NO: 648 to SEQ ID NO:

670.

14. The coding RNA according to any one of the preceding claims, wherein the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), optionally wherein substantially all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

15. The coding RNA according to any one of the preceding claims, wherein the coding sequence is a codon-modified coding sequence, wherein the amino acid sequence encoded by at least one codon-modified coding sequence is optionally unmodified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence, optionally wherein at least one codon-modified coding sequence is selected from a C-maximized coding sequence, a codon adaptation index (CAI)-maximized coding sequence, a human codon usage-adaptive coding sequence, a G / C content-modified coding sequence, and a G / C-optimized coding sequence, or any combination thereof.

16. The coding RNA according to any one of the preceding claims, wherein the coding RNA is an mRNA, which optionally comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 673 to SEQ ID NO: 695, SEQ ID NO: 698 to SEQ ID NO: 720, SEQ ID NO: 723 to SEQ ID NO: 745, SEQ ID NO: 748 to SEQ ID NO: 770, SEQ ID NO: 773 to SEQ ID NO: 795, SEQ ID NO: 798 to SEQ ID NO: 820, SEQ ID NO: 823 to SEQ ID NO: 845, SEQ ID NO: 848 to SEQ ID NO: 870, SEQ ID NO: 873 to SEQ ID NO: 895, SEQ ID NO: 898 to SEQ ID NO: 920, SEQ ID NO: 923 to SEQ ID NO: 945, SEQ ID NO: 948 to SEQ ID NO: 970, SEQ ID NO: SEQ ID NO: 1170, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1198 to SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1270, SEQ ID NO: 1173 to SEQ ID NO: 1195, SEQ ID NO: 1220, SEQ ID NO: 1223 to SEQ ID NO: 1245, SEQ ID NO: 1248 to SEQ ID NO: 1276, or a nucleic acid sequence that is identical or has at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or a fragment or variant thereof.

17. A pharmaceutical composition comprising the encoding RNA according to any one of claims 1 to 16.

18. The pharmaceutical composition of claim 17, further comprising a lipid-based carrier, wherein the lipid-based carrier is a lipid nanoparticle (LNP).

19. A vaccine comprising the encoding RNA according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 17 or 18.

20. A kit or kit set comprising the encoding RNA according to any one of claims 1 to 16, the pharmaceutical composition according to any one of claims 17 or 18 and / or the vaccine according to claim 19, optionally comprising a liquid carrier for dissolution, and optionally, technical instructions providing information on the administration and dosage of the components.

21. The encoding RNA according to any one of claims 1 to 16, the pharmaceutical composition according to any one of claims 17 or 18, the vaccine according to claim 19 or the kit or kit set according to claim 19 for use as a medicament.

22. The encoding RNA according to any one of claims 1 to 17, the pharmaceutical composition according to any one of claims 18 or 19, the vaccine according to claim 20, or the kit or kit set according to claim 21, for use in treating or preventing one or more symptoms associated with urinary tract infection (UTI) in a subject in need thereof.

23. The coding RNA according to any one of claims 1 to 16, the pharmaceutical composition according to any one of claims 17 or 18, the vaccine according to claim 19 or the kit or kit set according to claim 20 for use in the treatment or prevention of a disease caused by Escherichia coli.

24. A method for treating or preventing a disorder, wherein the method comprises administering to a subject in need thereof an effective amount of an encoding RNA according to any one of claims 1 to 16, a pharmaceutical composition according to any one of claims 17 or 18, a vaccine according to claim 21, or a kit or kit set according to claim 20.

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