Nucleic Acid-Based Vaccines

Abstract

The present disclosure is directed to coding RNAs encoding antigenic polypeptides selected from or derived from Escherichia coli FimH. The present disclosure is also directed to compositions and vaccines comprising the aforementioned coding RNAs. Furthermore, the present disclosure relates to kits, particularly kits of parts comprising the coding RNAs, or compositions, or vaccines. The present disclosure is also directed to methods of treating or preventing disorders caused by E. coli.

Description

[Technical field]

[0001] (Sequence Listing) This application contains an ST.26 Sequence Listing in XML file format, submitted electronically (created May 19, 2023), which is incorporated by reference herein in its entirety. Additional sequences shorter than 10 specifically defined nucleotides or 4 specifically defined amino acids are disclosed in Table 13.

[0002] The present disclosure is directed to coding RNA encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH. The present disclosure is also directed to pharmaceutical compositions, vaccines, kits or kits of parts suitable for use in the treatment and / or prevention of disease, particularly urinary tract infections (UTIs). [Background technology]

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

[0004] The type 1 pilin tip-localized adhesin FimH (type 1 fimbrial D-mannose-specific adhesin) enables 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 linked by a 5-amino acid linker: an N-terminal lectin domain (FimHL) that binds mannose on urothelial cell receptors, and a C-terminal pyrin domain (FimHP). The pyrin domain (FimHP) has an (Ig)-like fold but lacks the seventh C-terminal β-strand. The absence of the strand creates a deep groove on the surface of FimHP, exposing its hydrophobic core and rendering FimH unstable when expressed without a chaperone. In the chaperone:subunit complex, FimHP interacts noncovalently with a donor strand, either from the periplasmic chaperone FimC or from the subsequent subunit of the assembled pilus (FimG), simultaneously stabilizing the pilus subunit and covering its interaction surface in processes known as donor strand complementation and donor strand exchange, respectively.

[0006] The lectin domain (FimHL) is known to adopt two conformations with different mannose-binding affinities: a high-affinity conformation also called the relaxed (R) state, and a low-affinity conformation also called the tension (T) state. The in vivo conformation of FimH is affected by flow conditions, and shear stress conditions are known to induce the high mannose-binding conformation.

[0007] Antibodies that bind to FimH inhibit bacterial adhesion to the urinary tract, thereby preventing colonization and promoting bacterial clearance (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 improve adhesion inhibition to the bladder (Tchesnokova et al. Infect Immun. 2011 Oct;79(10):3895-904). The transfer of serum IgG to the urogenital tract appears to be involved in the inhibition of bacterial adhesion.

[0008] Therefore, FimH is considered a promising vaccine antigen. However, it is difficult to produce FimH on a commercial scale because it needs to be produced in sufficient quantities and in a conformation that can elicit functional antibodies.

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

[0010] Alternative strategies have also been reported to recombinantly produce FimH in a functional conformation, such as engineering 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 chain peptide of FimG (Sauer MM, et al. Nat Commun. 2016 Mar 7;7:10738), or mammalian cell expression of FimH stabilized by the donor chain peptide of FimG.

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

[0012] In a first aspect of the invention, a coding RNA is provided that comprises 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 fimbria D-mannose specific adhesin (FimH). In one embodiment, the E. coli FimH comprises an amino acid sequence identical to, or 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 SEQ ID NOs: 177-186, 247-256, or is an immunogenic fragment or variant thereof.

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

[0014] In one embodiment, the donor chain peptide comprises or consists of the 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, such as 1, 2, 3 or 4, single amino acid mutations compared to SEQ ID NO: 338. In one embodiment, the coding sequence further encodes a peptide linker element, optionally wherein the coding sequence encodes, from N-terminal to C-terminal, the following elements: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker element; and a donor chain 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 further encodes an antigen clustering domain, optionally wherein the antigen clustering domain is selected from or derived from ferritin or lumazine synthase. In some additional embodiments, the amino acid sequence of the antigen clustering domain is identical to, or 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 amino acid sequences SEQ ID NOs: 457-459, 443, 444, or a fragment or variant thereof.

[0017] In certain embodiments, the coding sequence further encodes a signal peptide, optionally the signal peptide is or is derived from Immunoglobulin E (IgE) or Immunoglobulin Kappa (IgK). In some additional embodiments, the amino acid sequence of the aforementioned signal peptide 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 the amino acid sequence of any one of SEQ ID NOs: 394, 395, or a fragment or variant thereof.

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

[0019] In some embodiments, the coding sequence encodes an amino acid sequence that is identical to, or 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 SEQ ID NOs: 177-186, 247-256, 498-520, 1277, or an immunogenic fragment or immunogenic variant thereof. In some embodiments, the coding sequence comprises a nucleic 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 any one of SEQ ID NOs: 187-246, 257-316, 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, 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-methylpseudouridine (m1ψ), and optionally, essentially all uracil nucleotides are replaced with pseudouridine (ψ) and / or N1-methylpseudouridine (m1ψ) nucleotides. In some embodiments, the coding sequence is a codon-modified coding sequence, wherein the amino acid sequence encoded by the at least one codon-modified coding sequence is optionally unmodified compared to the amino acid sequence encoded by a corresponding wild-type coding sequence, and optionally, the at least one codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage adapted 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 the sequences of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 11 The nucleic acid sequence of any one of the nucleic acids 48-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1276, or a fragment or variant thereof, is identical to, or comprises or consists of, 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, the nucleic acid sequence of any one of the nucleic acids 48-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1276, or a fragment or variant thereof.

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

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

[0024] In a fourth aspect, a kit or kit of parts is provided comprising the coding RNA, pharmaceutical composition, and / or vaccine of the present disclosure, optionally including a liquid vehicle for solubilization, and optionally including technical instructions providing information regarding administration and dosing of the components.

[0025] In a further aspect, the coding RNA, pharmaceutical composition, vaccine, or kit or kit of parts of the disclosure is provided for use as a medicament.In one embodiment, the coding RNA, pharmaceutical composition, vaccine, or kit or kit of parts of the disclosure is for use in treating or preventing one or more symptoms associated with urinary tract infection (UTI) in a subject in need thereof.

[0026] In a further aspect, there is provided a coding RNA, a pharmaceutical composition, a vaccine, or a kit or kit of parts of the disclosure for use as a medicament. In one embodiment, the coding RNA, the pharmaceutical composition, the vaccine, or the kit or kit of parts of the disclosure is for use in the treatment or prevention of a disease caused by E. coli.

[0027] In a further aspect, a method for treating or preventing a disorder is provided, comprising administering to a subject in need thereof an effective amount of the coding RNA, pharmaceutical composition, vaccine, or kit or kit of parts of the present disclosure. In one embodiment, the method induces antibodies capable of inhibiting bacterial adhesion. [Brief description of the drawings]

[0028] [Figure 1A] (A)(B): mRNA constructs encoding different E. coli FimH antigen designs were expressed and partially secreted from mammalian cells using Western blot analysis. Experiments were performed as described in Example 2.1. [Figure 1B] (A)(B): mRNA constructs encoding different E. coli FimH antigen designs were expressed and partially secreted from mammalian cells using Western blot analysis. Experiments were performed as described in Example 2.1. [Figure 2A] (A)-(F) show that formulated mRNA constructs encoding different E. coli FimH antigen designs induced humoral immune responses in mice. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 2B] (A)-(F) show that formulated mRNA constructs encoding different E. coli FimH antigen designs induced humoral immune responses in mice. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 2C] (A)-(F) show that formulated mRNA constructs encoding different E. coli FimH antigen designs induced humoral immune responses in mice. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 2D] (A)-(F) show that formulated mRNA constructs encoding different E. coli FimH antigen designs induced humoral immune responses in mice. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 2E] (A)-(F) show that formulated mRNA constructs encoding different E. coli FimH antigen designs induced humoral immune responses in mice. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 2F] (A)-(F) show that formulated mRNA constructs encoding different E. coli FimH antigen designs induced humoral immune responses in mice. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Diagram 3]FIG. 1 shows CD4+ and CD8+ T cell responses induced by vaccinating mice with mRNA constructs encoding different E. coli FimH antigen designs as described in Example 2.4. [Figure 4A] (A)-(C): Dose response of rats vaccinated with formulated mRNA constructs encoding different E. coli FimH antigen designs. Serum and urinary IgG titers were assessed by ELISA as described in Example 3.1. [Figure 4B] (A)-(C): Dose response of rats vaccinated with formulated mRNA constructs encoding different E. coli FimH antigen designs. Serum and urinary IgG titers were assessed by ELISA as described in Example 3.1. [Figure 4C] (A)-(C): Dose response of rats vaccinated with formulated mRNA constructs encoding different E. coli FimH antigen designs. Serum and urinary IgG titers were assessed by ELISA as described in Example 3.1. [Figure 5A] (A)(B): mRNA constructs encoding E. coli FimH antigen designs containing uridine, ψ, or m1ψ were expressed and partially secreted from mammalian cells using Western blot analysis. Experiments were performed as described in Example 4.1. [Figure 5B] (A)(B): mRNA constructs encoding E. coli FimH antigen designs containing uridine, ψ, or m1ψ were expressed and partially secreted from mammalian cells using Western blot analysis. Experiments were performed as described in Example 4.1. [Figure 6A] (A)-(C): Formulated mRNA constructs encoding E. coli FimH antigen designs containing uridine, ψ, or m1ψ induced humoral immune responses in rats. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 6B](A)-(C): Formulated mRNA constructs encoding E. coli FimH antigen designs containing uridine, ψ, or m1ψ induced humoral immune responses in rats. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. [Figure 6C] (A)-(C): Formulated mRNA constructs encoding E. coli FimH antigen designs containing uridine, ψ, or m1ψ induced humoral immune responses in rats. Serum and urinary IgG titers were assessed by ELISA as described in Example 2.2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] <Definition> For clarity and readability, the following definitions are provided. The technical features mentioned for these definitions can be read into each embodiment of the present invention. Additional definitions and explanations may be provided specifically in the context of these embodiments.

[0030] Percentages in numerical contexts should be understood as relative to the total number of the respective item. In other cases, unless the context dictates otherwise, percentages should be understood as weight percent (wt.-%).

[0031] About: The term "about" is used when the determinant or value does not need to be identical, i.e., 100% identical. Thus, "about" means that the determinant or value may vary by 1% to 20%, for example, 1% to 10%, particularly 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. A person skilled in the art knows that, for example, certain parameters or determinants may vary slightly based on how the parameter was determined. For example, if a determinant or value is defined herein as having a length of, for example, "about 100 nucleotides", the length may vary by 1% to 20%. Thus, a person skilled in the art knows that in that specific example, the length may vary by 1 to 20 nucleotides. Thus, a length of "about 100 nucleotides" can encompass sequences ranging from 80 to 120 nucleotides.

[0032] Adaptive immune response: The term "adaptive immune response" as used herein will be recognized and understood by those skilled in the art and is intended to refer, for example, to an antigen-specific response of the immune system (adaptive immune system). Antigen specificity allows for the generation of a response tailored to a particular pathogen or pathogen-infected cell. The ability to mount such a tailored response is typically maintained in the body by "memory cells" (B cells).

[0033] Antigen: The term "antigen" as used herein will be recognized and understood by those skilled in the art and is intended to refer to a substance that can be recognized by the immune system, e.g., the adaptive immune system, and can elicit an antigen-specific immune response, e.g., by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen can be or consist of a peptide or protein that can be presented to T cells by MHC. Also understood as antigens are fragments, variants, and derivatives of peptides or proteins that contain at least one epitope.

[0034] Antigenic Peptide, Polypeptide, or Protein: The term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by those of skill in the art and is intended to refer to, for example, a peptide, protein derived from an (antigenic or immunogenic) protein that stimulates the body's adaptive immune system to provide an adaptive immune response. Thus, an antigenic / immunogenic peptide or protein contains at least one epitope (as defined herein) or antigen (as defined herein) of the protein from which it is derived.

[0035] Cationic: Unless a different meaning is clear from the particular context, the term "cationic" means that the respective structure has a positive charge, either permanently or not permanently but in response to certain conditions, such as pH. Thus, the term "cationic" includes both "permanently cationic" and "cationizable". The term "permanently cationic" means, for example, that the respective compound, group, or atom is positively charged at all pH values ​​or hydrogen ion activities of its environment. Typically, the positive charge is due to the presence of a quaternary nitrogen atom. Compounds with multiple such positive charges are called persistent polycations.

[0036] Cationizable: The term "cationizable" as used herein means that a compound, or group or atom, is positively charged when the pH of its environment is low, and uncharged when the pH is high. Also, in non-aqueous environments where the pH value cannot be determined, a cationizable compound, group or atom is positively charged when the hydrogen ion concentration is high, and uncharged when the hydrogen ion concentration or activity is low. Depending on the individual properties of a cationizable or polycationizable compound, in particular the pKa of each cationizable group or atom, at what pH or hydrogen ion concentration it becomes charged or uncharged. In a dilute aqueous environment, the proportion of a cationizable compound, group or atom that has a positive charge can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, in some embodiments, if the compound or moiety is cationizable, it is preferred that it is positively charged at a pH value of about 1-9, preferably 4-9, 5-8, or even 6-8, such as a pH value of 9 or less, 8 or less, or 7 or less, such as a physiological pH value of about 7.3-7.4, i.e., under physiological conditions, particularly physiological salt conditions of cells in vivo. In other embodiments, it is preferred that the cationizable compound or moiety is primarily neutral at physiological pH values, such as about 7.0-7.4, but positively charged at lower pH values. In some embodiments, the pKa range of the cationizable compound or moiety is about 5 to about 7.

[0037] Coding sequence / coding region: The terms "coding sequence" or "coding region" as used herein, and the corresponding abbreviation "cds", will be recognized and understood by those skilled in the art, and are intended to refer to a sequence of several nucleotide triplets that can be translated, for example, into a peptide or protein. A coding sequence in the context of this disclosure can be an RNA sequence that begins with a start codon and ends, for example, with a stop codon, and consists of a number of nucleotides divisible by 3.

[0038] Derived from: The term "derived from" as used throughout the specification in the context of nucleic acids, i.e., relates to a nucleic acid that is "derived from" (another) nucleic acid, that is derived from (another) nucleic acid, shares, 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 with the nucleic acid from which it is derived. Those skilled in the art will recognize that sequence identity is usually calculated for nucleic acids of the same type, i.e., DNA or RNA sequences. Thus, when DNA is "derived" from RNA, or RNA is "derived" from DNA, it is understood that as a first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by replacing uracil (U) with thymine (T) throughout the sequence), or vice versa, the DNA sequence is converted to the corresponding RNA sequence (particularly by replacing T with U throughout the sequence). The sequence identity of the DNA sequence or the sequence identity of the RNA sequence is then determined. For example, a nucleic acid "derived" from a nucleic acid also refers to a nucleic acid that has been modified compared to the nucleic acid from which it is derived, for example to further increase the stability of the RNA and / or to extend and / or increase the production of a protein. In the context of amino acid sequences (e.g., antigenic peptides or proteins), the term "derived from" means that an amino acid sequence derived from an (another) amino acid sequence shares, 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 with the amino acid sequence from which it is derived.

[0039] Donor chain peptide: As used throughout this specification, the term "donor chain peptide" refers to the portion of a FimC or FimG polypeptide that interacts with FimHP in vivo or in vitro, occupies the groove, and completes the atypical Ig fold of FimHP by running parallel to the subunit C-terminal F strand.

[0040] Fragment: The term "fragment" as used throughout the present specification in the context of a nucleic acid sequence (e.g., RNA or DNA) or an amino acid sequence is typically a shorter portion of the full-length sequence of, for example, a nucleic acid sequence or an amino acid sequence. Thus, a fragment typically consists of a sequence identical to a corresponding stretch in the full-length sequence. A particular fragment of a sequence in the context of the present disclosure consists of a contiguous stretch of nucleotides or amino acids corresponding to a contiguous stretch of entities in the molecule from which the fragment is derived, which stretch represents at least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the entire (i.e., full-length) of the molecule from which the fragment is derived (e.g., a viral protein). The term "fragment" as used throughout the present specification in the context of a protein or peptide can typically consist of a protein or peptide sequence as defined herein, which sequence is N-terminally and / or C-terminally truncated with respect to its amino acid sequence, compared to the amino acid sequence of the original protein. The term "fragment" as used throughout the present specification in the context of an RNA sequence can typically include an RNA sequence that is 5'-terminally and / or 3'-terminally truncated compared to a reference RNA sequence. Such truncations may therefore occur either at the amino acid level or, correspondingly, at the nucleic acid level. Thus, sequence identity with respect to such fragments as defined herein may refer, for example, to the entire protein or peptide as defined herein, or to the entire (encoding) nucleic acid molecule of such protein or peptide. Fragments of proteins or peptides may contain at least one epitope of those proteins or peptides.

[0041] Identity (of a sequence): 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 those of skill in the art and is intended to refer to, for example, the percentage by which two sequences are identical. To determine the percentage by which two sequences are identical, e.g., a nucleic acid sequence or an amino acid (aa) sequence as defined herein, e.g., an aa sequence encoded by a nucleic acid sequence as defined herein or the aa sequence itself, the sequences can be aligned for subsequent comparison with each other. Thus, for example, a position of a first sequence can be compared to a corresponding position of a second sequence. If a position of the first sequence is occupied by the same residue as a position of the second sequence, the two sequences are identical at this position. If not, the sequences differ at this position. If an insertion occurs in the second sequence compared to the first sequence, a gap can be inserted into the first sequence and further alignment can be performed. If a deletion occurs in the second sequence compared to the first sequence, a gap can be inserted into the second sequence and further alignment can be performed. The percentage that two sequences are identical is then a function of the number of identical positions divided by the total number of positions, including positions that occur in only one sequence. The percentage that two sequences are identical can be determined using an algorithm, such as the algorithm built into the BLAST program.Sequence identity can be determined using the EMBOSS Water sequence alignment tool at the EMBL-EBI website https: / / www.ebi.ac.uk / Tools / psa / emboss_water / with parameters gap open=12, gap extend=1, and matrix=BLOSUM62 for protein sequences or matrix=fullDNA for DNA / RNA sequences, or the EMBOSS Needle sequence alignment tool at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / with default parameters (e.g., gap open=10, gap extend=0.5, end gap penalty=false, end gap open=10, and end gap extend=0.5, and matrix=BLOSUM62 for protein sequences or matrix=fullDNA for DNA / RNA sequences). Unless otherwise specified, when the Application refers to sequence identity to a particular reference sequence, it is intended that the identity is calculated over the entire length of that reference sequence. Immunogen: The term "immunogen" or "immunogenic" will be recognized and understood by those of skill in the art and is intended to refer to a compound capable of stimulating / inducing, for example, an (adaptive) immune response. Immunogens can be peptides, polypeptides, or proteins.

[0042] Immune response: The term "immune response" will be recognized and understood by those of skill in the art and is intended to refer, for example, to a specific reaction of the adaptive immune system to a particular antigen (a so-called specific or adaptive immune response), or a non-specific reaction of the innate immune system (a so-called non-specific or innate immune response), or a combination thereof.

[0043] Lipidoids: Lipidoids, also called lipidoid compounds, are lipid-like compounds, i.e. amphipathic compounds that have lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoid compounds.

[0044] Nucleic acid, nucleic acid molecule: The term "nucleic acid" or "nucleic acid molecule" as used herein will be recognized and understood by those skilled in the art. The term "nucleic acid" or "nucleic acid molecule" refers specifically to DNA (molecule) or RNA (molecule). The term is used synonymously with the term polynucleotide. For example, a nucleic acid or nucleic acid molecule is a polymer that comprises or consists of nucleotide monomers covalently linked to each other by sugar / phosphate backbone phosphodiester bonds. The term "nucleic acid" or "nucleic acid molecule" also encompasses modified nucleic acids (molecules), such as base-, sugar- or backbone-modified DNA or RNA (molecule) as defined herein.

[0045] Nucleic acid sequence, DNA sequence, RNA sequence: The terms "nucleic acid sequence", "DNA sequence", "RNA sequence" will be recognized and understood by those of skill in the art and refer to a particular particular order of a sequence of nucleotides, for example.

[0046] RNA species: In the context of the present disclosure, the term "RNA species" is not limited to mean one single molecule, but is understood to constitute a collection of essentially identical RNA molecules. It can therefore relate to a plurality of essentially identical RNA molecules.

[0047] RNA: The term "RNA" is an abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer made up of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP) monomers or their analogs, which are linked together along a so-called backbone. The backbone is usually formed by a phosphodiester bond between the sugar, i.e. the ribose, of a first monomer and the phosphate moiety of a second adjacent monomer. The specific order of the monomers, i.e. the order of the bases attached to the sugar / phosphate backbone, is called the RNA sequence. In general, RNA can be obtained by transcription of a DNA sequence, for example 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.

[0048] RNA in vitro transcription: The term "RNA in vitro transcription" or "in vitro transcription" refers to a process in which RNA is synthesized in vitro in a cell-free system. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which is typically a linear DNA template (e.g. linearized plasmid DNA or PCR product). The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5 RNA polymerase. In one embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme before being subjected to RNA in vitro transcription. Reagents typically used in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has high binding affinity for a respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine, and uracil); optionally, a cap analog as defined herein; optionally, modified nucleotides as defined herein; a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5 RNA polymerase) capable of binding to the promoter sequence in the DNA template; optionally, a ribonuclease (RNase) inhibitor to inactivate potentially contaminating RNases; optionally, pyrophosphatase; MgCl 2 a buffer (TRIS or HEPES) to maintain a suitable pH value, and may also include an antioxidant (e.g., DTT), and / or a polyamine such as spermidine.

[0049] Variant (of a sequence): The term "variant" as used throughout the present specification in the context of a nucleic acid sequence will be recognized and understood by those skilled in the art and is intended to refer to a variant of a nucleic acid sequence derived, for example, from another nucleic acid sequence. For example, a variant of a nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and / or substitutions compared to the nucleic acid sequence from which the variant is derived. A variant of a nucleic acid sequence may be at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleic acid sequence from which it is derived. A variant is a functional variant in the sense that it retains at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the function of the sequence from which it is derived. A "variant" of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of at least 10, 20, 30, 50, 75 or 100 nucleotides of such a nucleic acid sequence.

[0050] The term "variant" as used throughout this specification in the context of a protein or peptide is intended to refer to a variant of a protein or peptide having an amino acid sequence that differs from the original sequence in one or more mutations / substitutions, such as, for example, one or more substituted, inserted and / or deleted amino acids. Suitably, these fragments and / or variants have the same or equivalent specific antigenic properties (immunogenic variants, antigenic variants). Insertions and substitutions are possible, especially at sequence positions that do not cause alterations in the three-dimensional structure or affect the binding region. Conformational changes due to insertions or deletions can be easily determined, for example, using CD spectra (circular dichroism spectra). A "variant" of a protein or peptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Alternatively, a "variant" of a protein or polypeptide may have 1-20, such as 1-10, 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 such protein or peptide. By mutation, we mean or include substitutions, insertions or deletions. In one embodiment, a variant of a protein includes a functional variant of a protein, which in the context of the present disclosure means that the variant exerts essentially the same or at least 40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity of the protein from which it is derived.

[0051] <Detailed Description of the Invention> When referring to "SEQ ID NO" of other patent applications or patents, the sequence, e.g., amino acid sequence or nucleic acid sequence, is expressly incorporated herein by reference. For "SEQ ID NO" provided herein, the information provided under "feature key", i.e., "source" (for nucleic acid or protein) or "misc_feature" (for nucleic acid) or "REGION" (for protein) (in sequence listing according to WIPO ST.26 standard) is also expressly included herein in its entirety. When reference is made to "SEQ ID NO" in the context of an RNA sequence, the skilled artisan will be able to understand and derive the RNA sequence from the referenced SEQ ID NO, even when a DNA sequence is provided. When reference is made to "SEQ ID NO" in the context of a DNA sequence, the skilled artisan will be able to understand and derive the respective DNA sequence from the referenced SEQ ID NO, even when an RNA sequence is provided.

[0052] The present inventors have overcome the challenge of producing E. coli recombinant polypeptides by administering an RNA vaccine that is or encodes an antigenic polypeptide derived from E. coli FimH. The present inventors have further overcome the challenge of raising a rapid and robust immune response against E. coli FimH.

[0053] (1: RNA encoding an antigenic polypeptide of E. coli) In a first aspect, a coding RNA is provided that includes 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 fimbria D-mannose specific adhesin (FimH).

[0054] It should be noted that certain 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 (compositions of the disclosure), the third aspect (vaccines of the disclosure), the fourth aspect (kits of the disclosure or kits of parts of the disclosure), or further aspects, including medical uses and methods of treatment.

[0055] The term "coding RNA" as used herein will be recognized and understood by those of skill in the art and is intended to refer to RNA that includes coding sequences ("cds") that include, for example, several nucleotide triplets, which can be translated (e.g., upon administration to a cell or organism) into a peptide or protein.

[0056] The E. coli FimH of the present disclosure can be selected from or derived from any one of any strain of Escherichia coli, such as, for example, any one 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 NA139, E. coli NA114, E. coli IHE3034, E. coli 789, E. coli F11, and E. coli UTI89.

[0057] In one embodiment, the E. coli FimH of the present disclosure comprises, consists of, or is derived from the amino acid sequence of SEQ ID NO: 177-186, 247-256. In one embodiment, the E. coli FimH comprises an amino acid sequence identical to, or 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 SEQ ID NO: 177-186, 247-256. In one embodiment, the E. coli FimH is an immunogenic fragment or immunogenic variant of SEQ ID NO: 177-186, 247-256. In one embodiment, the E. coli FimH comprises an amino acid sequence identical to 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 identical to 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.

[0058] In some embodiments, glycosylation sites in the encoded amino acid sequence are mutated / substituted. This means that the encoded amino acids that can be glycosylated after the coding RNA is translated, for example, when administered in vivo, are replaced with different amino acids. Thus, at the nucleic acid level, codons that code for amino acids that are known or predicted to be N-glycosylated or O-glycosylated are replaced with amino acids that are not or are not easily glycosylated, such as serine (S, Ser), aspartic acid (D, Asp), alanine (A, Ala) or glutamine (Q, Gln).

[0059] N-glycosylation or O-glycosylation can be determined using any suitable means known to those skilled in the art, for example using the NetNGlyc 1.0 and NetOGlyc 4.0 servers (accessible 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.

[0060] In one embodiment, the antigenic polypeptide does not contain (or is modified to not contain) a glycosylation site at one or more positions selected from the group consisting of positions 28, 91, 228, 249, and 256 relative to SEQ ID NO: 177, or positions of SEQ ID NOs: 178-186, 247-256 corresponding to those positions in SEQ ID NO: 177. In one embodiment, the polypeptide contains one or more of the following amino acid substitutions: N28S, N91D, N249D, N256D relative to SEQ ID NO: 177, or contains, for example, one, two, three or four of those amino acid substitutions at positions of SEQ ID NOs: 178-186, 247-256 corresponding to those positions in SEQ ID NO: 177.

[0061] 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 that context, the antigenic polypeptide comprises an amino acid selected from the group consisting of 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 positions 247-256 of SEQ ID NO: 178-186, corresponding to this position of SEQ ID NO: 177. In one embodiment, the polypeptide comprises an F165V substitution of SEQ ID NO: 177, or at positions 247-256 of SEQ ID NO: 178-186, corresponding to this position of SEQ ID NO: 177. This mutation is reported in WO2021144369, the contents of which are incorporated herein by reference, and is intended to lock the FimH lectin domain into a low mannose-binding affinity conformation.

[0062] In some embodiments, the encoded FimH comprises one or more amino acid substitutions selected from the group consisting of N28S, V48C, L55C, N91S, N249Q, N256D, F165V of SEQ ID NO: 177, or positions 247-256 of SEQ ID NO: 178-186 corresponding to these positions of SEQ ID NO: 177; for example, the encoded FimH comprises amino acid substitutions of N28S, N91S, N249Q; N28S, N91S, N249Q, N256D; N28S, V48C, L55C, N91S, N249Q; or F165V of SEQ ID NO: 177, or positions 247-256 of SEQ ID NO: 178-186 corresponding to these positions of SEQ ID NO: 177.

[0063] In some embodiments, the encoded FimH comprises one or more amino acid substitutions at a position selected from the group consisting of 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 in SEQ ID NO:247, or the positions in SEQ ID NOs:177-186, 248-256 corresponding to these positions in SEQ ID NO:247.

[0064] In some embodiments, the encoded FimH is 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;T86C;L107C;Y108C;L109C;V112C in SEQ ID NO:247. ; S113C; A115V; G116C; V118C; A119C; A119N; A119S; A119T; A119D; A119E; A119K; A119R; A127C; L129C; Q133K; F144C; V154C; V156C; P157C; T158C; V163I; and V185I, or the positions in SEQ ID NOs: 177-186, 248-256 corresponding to these positions in SEQ ID NO: 247, or any combination thereof.

[0065] In some embodiments, the encoded FimH comprises the amino acids: 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 L109C; N33C and L109C; C and P157C;V35C and L107C;V35C and L109C;S62C and T86C;S62C and L129C;Y64C and L68C;Y64C and A127C;L68C and F71C;V112C and T158C;S113C and G116C;S113C and T158C;V118C and V156C;A119C and V155C;L34N and V2 7A;L34S and V27A;L34T and V27A;L34D and V27A;L34E and V27A;L34K and V27A;L34R and V27A;A119N and V27A;A119S and V27A;A119T and V27A;A119D and V27A;A119E and V27A;A119K and V27A;A119R and V27A;G15A and V27 G15A, G16A and V27A; G65A and V27A; V27A and Q133K; and G15A, G16A, V27A and Q113K; or positions in SEQ ID NOs: 177-186, 248-256 which correspond to these positions in SEQ ID NO: 247.

[0066] 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 of SEQ ID NO: 177-186, 248-256 which correspond to these positions of SEQ ID NO: 247.

[0067] <Specific antigen design> According to various embodiments, the coding sequence of the RNA encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, and one or more additional peptide or protein elements, in some embodiments, the one or more additional peptide or protein elements are heterologous.

[0068] Suitably, the further peptide or protein element may stabilize the FimH subunits (e.g., via a donor chain peptide) and / or shield their interaction surface. In addition, the further peptide or protein element may facilitate secretion of the encoded antigenic peptide or protein of the present disclosure (e.g., via a secretory signal sequence). In addition, the further peptide or protein element may facilitate anchoring of the encoded antigenic peptide or protein of the present disclosure to a cell membrane (e.g., via a transmembrane element) or facilitate formation of an antigen complex (e.g., via a multimerization domain or an antigen clustering domain).

[0069] Suitably, the coding sequence further encodes one or more peptide or protein elements selected from a donor chain peptide, a signal peptide, a helper epitope, an antigen clustering domain, or a transmembrane domain, hi some embodiments, the coding sequence encodes one or more additional peptide or protein elements and a peptide linker.

[0070] <Donor chain peptide> In some embodiments, the coding RNA encodes an antigenic protein selected from or derived from Escherichia coli FimH, and further encodes a donor strand peptide.

[0071] In some embodiments, the coding sequence encodes, from N-terminus to C-terminus, the following elements: an antigenic polypeptide selected from or derived from Escherichia coli FimH; and a donor chain peptide.

[0072] In one embodiment, the donor strand peptide comprises or consists of the 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, such as 1, 2, 3 or 4, single amino acid mutations compared to SEQ ID NO: 338 or SEQ ID NO: 339.

[0073] In one embodiment, the donor chain peptide comprises or consists of SEQ ID NO: 338. It may be particularly preferred in the context of the present disclosure that the donor chain peptide comprises or consists of SEQ ID NO: 338 such that the polypeptide of the disclosure is in a low mannose binding affinity conformation.

[0074] <Peptide linker> In protein constructs composed of multiple elements, the protein elements are often separated by peptide linkers, which may be advantageous as they allow for proper folding and thereby proper function of the individual elements.

[0075] When used in the context of the present disclosure, such linkers are particularly useful when encoded by a nucleic acid that codes for at least two protein elements, such as an antigenic polypeptide and at least one additional peptide or protein element. In that case, the linker is typically located on the polypeptide chain between the polypeptide of interest and the multiple additional protein elements. When the coding sequence codes for multiple additional peptide or protein elements, the linker can be suitably placed between each additional peptide or protein element. At the nucleic acid level, the coding sequence of such linkers is typically placed in the 5' or 3' reading frame of the coding sequence of the polypeptide or protein of interest, or between the coding regions of the individual peptide or protein elements.

[0076] 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 (e.g., glycine) or polar (e.g., serine or threonine) amino acids. As Chen et al. (Adv Drug Deliv Reb. 2013; 65(10): 1357-1369) state, the small size of these amino acids allows flexibility and fluidity in the binding functional domain. 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, thus reducing the interactions between the linker and the protein moiety. Inflexible linkers generally maintain the distance between protein domains and are based on helical structures or have proline-rich sequences.

[0077] A typical sequence of a flexible linker is composed of repeats of the amino acids glycine (G, Gly) and serine (S, Ser). For example, the linker can have the following sequences: GS, GSG, SGG, GGS, SGS, GSS, SSG. In some embodiments, the same sequence is repeated multiple times (e.g., 2, 3, 4, 5 or 6 times) to create a longer linker. In other embodiments, a single amino acid residue such as S or G can be used as a linker.

[0078] At the nucleic acid level, particularly the RNA level, any nucleotide sequence portion encoding any of the linkers used in the present disclosure can be employed. Due to the degenerate genetic code, for most of the polypeptides of SEQ ID NOs: 352-358, multiple specific nucleic acid sequences are considered to encode each of the polypeptides listed. While such nucleic acids may generally be used in the context of the present disclosure, it is preferred that the nucleic acid sequence encoding the polypeptide sequence is selected such that the sequence is codon-optimized according to the general guidance provided herein.

[0079] In some embodiments, the coding sequence encodes an antigenic protein selected from or derived from Escherichia coli FimH, a donor chain peptide, and a peptide linker.

[0080] In one embodiment, the coding sequence encodes, from N-terminus to C-terminus, the following elements: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker; and a donor chain peptide.

[0081] Where the coding sequence encodes, from N-terminal to C-terminal, the following elements: an antigenic polypeptide selected from or derived from Escherichia coli FimH; a peptide linker; and a donor chain peptide, it is particularly preferred that the peptide linker comprises or consists of: (i) PGDGN [SEQ ID NO: 352], or a variant or fusion thereof; or (ii) GGGGSGG [SEQ ID NO: 353], or a variant or fusion thereof; or (iii) GGGGSGGGGSGGGS [SEQ ID NO: 354], or a variant or fusion thereof; or (iv) SGG [SEQ ID NO: 355], or a variant or fusion thereof; or (v) SGM [SEQ ID NO: 356], or a variant or fusion thereof; or (vi) GGSGGSGGSGGSGGG [SEQ ID NO: 357], or a variant or fusion thereof, or (vii) GGSGGSGGSGGS [SEQ ID NO: 358], or a variant or fusion thereof.

[0082] In one embodiment, the peptide linker is a variant of any one of SEQ ID NOs: 352-358, optionally wherein the variant has 1 to 5, for example 1, 2, 3 or 4, single amino acid mutations compared to SEQ ID NOs: 352-358.

[0083] In one embodiment, the peptide linker comprises or consists of SEQ ID NO: 352. It may be particularly advantageous in the context of the present disclosure that the peptide linker located between the antigenic polypeptide and the donor chain peptide comprises or consists of SEQ ID NO: 352, thereby locking the polypeptide of the present disclosure into a low mannose binding affinity conformation.

[0084] <Conformation of antigenic polypeptide> In one embodiment, the antigenic polypeptide selected from or derived from E. coli FimH is in a low mannose binding affinity conformation or tension (T) state, e.g., K dmannose binding affinity of about 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1 mM, or no detectable mannose binding affinity. In one embodiment, the mannose binding affinity is about K d ≈300 μM or higher (i.e., has no detectable mannose binding affinity). The high mannose binding affinity conformation or relaxed (R) state of FimH is, for example, d This is known in the art to correspond to a mannose binding affinity of <1.2 μM.

[0085] Mannose binding can be determined using any suitable means known in the art, for example, surface plasmon resonance can be used to verify the binding, binding specificity and binding constants of FimH constructs with Man-BSA and Glc-BSA (negative control), see e.g. Rabbani S, et al. J Biol Chem. 2018 Feb 2;293(5):1835-1849.

[0086] The conformation of FimH can also be evaluated by measuring the binding of conformational antibodies using any suitable means known in the art, such as surface plasmon resonance. Exemplary antibodies can recognize epitopes that overlap differently with the mannose-binding pocket of FimH, such as antibodies that bind to epitopes that overlap with the mannose-binding pocket, such as epitopes that are restricted to only one loop of the mannose-binding pocket. Exemplary antibodies are those disclosed in WO2016183501, or those disclosed 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.

[0087] <Signal peptide> In some embodiments, a coding sequence of the disclosure encodes at least one antigenic polypeptide selected from or derived from Escherichia coli FimH, and further encodes a signal peptide.

[0088] 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).

[0089] In some embodiments, the signal peptide is selected from or derived from IgE, IgK, FimH, FimC, TPA, HSA, or HLA-A2, and the amino acid sequence of the aforementioned signal peptide is identical to or 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 amino acid sequences SEQ ID NOs: 394-400, or a fragment or variant of any of these.

[0090] In some embodiments, the signal peptide is heterologous. In some embodiments, the signal peptide is selected from or derived from IgE or IgK, and the amino acid sequence of the aforementioned signal peptide 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 any one of the amino acid sequences SEQ ID NOs: 394, 395, or a fragment or variant of any of these.

[0091] In embodiments in which the coding sequence of the present disclosure further encodes a signal peptide, it is particularly preferred to generate a fusion protein comprising an N-terminal signal peptide and a C-terminal peptide or a protein selected from or derived from Escherichia coli FimH, optionally including a peptide linker and a donor chain peptide, wherein the aforementioned C-terminal peptide or a protein selected from or derived from Escherichia coli FimH lacks an endogenous N-terminal secretory signal peptide, such as, for example, SEQ ID NOs: 247-256 and 1277.

[0092] Constructs including an N-terminal signal peptide may ideally improve the secretion of Escherichia coli FimH (encoded by the coding RNA of the first aspect). Improved secretion of Escherichia coli FimH following administration of the coding RNA of the first aspect may therefore be advantageous for the induction of a humoral immune response against the encoded Escherichia coli FimH antigenic protein.

[0093] Further suitable signal peptides are SEQ ID NOs: 1-1115 and SEQ ID NO: 1728 of published PCT patent application WO2017081082, which is incorporated herein by reference, or fragments or variants of these sequences, wherein the aforementioned secretory signal peptides are N-terminally fused to an antigenic polypeptide selected from or derived from Escherichia coli FimH or an immunogenic fragment or variant thereof lacking an endogenous secretory signal sequence.

[0094] Suitable examples of constructs containing an N-terminal signal sequence are SEQ ID NOs: 498 to 520. The corresponding nucleic acid sequences for each of the above constructs can be found in Table 1.

[0095] <Antigen clustering domain or multimerization domain> In various embodiments, the coding sequence of the disclosure encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH and further encodes an antigen clustering domain or a multimerization domain.

[0096] Suitably, the antigen clustering domain (multimerization domain) is selected from or derived from ferritin or lumazine synthase (LS, LumSynth).

[0097] In embodiments in which the coding sequence of the present disclosure further encodes an antigen clustering domain, it is particularly preferred to generate a fusion protein comprising an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally comprising a donor chain peptide and a (first) peptide linker, further comprising an antigen clustering domain, and optionally comprising a (second) peptide linker. Constructs comprising an antigen clustering domain may enhance antigen clustering and thus promote immune responses, for example, by multiple simultaneous binding events between clustered antigens and host cell receptors (for further details, see Lopez-Sagaseta, Jacinto, et al. “Self-assembling protein nanoparticles in the design of vaccines”. Computational and structural biotechnology journal 14 (2016):58-68). Additionally, such constructs may further comprise an N-terminal signal sequence (as defined above).

[0098] Lumazine synthase (LS, LumSynth) is an enzyme with particle-forming properties, present in a wide variety of organisms, and involved in the biosynthesis of riboflavin. Jardine et al. reported an attempt to enhance the immunoreactivity of recombinant gp120 against HIV infection by encapsulating lumazine synthase (LS, LumSynth) for the optimization of vaccine candidates (Jardine, Joseph, et al. "Rational HIV immunogen design to target specific germline B cell receptors". Science 340.6133 (2013):711-716). Constructs containing lumazine synthase allow the formation of multimeric nanoparticles, especially 60mer nanoparticles, displaying antigenic polypeptides, thus optimizing B cell activation.

[0099] In some embodiments, lumazine synthase may be used to promote antigen clustering and thus enhance the immune response of a coding sequence encoding the E. coli FimH antigen.

[0100] In some embodiments, the antigen clustering domain (multimerization domain) is selected from or derived from lumazine synthase (LS, LumSynth), and the amino acid sequence of the aforementioned antigen clustering domain is identical to, or 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 the amino acid sequences (SEQ ID NOs: 443, 444), fragments or variants of any of these.

[0101] Ferritin is a protein whose main function is intracellular iron storage. Nearly all living organisms produce ferritin, which is made up of 24 subunits that self-assemble into a quaternary structure with octahedral symmetry. Its self-assembly into nanoparticles makes it suitable for antigen delivery and exposure.

[0102] In some embodiments, ferritin may be used to promote antigen clustering and thus enhance the immune response of RNA encoding the E. coli FimH antigen.

[0103] In some embodiments, the antigen clustering domain (multimerization domain) is or is derived from ferritin, wherein the amino acid sequence of the antigen clustering domain described above is identical to, or 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 the amino acid sequences (SEQ ID NOs: 457-459), any fragment or variant thereof.

[0104] In some embodiments, the coding sequence encodes from N-terminal to C-terminal the following elements: an optional signal peptide, an antigen clustering domain selected from or derived from lumazine synthase or ferritin as defined herein, a (second) peptide linker, and an antigenic polypeptide selected from or derived from E. coli FimH, optionally further comprising a (first) peptide linker, and a donor chain peptide as defined herein.

[0105] In some alternative embodiments, the coding sequence encodes from N-terminal to C-terminal the following elements: an optional signal peptide, an antigenic polypeptide selected from or derived from E. coli FimH, optionally comprising a (first) peptide linker and a donor chain peptide, a (second) peptide linker, and an antigen clustering domain, preferably selected from or derived from lumazine synthase or ferritin as defined herein.

[0106] In one embodiment, the (first) peptide linker comprises or consists of any one of SEQ ID NOs: 352-354 or a variant thereof, optionally, the variant has 1-5, e.g. 1, 2, 3 or 4 single amino acid mutations compared to SEQ ID NOs: 352-354. 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 NOs: 355-358 or a variant thereof, optionally, the variant has 1-5, e.g. 1, 2, 3 or 4 single amino acid mutations compared to SEQ ID NOs: 355-358. In one embodiment, the (second) peptide linker comprises or consists of SEQ ID NO: 355.

[0107] Suitable examples of constructs comprising a heterologous antigen clustering domain are SEQ ID NOs: 507 to 510, 512 to 520. The corresponding nucleic acid sequence of each of the above constructs can be found in Table 1.

[0108] In various embodiments, the coding sequence of the present disclosure encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH and further encodes a transmembrane domain. In one embodiment, the transmembrane domain is heterologous. The heterologous transmembrane domain may facilitate membrane anchoring of the encoded E. coli FimH antigenic polypeptide, thereby enhancing the immune response, particularly the cellular immune response.

[0109] Suitably the transmembrane domain is or is derived from an influenza HA transmembrane domain, for example influenza A HA H1N1, more for example H1N1 / A / Netherlands / 602 / 2009, HA, aa521-566, GenBank Acc. No.: ACQ45338.1, (SEQ ID NO: 478).

[0110] Further preferred transmembrane domains include: Human immunodeficiency virus 1, Env, aa19-35, BAF32550.1, AB253679.1; Human immunodeficiency virus 1, Env, aa515-536, BAF32550.1, AB253679.1; Human immunodeficiency virus 1, Env, aa680-702, BAF32550.1, AB253679.1; Equine infectious anemia virus 1, Env, aa450-472, AAC03762.1, AF016316.1; Equine infectious anemia virus, Env, aa614-636, AAC03762.1, AF016316.1; Equine infectious anemia virus, Env, aa798-819, AAC03762.1, AF016316.1; murine leukemia virus, Env, aa601-623, AAA46526.1, M93052.1; mouse mammary tumor virus, Env, aa457-479, BAA03768.1, D16249.1; mouse mammary tumor virus, Env, aa624-646, NP_056883.1, NC_001503.1; vesicular stomatitis virus, G, aa477-499, CAA24525.1, V01214.1; rabies virus, G, aa460-479, AEV43288.1, derived from JN234423.1 (Env: envelope glycoprotein; G: glycoprotein).

[0111] In an embodiment in which the coding sequence of the present disclosure further codes for a heterologous transmembrane domain, it is particularly preferred to generate a fusion protein comprising an N-terminal peptide or protein comprising an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally comprising a donor chain peptide and a (first) peptide linker (as defined above) between the antigenic polypeptide and the donor chain peptide; and a C-terminal heterologous transmembrane domain, and optionally a (second) peptide linker (as defined above) between the N-terminal peptide and the C-terminal peptide. A construct comprising a heterologous transmembrane domain may promote membrane anchoring of an antigen and thus promote an immune response, particularly a cellular immune response, of an RNA encoding an antigenic polypeptide. In addition, such a construct may further comprise an N-terminal secretion signal sequence (as defined above). Alternatively, the transmembrane domain may be present at the N-terminus.

[0112] Further transmembrane elements / domains may be selected from the list of amino acid sequences according to SEQ ID NOs: 1228 to 1343 of patent application WO2017081082, or fragments or variants of these sequences, which are incorporated herein by reference.

[0113] In some embodiments, the coding sequence encodes, from N-terminal to C-terminal, the following elements: a secretory signal peptide, an antigenic polypeptide selected from or derived from Escherichia coli FimH, optionally including a (first) peptide linker and a donor chain peptide, an antigenic peptide, a (second) peptide linker, and a heterologous transmembrane domain.

[0114] A suitable example of a construct containing a heterologous transmembrane element is SEQ ID NO: 511. The corresponding nucleic acid sequence of the construct can be found in Table 1.

[0115] In various embodiments of the invention, the coding sequence encodes, for example, the following elements from N-terminus to C-terminus: a) a signal peptide, an antigenic polypeptide as defined herein; b) a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, a donor chain peptide; c) an antigen clustering domain, a peptide linker, an antigenic polypeptide as defined herein, a peptide linker, a donor chain peptide; d) signal peptides, antigen clustering domains, peptide linkers, antigenic polypeptides as defined herein, peptide linkers, donor chain peptides; e) a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, a donor chain peptide, a peptide linker, an antigen clustering domain; or f) signal peptide, antigenic polypeptide as defined herein, peptide linker, donor chain peptide, peptide linker, transmembrane domain.

[0116] In some embodiments, the coding sequence encodes, for example, the following elements from N-terminal to C-terminal: a signal peptide, an antigenic polypeptide as defined herein, a peptide linker, and a donor chain peptide; optionally, the signal peptide is selected from SEQ ID NOs: 394-400, optionally the signal peptide is SEQ ID NO: 395; the antigenic polypeptide is selected from SEQ ID NOs: 247-256, optionally the antigenic polypeptide is SEQ ID NO: 247; the peptide linker is selected from SEQ ID NOs: 352-354, optionally the peptide linker is SEQ ID NO: 352; the donor chain peptide is selected from SEQ ID NOs: 338, 339, optionally the donor chain peptide is SEQ ID NO: 338.

[0117] In some embodiments, the coding sequence encodes, e.g., from N-terminal to C-terminal, the following elements: a signal peptide, an antigenic polypeptide as defined herein, a (first) peptide linker, a donor chain peptide, a (second) peptide linker; and an antigen clustering domain; optionally, the signal peptide is selected from SEQ ID NOs: 394-400, optionally the signal peptide is SEQ ID NO: 394; the antigenic polypeptide is selected from SEQ ID NOs: 247-256, optionally the antigenic polypeptide is SEQ ID NO: 247; (the first ) the peptide linker is selected from SEQ ID NOs: 352-354, optionally wherein the (first) peptide linker is SEQ ID NO: 352; the donor chain peptide is selected from SEQ ID NOs: 338, 339, optionally wherein the donor chain peptide is SEQ ID NO: 338; the (second) peptide linker is selected from SEQ ID NOs: 355-358, optionally wherein the (second) peptide linker is SEQ ID NO: 355; the antigen clustering domain is selected from SEQ ID NOs: 443, 444, 457-459, optionally wherein the peptide linker is SEQ ID NO: 444 or 459.

[0118] The preferred sequences as defined above are shown in Table 1, where each row corresponds to a preferred sequence. Column A of Table 1 is a brief description of the preferred antigen constructs. Column B of Table 1 shows the protein (amino acid) SEQ ID NO of the respective antigen construct. Column C of Table 1 shows the SEQ ID NO of the corresponding wild type 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 matched nucleic acid coding sequence (opt3, human). Column F of Table 1 provides the SEQ ID NO of further codon optimized coding sequences (opt4, main or opt5, gc mod).

[0119] In particular, this specification expressly includes the information provided under "feature key" in the ST.26 sequence listing of this application, i.e., "source" (for nucleic acids or proteins) or "misc_feature" (for nucleic acids) or "REGION" (for proteins). RNA constructs comprising the coding sequences of Table 1, e.g., mRNA sequences comprising the coding sequences of Table 1, are provided in Table 3.

[0120] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0121] Preferred coding sequences: According to some embodiments, the coding RNA of the present disclosure comprises a coding sequence that encodes 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 that encodes at least one antigenic protein as defined herein, or fragments and variants thereof, can be understood as a suitable coding sequence and thus can be included in the nucleic acid of the present disclosure.

[0122] In some embodiments, the coding 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 encoding a fragment of any one of SEQ ID NOs: 177-186, 247-256, 498-520, 1277 or a variant thereof. At the RNA level, it must be understood that any sequence encoding 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 any one of SEQ ID NOs: 177-186, 247-256, 498-520, 1277, or a fragment or variant thereof, can be selected and accordingly understood as a suitable coding sequence of the present disclosure.

[0123] In some embodiments, the coding sequence encodes an amino acid sequence that is identical to, or 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 SEQ ID NOs: 498-520, 1277, or an immunogenic fragment or immunogenic variant thereof.

[0124] In some embodiments, the coding sequence encodes an amino acid sequence that is identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 504, 508, and 509, or an immunogenic fragment or immunogenic variant thereof.

[0125] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or 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 SEQ ID NOs: 187-246, 257-316, 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or variant of any of these sequences.

[0126] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence according to any one of SEQ ID NOs: 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or variant thereof:

[0127] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence according to any one of SEQ ID NOs: 529, 533, 534, 554, 558, 559, 579, 583, 584, 604, 608, 609, 629, 633, 634, 654, 658, 659, or a fragment or variant of any of these sequences.

[0128] In some embodiments, the coding RNA of the first aspect is an artificial RNA.

[0129] The term "artificial RNA" as used herein is intended to refer to RNA that does not exist in nature. In other words, artificial RNA can be understood as a non-natural RNA molecule. Such an RNA molecule can be non-natural due to its individual sequence (e.g., G / C content modified coding sequence, UTR) and / or due to other modifications, such as structural modifications of nucleotides. Typically, artificial RNA is designed and / or generated by genetic engineering to correspond to a desired artificial sequence of nucleotides. In this context, artificial RNA is a sequence that does not exist in nature, i.e., a sequence that differs from a wild-type or reference sequence / naturally occurring sequence by at least one nucleotide (e.g., via a codon modification as further defined below). The term "artificial RNA" is not limited to mean a "single molecule", but is understood to include a collection or a plurality of essentially identical RNA molecules.

[0130] In some embodiments, the coding RNA is a modified and / or stabilized RNA.

[0131] According to some embodiments, the coding RNA may be provided as a "stabilized RNA", i.e. an RNA that exhibits improved resistance to degradation in vivo, and / or an RNA that exhibits improved stability in vivo, and / or an RNA that exhibits improved translatability in vivo. The term "stabilized RNA" refers to an RNA that has been modified to be more stable against degradation or degradation, e.g., by environmental agents or enzymatic digests, e.g., exonuclease or endonuclease degradation, as compared to an RNA without such modification. In one embodiment, the stabilized RNA in the context of the present disclosure is stabilized in a cell, e.g., a prokaryotic or eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. The stabilizing effect may also be exerted outside the cell, e.g., in a buffer solution, for storage of a composition comprising the stabilized RNA.

[0132] The coding RNA of the present disclosure may be provided as a "stabilizing RNA."

[0133] Below we describe suitable modifications / adaptations that can "stabilize" RNA.

[0134] In some embodiments, the coding RNA comprises at least one codon-modified coding sequence.

[0135] In some embodiments, at least one coding sequence of the coding RNA is a codon-modified coding sequence. Advantageously, the amino acid sequence encoded by at least one codon-modified coding sequence is unmodified compared to the amino acid sequence encoded by the corresponding wild-type or reference coding sequence.

[0136] The term "codon-modified coding sequence" refers to a coding sequence that differs in at least one codon (a triplet of nucleotides that codes for one amino acid) compared to the corresponding wild-type or reference coding sequence. Advantageously, a codon-modified coding sequence in the context of the present disclosure may exhibit improved resistance to degradation in vivo, and / or improved stability in vivo, and / or improved translatability in vivo. Codon modification in its broadest sense takes advantage of the degeneracy of the genetic code, where multiple codons may code for the same amino acid, and may be used interchangeably to optimize / modify coding sequences for in vivo applications.

[0137] In some embodiments, the coding sequence of the coding RNA is a codon-modified coding sequence, and the codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage compatible coding sequence, a G / C content modified coding sequence, and a G / C optimized coding sequence, or any combination thereof.

[0138] In some embodiments, the coding sequence of the coding RNA has a G / C content of at least about 50%, 55%, or 60%. In certain 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%.

[0139] When transfected into a mammalian host cell, the coding RNA comprising the codon-modified coding sequence has stability for between 12-18 hours, or for more than 18 hours, e.g., 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or for more than 72 hours, and is expressible by the mammalian host cell (e.g., muscle cell). RNA detection methods are known in the art.

[0140] 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, or e.g., at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 100%, or at least 200% greater than, the amount of protein obtained by a naturally occurring or wild-type or reference coding sequence transfected into a mammalian host cell.

[0141] In some embodiments, the coding RNA may be modified, wherein the C content of at least one coding sequence may be increased, e.g., maximized, compared to the C content of the corresponding wild-type or reference coding sequence (referred to herein as "C-maximized coding sequence"). The generation of C-maximized nucleic acid sequences may be suitably carried out using the modified method according to WO2015062738. In this context, the disclosure of WO2015062738 is incorporated herein by reference.

[0142] In some embodiments, the coding RNA may be modified, wherein the G / C content of at least one coding sequence may be optimized compared to the G / C content of the corresponding wild-type or reference coding sequence (herein referred to as "G / C-optimized coding sequence"). "Optimized" in this context refers, for example, to a coding sequence in which the G / C content is essentially increased to the highest possible G / C content. The generation of nucleic acid sequences with optimized G / C content 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 indicated by the abbreviation "opt1" or "gc".

[0143] In some embodiments, the coding RNA may be modified, where the codons in at least one coding sequence may be adapted to human codon usage (referred to herein as "human codon usage adapted coding sequence"). Codons encoding the same amino acid occur at different frequencies in humans. Thus, the coding sequence of the RNA is modified so that the frequency of codons encoding the same amino acid corresponds to the natural occurrence frequency of that codon according to human codon usage. For example, for the amino acid Ala, the wild type or reference coding sequence is adapted, for example, to use the codon "GCC" at a frequency of 0.40, the codon "GCT" at a frequency of 0.28, the codon "GCA" at a frequency of 0.22, and the codon "GCG" at a frequency of 0.10 (see, for example, Table 2 of published PCT patent application WO2021156267, which is incorporated herein by reference). Thus, such a procedure (exemplified for alanine) is applied for each amino acid encoded by the coding sequence of the RNA to obtain a human codon usage-compatible coding sequence, which is designated by the abbreviation "opt3" or "human".

[0144] In some embodiments, the coding RNA may be modified, where the G / C content of at least one coding sequence may be modified compared to the G / C content of the corresponding wild-type or reference coding sequence (herein referred to as "G / C modified coding sequence"). In this context, the term "G / C optimized" or "G / C content modified" refers to a nucleic acid that contains modified, e.g., increased, number of guanosine and / or cytosine nucleotides compared to the corresponding wild-type or reference coding sequence. Such an increase in number may occur by replacing codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. Advantageously, RNA sequences with increased G / C content are more stable or show better expression than sequences with increased A / U. For example, the G / C content of the coding sequence of the RNA is increased by at least 10%, 20%, 30%, e.g., at least 40%, compared to the G / C content of the coding sequence of the corresponding wild-type or reference nucleic acid sequence (herein referred to as "opt5" or "gc mod").

[0145] In some embodiments, the coding RNA may be modified, in which the codon adaptation index (CAI) may be increased or, for example, maximized in at least one coding sequence (referred to herein as a "CAI-maximized coding sequence"). It is preferred that all codons of the wild-type or reference nucleic acid sequence that are relatively rare, for example, in humans, are replaced with respective codons that are frequent, for example, in humans, and the frequent codons code for the same amino acid as the relatively rare codon. Preferably, the most frequent codon is used for each amino acid of the encoded protein (see Table 2 of published PCT patent application WO2021156267). Preferably, the RNA comprises at least one coding sequence, and the codon adaptation index (CAI) of the 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 the at least one coding sequence is 1 (CAI=1). For example, for the amino acid Ala, the wild-type or reference coding sequence can be adapted so that the most frequent human codon "GCC" is always used for the aforementioned amino acid. Thus, such a procedure (as exemplified for alanine) can be applied for each amino acid encoded by the coding sequence of the nucleic acid to obtain a CAI-maximized coding sequence (referred to herein as "opt4" or "main").

[0146] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or 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 SEQ ID NOs: 197-246, 267-316, 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or variant thereof.

[0147] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or variant thereof.

[0148] In some embodiments, at least one coding sequence of an RNA of the disclosure is a G / C optimized coding sequence.

[0149] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or 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 SEQ ID NOs: 197-206, 207-216, 237-246, 267-276, 277-286, 307-316, 523-545, 548-570, 648-670, or a fragment or variant thereof.

[0150] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 523-545, 548-570, 648-670, or a fragment or variant thereof.

[0151] In some embodiments, the coding RNA of the first aspect comprises a coding sequence comprising at least one of the nucleic acid sequences identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence according to any one of SEQ ID NOs: 529, 533, 534, 554, 558, 559, 654, 658, 659, or a fragment or variant of any of these sequences.

[0152] In some embodiments, the coding sequence contains multiple stop codons to allow for sufficient termination of translation. In particular embodiments, the coding sequence contains two or three stop codons to allow for sufficient termination of translation. These multiple stop codons can optionally be placed in alternative reading frames.

[0153] UTR: The RNA of the first embodiment comprises at least one untranslated region (UTR).

[0154] The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those skilled in the art and is intended to refer to a portion of a nucleic acid molecule that is typically located, for example, 5' or 3' of a coding sequence. A UTR is not translated into a protein. A UTR may be part of an RNA. A UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements are, for example, ribosome binding sites, miRNA binding sites, promoter elements, etc.

[0155] In some embodiments, the coding RNA comprises a protein coding region ("coding sequence" or "cds"), and a 5'-UTR and / or a 3'-UTR. Of note, the UTR may carry regulatory sequence elements that determine the turnover, stability, and localization of the RNA. In addition, the UTR may carry sequence elements that promote translation. For medical applications, it is paramount for the therapeutic effect that the RNA is translated into at least one peptide or protein. Certain combinations of 3'-UTR and / or 5'-UTR may enhance the expression of the operably linked coding sequence that encodes a peptide or protein as defined herein. RNA molecules carrying the aforementioned combinations of UTRs advantageously allow for rapid and transient expression of antigenic peptides or proteins after administration to a subject, for example after intramuscular administration. Thus, the RNA of the present disclosure that includes certain combinations of 3'-UTR and / or 5'-UTR are particularly suitable for administration as a vaccine, and in particular for administration to the muscle, dermis, or epidermis of a subject.

[0156] Suitably, the coding RNA comprises at least one 5'-UTR and / or at least one 3'-UTR. The aforementioned 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.

[0157] In some embodiments, the coding RNA of the present disclosure comprises at least one 3'-UTR.

[0158] The term "3'-untranslated region" or "3'-UTR" will be recognized and understood by those of skill in the art and is intended to refer to, for example, a portion of an RNA molecule that is located 3' (i.e., downstream) of a coding sequence and is not translated into protein. The 3'-UTR may be a portion of a nucleic acid located between a coding sequence and an (optional) terminal poly(A) sequence. The 3'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements are, for example, ribosome binding sites, miRNA binding sites, etc.

[0159] Optionally, the coding RNA comprises at least one 3'-UTR, which may be derived from a gene associated with an RNA that has enhanced half-life (ie, provides a stable RNA).

[0160] In some embodiments, the 3'-UTR comprises one or more of a polyadenylation signal, a binding site for a protein that affects nucleic acid stability at a subcellular location, or one or more miRNAs or binding sites for miRNAs.

[0161] MicroRNAs (or miRNAs) are non-coding RNAs approximately 19-25 nucleotides long that bind to the 3'-UTR of RNA molecules and downregulate gene expression by reducing RNA stability or inhibiting translation. For example, microRNAs are known to regulate RNA and thus protein expression, for example 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), bone marrow 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). The RNA may include one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA, such as, for example, those taught in US20050261218 and US20050059005, which are incorporated herein by reference.

[0162] Thus, to tailor expression of the RNA to a desired cell type or tissue (e.g., muscle cells), the miRNA, or a binding site for the miRNA as defined above, can be removed from or introduced into the 3'-UTR.

[0163] In some embodiments, the coding RNA comprises at least one 3'-UTR, and at least one 3'-UTR comprises a nucleic acid sequence derived from or selected from the 3'-UTR of a gene selected from PSMB3, alpha-globin, ALB7, CASP1, COX6B1, FIG4, GNAS, NDUFA1 and RPS9, or a homolog, fragment or variant of any one of these genes.

[0164] In some embodiments, PSMB3, alpha-globin, ALB7, CASP1, COX6B1, FIG4, GNAS, NDUFA1 or RPS9 comprises or consists of a nucleic acid sequence identical to or 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 SEQ ID NOs: 67-90, 109-120, or a fragment or variant thereof.

[0165] In one embodiment, the coding RNA comprises a 3'-UTR derived from or selected from the PSMB3 gene.

[0166] In one embodiment, the 3'-UTR derived from or selected from PSMB3 comprises or consists of a nucleic acid sequence identical to, or 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 SEQ ID NOs: 67, 68, 109-120, or a fragment or variant thereof.

[0167] In other embodiments, the coding RNA comprises a 3'-UTR that comprises or consists of a nucleic acid sequence identical to 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: 91-108 or a fragment or variant thereof.

[0168] In another embodiment, the coding RNA comprises a 3'-UTR as described in WO2016107877, the disclosure of which regarding 3'-UTR sequences is incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016107877, or fragments or variants of these sequences. In another embodiment, the RNA comprises a 3'-UTR as described in WO2017036580, the disclosure of which regarding 3'-UTR sequences is incorporated herein by reference. Suitable 3'-UTRs are SEQ ID NOs: 152-204 of 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 regarding 3'-UTR sequences is incorporated herein by reference. Particularly suitable 3'-UTRs are nucleic acid sequences according to SEQ ID NOs: 20-36 of WO2016022914, or fragments or variants of these sequences.

[0169] In some embodiments, the coding RNA of the present disclosure comprises at least one 5'-UTR.

[0170] The term "5'-untranslated region" or "5'-UTR" will be recognized and understood by those skilled in the art and is intended to refer, for example, to a portion of an RNA located 5' (i.e., "upstream") of a coding sequence and not translated into a protein. A 5'-UTR may be a portion of a nucleic acid 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. A 5'-UTR may contain elements for controlling gene expression, also called regulatory elements. Such regulatory elements are, for example, ribosome binding sites, miRNA binding sites, etc. A 5'-UTR may be modified, for example, by enzymatically or co-transcriptionally adding a 5'-cap structure (e.g., in the case of mRNA, as defined below).

[0171] Optionally, the coding RNA comprises at least one 5'-UTR, which may be derived from a gene associated with an RNA that has enhanced half-life (ie, provides a stable RNA).

[0172] In some embodiments, the 5'-UTR contains one or more binding sites for proteins that affect RNA stability or RNA location within a cell, or one or more binding sites for miRNAs or miRNAs (as defined above).

[0173] Thus, to tailor expression of a nucleic acid to a desired cell type or tissue (e.g., muscle cells), miRNAs or binding sites for miRNAs as defined above can be removed from or introduced into the 5'-UTR.

[0174] In some embodiments, the coding RNA comprises at least one 5'-UTR, and at least one 5'-UTR is derived from or selected from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, including a nucleic acid sequence selected therefrom, or a homolog, fragment or variant of any one of these genes.

[0175] In some embodiments, HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2 comprise or consist of a nucleic acid sequence identical to, or 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 SEQ ID NOs: 1-32, 65, 66, or a fragment or variant of any of these.

[0176] In one embodiment, the coding RNA comprises a 5'-UTR derived from or selected from the HSD17B4 gene.

[0177] In one embodiment, the 5'-UTR derived from or selected from HSD17B4 comprises or consists of a nucleic acid sequence identical to or 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 SEQ ID NOs: 1, 2, 65, 66, or a fragment or variant thereof.

[0178] In other embodiments, the coding RNA comprises a 5'-UTR that comprises or consists of a nucleic acid sequence identical to 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: 33-64 or a fragment or variant thereof.

[0179] In another embodiment, the coding RNA comprises a 5'-UTR as described in WO2013143700, the disclosure of which regarding 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, 1395, 1421 and 1422 of WO2013143700, or fragments or variants of these sequences. In another embodiment, the coding RNA comprises a 5'-UTR as described in WO2016107877, the disclosure of which regarding 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 25-30 and 319-382 of WO2016107877, or fragments or variants of these sequences. In another embodiment, the RNA comprises a 5'-UTR as described in WO2017036580, the disclosure of which regarding 5'-UTR sequences is incorporated herein by reference. Particularly suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 of WO2017036580, or fragments or variants of these sequences. In another embodiment, the RNA comprises a 5'-UTR as described in WO2016022914, the disclosure of which regarding 5'-UTR sequences is incorporated herein by reference. Suitable 5'-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 of WO2016022914, the disclosure of which regarding 5'-UTR sequences is incorporated herein by reference, or fragments or variants of these sequences.

[0180] In some embodiments, the coding RNA of the present disclosure comprises a coding sequence as defined herein that encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH and a 3'-UTR and / or a 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 (UBQLN2 / RPS9), b-3 (UBQLN2 / RPS9), b-4 (UBQLN2 / RPS9), b-5 (UBQLN2 / RPS9), b-6 (UBQLN2 / RPS9), b-7 (UBQLN2 / RPS9), b-8 (UBQLN2 / RPS9), b-9 (UBQLN2 / RPS9), b-10 (UBQLN2 / RPS9), b-11 (UBQLN2 / RPS9), b-12 (UBQLN2 / RPS9), b-13 (UBQLN2 / RPS9), b-14 (UBQLN2 / RPS9), b-15 (UBQLN2 / RPS9), b-16 (UBQLN2 / RPS9), b-17 (UBQLN2 / RPS9), b-18 (UBQLN2 / RPS9), b-19 (UBQLN2 / RPS9), b-20 (UBQLN2 / RPS9), b-21 (UBQLN2 / RPS9), b-22 (UBQLN2 / RPS9), b-23 (UBQLN2 / RPS9), b-24 (UBQLN2 / RPS9), b-25 (UBQL -2(ASAH1 / RPS9), b-3(HSD17B4 / RPS9), b-4(HSD17B4 / CASP1), b-5(NOSIP / COX6B1), c-1(NDUFA4 / RPS9), c-2(NOSIP / NDUFA1), c-3(NDUFA 4 / COX6B1), c-4(NDUFA4 / NDUFA1), c-5(ATP5A1 / PSMB3), d-1(Rpl31 / PSMB3), d-2(ATP5A1 / CASP1), d-3(SLC7A3 / GNAS), d-4(HSD17B4 / NDUF A1), 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(alpha-globin genes).

[0181] In some embodiments, the coding RNA comprises a coding sequence as defined herein that encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH and HSD17B4 5'-UTR and PSMB3 3'-UTR (HSD17B4 / PSMB3(a-1)). This embodiment has been shown by the inventors to be particularly beneficial for inducing an immune response against Escherichia coli FimH.

[0182] In various embodiments, the coding RNA of the present disclosure is monocistronic, bicistronic, or multicistronic.

[0183] In some embodiments, the coding RNA of the present disclosure is monocistronic.

[0184] The term "monocistronic" will be recognized and understood by those of skill in the art and is intended to refer, for example, to an RNA that contains only one coding sequence. As used herein, the terms "bicistronic" or "multicistronic" are intended to refer, for example, to an RNA that contains two (bicistronic) or more (multicistronic) coding sequences.

[0185] In some embodiments, the A / U (A / T) content of the environment of the ribosome binding site of the RNA is increased compared to the A / U (A / T) content of the environment of the ribosome binding site of the respective wild-type or reference nucleic acid. This modification increases the efficiency of ribosome binding to the RNA, which is therefore beneficial for the efficient translation of the RNA into antigenic peptides or proteins.

[0186] Thus, in one embodiment, the coding RNA comprises a ribosome binding site, also referred to as a "Kozak sequence", that is identical or at least 80%, 85%, 90%, 95% identical to any one of SEQ ID NOs: 128-135, or a fragment or variant of any of these, or a fragment or variant of any of these.

[0187] Poly(N) sequences, histone stem loops: In some embodiments, the coding 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.

[0188] In some embodiments, the coding RNA of the present disclosure comprises at least one poly(A) sequence.

[0189] The term "poly(A) sequence", "poly(A) tail" or "3'-poly(A) tail" as used herein will be recognized and understood by those skilled in the art and is intended to mean a sequence of adenosine nucleotides located at the 3'-end of a linear RNA, typically up to about 1000 adenosine nucleotides. For example, the poly(A) sequences described above are essentially homopolymeric, e.g., a poly(A) sequence of 100 adenosine nucleotides has a length of essentially 100 nucleotides. In other embodiments, the poly(A) sequence is interrupted by at least one nucleotide different from adenosine nucleotides, e.g., a poly(A) sequence of 100 adenosine nucleotides may have a length of more than 100 nucleotides (including 100 adenosine nucleotides and, in addition, at least one nucleotide--or stretch of nucleotides--different from adenosine nucleotides as described above).

[0190] In some embodiments, at least one poly(A) sequence can comprise from about 40 to about 500 adenosine nucleotides, from about 40 to about 200 adenosine nucleotides, from about 40 to about 150 adenosine nucleotides, such as from about 60 to about 150 adenosine nucleotides.

[0191] In some embodiments, at least one poly(A) sequence can comprise from about 40 to about 500 consecutive adenosine nucleotides, from about 40 to about 200 consecutive adenosine nucleotides, from about 40 to about 150 consecutive adenosine nucleotides, for example, from about 60 to about 150 consecutive adenosine nucleotides.

[0192] Suitably, the length of the poly(A) sequence may be at least about 10, 50, 64, 75, 100, 200, 300, 400, or 500 or more adenosine nucleotides, such as consecutive adenosine nucleotides.

[0193] In some embodiments, at least one poly(A) sequence comprises about 100 adenosine nucleotides (A100), such as about 100 consecutive adenosine nucleotides.

[0194] In a further embodiment, 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 about 10 non-adenosine nucleotides (A30-N10-A70).

[0195] Advantageously, the poly(A) sequence defined herein may be located directly at the 3'-end of the RNA. In some embodiments, the 3'-terminal nucleotide (i.e. the last 3'-terminal nucleotide of the polynucleotide chain) is the 3'-terminal A nucleotide of at least one poly(A) sequence. The term "directly located at the 3'-end" should be understood to mean located exactly at the 3'-end. In other words, the 3'-end of the RNA consists of a poly(A) sequence that terminates with an A.

[0196] Terminating with adenosine nucleotides can reduce the induction of interferon, such as IFNα, by the RNA of the present disclosure, for example when administered as a vaccine. This is particularly important because the induction of interferon, such as IFNα, is believed to be one of the major factors for inducing fever in vaccinated subjects.

[0197] Thus, in some embodiments, the coding RNA of the present disclosure comprises a poly(A) sequence of about 100 consecutive adenosine nucleotides, the aforementioned poly(A) sequence being located directly at the 3'-end of the RNA, and optionally, the 3'-terminal nucleotide is an adenosine.

[0198] In some embodiments, the poly(A) sequence of the RNA is obtained from a DNA template during RNA in vitro transcription.In other embodiments, the poly(A) sequence is obtained in vitro by the general method of chemical synthesis, without necessarily being transcribed from a DNA template.In other embodiments, the poly(A) sequence is generated by enzymatic polyadenylation of RNA (after RNA in vitro transcription), for example using immobilized poly(A) polymerase, according to the methods and means as described in WO2016174271, which is incorporated herein by reference.

[0199] In some embodiments, the coding RNA comprises at least one poly(A) sequence obtained by enzymatic polyadenylation, and the majority of the RNA molecule comprises from about 100 (+ / -20) to about 500 (+ / -100) adenosine nucleotides, for example from about 100 (+ / -20) to about 200 (+ / -40) adenosine nucleotides.

[0200] In some embodiments, the coding RNA comprises at least one poly(A) sequence derived from a template DNA and 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.

[0201] In some embodiments, the coding RNA comprises at least one polyadenylation signal.

[0202] 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 the 3'-UTR.

[0203] As used herein, the term "poly(C) sequence" refers to a sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises from about 10 to about 200 cytosine nucleotides, from about 10 to about 100 cytosine nucleotides, from about 20 to about 70 cytosine nucleotides, from about 20 to about 60 cytosine nucleotides, or from about 10 to about 40 cytosine nucleotides. In one embodiment, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0204] In some embodiments, the coding 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.

[0205] In some embodiments, the coding RNA of the present disclosure comprises at least one histone stem loop (hSL) or histone stem loop structure. hSL in the context of the present disclosure can be located in the UTR region, for example, the 3'-UTR.

[0206] The term "histone stem loop" (hSL) is intended to refer to nucleic acid sequences that form stem-loop secondary structures found primarily in histone mRNAs.

[0207] The histone stem loop sequence / structure may suitably be selected from the hSL sequences disclosed in WO2012019780, the disclosure of which is incorporated herein by reference with respect to histone stem loop sequences / histone stem loop structures. The hSL sequences that may be used within 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 the specific formulas (Ia) or (IIa) of WO201019780.

[0208] In some embodiments, the coding RNA comprises at least one hSL, wherein said hSL comprises or consists of an RNA sequence identical to any one of SEQ ID NOs: 136, 137, or a fragment thereof or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0209] In an alternative embodiment, the coding RNA does not comprise a histone stem loop as defined herein.

[0210] In some embodiments, the coding 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 at least one hSL as defined herein.

[0211] In some embodiments, the coding RNA comprises at least one 3'-terminal sequence element that comprises or consists of an RNA sequence that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of SEQ ID NOs: 138-172, or a fragment or variant of these sequences.

[0212] In some embodiments, the coding RNA comprises a 3'-terminal sequence element comprising hSL as defined herein, followed by a poly(A) sequence comprising about 100 consecutive adenosines.

[0213] In some embodiments, the coding RNA comprises an RNA sequence that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or consists of, SEQ ID NO: 144, or a fragment or variant thereof.

[0214] In some embodiments, the coding RNA comprises a 5'-terminal sequence element that comprises or consists of an RNA sequence that is identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, any one of SEQ ID NOs: 121-127, or a fragment or variant of these sequences.

[0215] In some embodiments, the coding RNA comprises a 5'-end sequence element that comprises or consists of an RNA sequence identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO: 122, or a fragment or variant thereof.

[0216] Such a 5'-terminal sequence element may, for example, comprise a binding site for T7 RNA polymerase. Furthermore, the first nucleotide of the aforementioned 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.

[0217] Cap Construction: Preferably, the coding RNA comprises a 5'-cap structure, which preferably stabilizes the RNA and / or enhances expression of the encoded antigen and / or reduces stimulation of the innate immune system (after administration to a subject, e.g. a human subject).

[0218] Thus, in some embodiments, the coding RNA comprises a 5'-cap structure, such as m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure.

[0219] The term "5'-cap structure" as used herein will be recognized and understood by those of skill in the art and is intended to refer to a 5' modified nucleotide, particularly a guanine nucleotide, placed at the 5'-end of an RNA, such as an mRNA. For example, the 5'-cap structure is linked to the RNA via a 5'-5'-triphosphate linkage.

[0220] 5'-cap structures that may be suitable in the context of the present disclosure are cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), cap2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0221] The 5'-cap (cap0 or cap1) structure can be formed during chemical RNA synthesis or during RNA in vitro transcription using a cap analog (co-transcriptional capping).

[0222] The term "cap analog" as used herein will be recognized and understood by those of skill in the art and is intended to refer to a non-polymerizable di- or trinucleotide that has a cap functionality that, for example, when incorporated at the 5'-end of a nucleic acid molecule, facilitates translation or localization and / or prevents degradation of the RNA molecule. Non-polymerizable means that the cap analog 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 the group consisting of 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). Further suitable cap analogs are described in WO2008016473, WO2008157688, WO2009149253, WO2011015347, WO2013059475, WO2017066793, WO2017066781, WO2017066791, WO2017066789, WO2017053297, WO2017066782, WO2018075827 and WO2017066797, the disclosures of which are incorporated herein by reference.

[0223] In embodiments, the cap1 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 structures disclosed in claims 1-5 of WO2017053297 can be suitably used to co-transcriptionally generate a cap1 structure. Furthermore, any cap structure that can be derived from the structures defined in claim 1 or claim 21 of WO2018075827 can be suitably used to generate a cap1 structure. These disclosures are incorporated herein by reference.

[0224] In some embodiments, a 5'-cap structure may be added co-transcriptionally, preferably using a trinucleotide cap analog as defined herein, particularly in an RNA in vitro transcription reaction as defined herein.

[0225] In some embodiments, the coding RNA, particularly the mRNA, of the present disclosure comprises a cap1 structure.

[0226] In some embodiments, the cap1 structure of the RNA is formed through co-transcriptional capping using the trinucleotide cap analog m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG.

[0227] A particularly preferred cap1 analog in this context is m7G(5')ppp(5')(2'OMeA)pG.

[0228] In another embodiment, the cap1 structure of the RNA is formed using co-transcriptional capping with the trinucleotide cap analog 3'OMe-m7G(5')ppp(5')(2'OMeA)pG.

[0229] In an alternative embodiment, the 5'-cap structure is formed via enzymatic capping using a capping enzyme (e.g., vaccinia virus capping enzyme and / or a cap-dependent 2'-O methyltransferase) to generate a cap0 or cap1 or cap2 structure. In that context, the 5'-cap structure (cap0 or cap1) can be added using an immobilized capping enzyme and / or a cap-dependent 2'-O methyltransferase using the methods and means disclosed in published PCT patent application WO2016193226, which is incorporated herein by reference.

[0230] In some embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises a cap structure, e.g., a cap1 structure, as determined by a capping assay.

[0231] To determine the presence or absence of a cap structure, a capping assay can be used as described in published PCT application WO2015101416, in particular as described in claims 27-46 of published PCT application WO2015101416. Other capping assays that can be used to determine the presence or absence of a cap structure of an RNA are described in published PCT application WO2020127959, the disclosures of which are incorporated herein by reference.

[0232] Modified Nucleotides: According to various embodiments, the coding RNA of the present disclosure is a modified RNA, where modification refers to chemical modifications including backbone modifications as well as sugar or base modifications.

[0233] The modified RNA may include nucleotide analogs / modifications, such as backbone modifications, sugar modifications or base modifications. Backbone modifications in the context of the present disclosure are modifications in which the backbone phosphate of the nucleotide of the RNA is chemically modified. Sugar modifications in the context of the present disclosure are chemical modifications of the sugar of the nucleotide of the RNA. Furthermore, base modifications in the context of the present disclosure are chemical modifications of the base portion of the nucleotide of the RNA. In this context, the nucleotide analogs or modifications are selected from, for example, nucleotide analogs applicable to transcription and / or translation.

[0234] Thus, in some embodiments, the coding RNA of the present disclosure comprises at least one modified nucleotide.

[0235] In some embodiments, the at least one modified nucleotide is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-azauridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0236] Preferred in this context are pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), especially N1-methylpseudouridine (m1ψ).

[0237] In some embodiments, essentially all, eg, essentially 100%, of the uracils in the coding sequence (or complete RNA sequence) have a chemical modification, eg, a chemical modification at the 5-position of the uracil.

[0238] Incorporation of modified nucleotides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ) into the coding sequence (or the complete RNA sequence) can be advantageous since (if necessary) undesirable innate immune responses (upon administration of the RNA) can be modulated or reduced.

[0239] In alternative embodiments, the coding RNA of the present disclosure does not include modified nucleotides such as N1-methylpseudouridine (m1Ψ) or pseudouridine (ψ) substituted positions.

[0240] In some embodiments in that context, the coding RNA of the present disclosure comprises a coding sequence that consists only of G, C, A and U nucleotides and thus does not contain modified nucleotides.

[0241] Further RNA characteristics: In the context of the present disclosure, coding RNA provides a coding sequence that encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, which is translated into a (functional) antigen after administration (e.g., after administration to a subject, e.g., a human subject).

[0242] In the context of the present disclosure, the coding RNA can be any type of RNA that includes a coding sequence that encodes 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.

[0243] Optionally, the coding RNA comprises from about 50 to about 20000 nucleotides, or from about 500 to about 10000 nucleotides, or from about 1000 to about 10000 nucleotides, or for example from about 1000 to about 5000 nucleotides, or for example from about 2000 to about 5000 nucleotides.

[0244] In some embodiments, the coding RNA is selected from an mRNA, a coding self-replicating RNA, a coding circular RNA, a coding viral RNA, or a coding replicon RNA.

[0245] In an embodiment, the coding RNA is a circular RNA. As used herein, "circular RNA" or "circRNA" should be understood as an RNA construct that is linked to form a circle and thus does not contain a 3' or 5' end. In some embodiments, the aforementioned circRNA comprises a coding sequence that encodes an antigenic polypeptide selected from or derived from Escherichia coli FimH, as defined herein.

[0246] In some embodiments, the coding RNA is mRNA.

[0247] Suitable features that the mRNA of the disclosure optionally include 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 any chemical modifications as defined herein.

[0248] In some embodiments, the coding RNA is an in vitro transcribed RNA (e.g., an in vitro transcribed mRNA).

[0249] In some embodiments, the nucleotide mixture for RNA in vitro transcription comprises modified nucleotides as defined herein. In that context, suitable modified nucleotides may be selected from pseudouridine (ψ) or N1-methylpseudouridine (m1ψ). Preferably, uracil nucleotides in the nucleotide mixture are replaced (partially or completely) by pseudouridine (ψ) and / or N1-methylpseudouridine (m1ψ) to obtain modified RNA (e.g. modified mRNA).

[0250] In some embodiments, the nucleotide mixture used for RNA in vitro transcription does not include modified nucleotides as defined herein. In some embodiments, the nucleotide mixture used for RNA in vitro transcription includes only guanosine (G), cytidine (C), adenosine (A) and uridine (U) nucleotides, and optionally, cap analogs as defined herein to obtain unmodified RNA (e.g., unmodified mRNA).

[0251] In some embodiments, the nucleotide mixture (i.e., the proportion of each nucleotide in the mixture) used in 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.

[0252] In one embodiment, the coding RNA is freeze-dried (e.g., according to WO2016165831 or WO2011069586) to obtain a temperature-stable dry RNA. The RNA can also be dried using spray drying or spray freeze drying (e.g., according to WO2016184575 or WO2016184576) to obtain a temperature-stable RNA (powder). The disclosures of these are incorporated herein by reference.

[0253] In the context of the present disclosure (e.g., RNA-based vaccines), it may be required to provide GMP-grade RNA. GMP-grade RNA is produced using a manufacturing process approved by a regulatory agency. In some embodiments, the production of RNA is carried out under current Good Manufacturing Practice (GMP) and implements various quality control steps at DNA and RNA levels, such as 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.

[0254] In some embodiments, the coding RNA of the present disclosure is purified RNA, optionally purified mRNA.

[0255] The term "purified RNA" or "purified mRNA" as used herein should be understood as RNA that has a higher purity after a specific purification step (e.g., HPLC, TFF, oligo d(T) purification, precipitation step) than the starting material (e.g., in vitro transcribed RNA). Typical impurities that are essentially absent in purified RNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription, e.g., RNA polymerases, RNases, pyrophosphatases, restriction endonucleases, DNases), spermidine, BSA, aborted 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, for example, come from the fermentation procedure include bacterial impurities (bioburden, bacterial DNA) or impurities derived from the purification procedure (organic solvents, etc.). It is therefore desirable that the "purity of RNA" is as close as possible to 100%. Thus, as used herein, "purified RNA" has a purity of more than 75%, more than 80%, more than 85%, more particularly more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, and most preferably more than 99%. The degree of purity is determined, for example, by analytical HPLC, and the above percentages correspond to the ratio between the area of ​​the peak of target RNA and the total area of ​​all peaks, including peaks representing by-products. Alternatively, purity is determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0256] Suitably, purification of the coding RNA of the present disclosure may be carried out 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.

[0257] Optionally, the RNA has been 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, uncapped RNA and / or RNA fragments.

[0258] In an embodiment, the coding RNA of the present disclosure has a certain RNA integrity.

[0259] The term "RNA integrity" generally refers to whether or not a complete RNA sequence exists. Low RNA integrity may be due to, among other things, RNA degradation, RNA cleavage, incorrect or incomplete chemical synthesis of RNA, incorrect base pairing, integration of modified nucleotides or modification of already integrated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation, or incomplete RNA in vitro transcription. RNA is a fragile molecule and is easily degraded, which may occur, for example, by temperature, ribonucleases, pH, and other factors (e.g., nucleophilic attack, hydrolysis, etc.), which may reduce RNA integrity and thus its functionality.

[0260] Those skilled in the art can choose from a variety of different chromatographic or electrophoretic methods to determine the integrity of RNA. Chromatographic and electrophoretic methods (e.g., capillary gel electrophoresis) are well known in the art. When using chromatography (e.g., RP-HPLC), the analysis of RNA integrity can be based on determining the peak area (or "peak area") of expected full-length RNA (RNA with correct RNA length) in the corresponding chromatogram.

[0261] In an embodiment, the coding RNA of the present disclosure has an RNA integrity in the range of about 40% to about 100%. In an embodiment, the RNA has an RNA integrity in the range of about 50% to about 100%. In an embodiment, the RNA has an RNA integrity in the range of about 60% to about 100%. In an embodiment, the RNA has an RNA integrity in the range of about 70% to about 100%. In an embodiment, the RNA integrity is, for example, about 50%, about 60%, about 70%, about 80%, or about 90%. The RNA is preferably determined using analytical HPLC, for example analytical RP-HPLC.

[0262] In some embodiments, the coding RNA has an RNA integrity of at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80% or about 90%. RNA integrity is preferably determined using analytical HPLC, such as analytical RP-HPLC.

[0263] In some embodiments, the coding RNA is suitable for nasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal, or subcutaneous administration. In some embodiments, the coding RNA is suitable for intramuscular administration.

[0264] RNA constructs: In some embodiments, the coding RNA comprises at least the following elements, e.g., in the 5' to 3' direction: A) a 5'-cap structure, for example as defined herein; B) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein; C) the 5'-UTR and / or the 3'-UTR, e.g., as defined herein; D) at least one poly(A) sequence, for example as defined herein.

[0265] In various embodiments, the coding RNA, e.g., mRNA, comprises the following elements, e.g., in the 5' to 3' direction: A) a 5'-cap structure, for example as defined herein; B) a 5'-end initiation element, e.g., as defined herein; C) optionally, a 5'-UTR, e.g., as defined herein; D) a ribosome binding site, e.g., as defined herein; E) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein; F) a 3'-UTR, e.g., as defined herein; G) optionally, at least one poly(A) sequence, e.g., as defined herein; H) optionally at least one poly(C) sequence, e.g., as defined herein; I) optionally a histone stem loop, e.g., as defined herein; J) optionally a 3'-end sequence element, e.g., as defined herein; K) Optionally, a chemically modified nucleotide, for example as defined herein.

[0266] In various embodiments, the coding RNA, e.g., mRNA, comprises the following elements, e.g., in the 5' to 3' direction: A) 5'-cap structure; B) a 5'-UTR, for example selected from or derived from the 5'-UTR of the HSD17B4 gene; C) at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein; D) a 3'-UTR, for example selected from or derived from the 3'-UTR of the PSMB3 gene; E) optionally, a histone stem loop; and F) A poly(A) sequence, for example comprising about 100 A nucleotides.

[0267] In some embodiments, the mRNA comprises the following elements, e.g., in the 5'- to 3'-direction: A) a cap1 structure, e.g., as defined herein; B) the 5'-UTR, derived from the HSD17B4 gene as defined herein; C) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, for example a coding sequence constituting at least one of the nucleic acid sequences 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 a sequence according to any one of SEQ ID NOs: 187-246, 257-316, 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or fragment or variant thereof; D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein; E) optionally a histone stem loop, as defined herein; F) a poly(A) sequence, for example comprising about 100 A nucleotides; G) Optionally, a chemically modified nucleotide, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0268] In further embodiments, the mRNA comprises the following elements, e.g., in the 5'- to 3'-direction: A) a cap1 structure, e.g., as defined herein; B) The 5'-UTR from the HSD17B4 gene as defined herein; C) at least one cds encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH as defined herein, wherein the coding sequence comprising at least one of the nucleic acid sequences 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 a sequence according to any one of SEQ ID NOs: 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or fragment or variant thereof; D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein; E) optionally a histone stem loop, as defined herein; F) a poly(A) sequence, for example comprising about 100 A nucleotides; G) Optionally, a chemically modified nucleotide, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0269] In some embodiments, the mRNA comprises the following elements in the 5'- to 3'-direction: A) the cap1 structure, as defined herein; B) the 5'-UTR, derived from the HSD17B4 gene as defined herein; C) at least one cds encoding an antigenic polypeptide which is or is derived from Escherichia coli FimH as defined herein, e.g. the coding sequence constituting at least one of the nucleic acid sequences 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 a sequence according to any one of SEQ ID NOs: 529, 554, 579, 604, 629, 654, or a fragment or fragment or variant thereof; D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein; E) optionally a histone stem loop, as defined herein; F) a poly(A) sequence comprising about 100 A nucleotides, e.g., representing the 3' end; G) Optionally, a chemically modified nucleotide, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0270] In some embodiments, the mRNA comprises the following elements in the 5' to 3' direction: A) the cap1 structure, as defined herein; B) the 5'-UTR, derived from the HSD17B4 gene as defined herein; C) at least one cds encoding an antigenic polypeptide which is or is derived from Escherichia coli FimH as defined herein, e.g. the coding sequence constituting at least one of the nucleic acid sequences 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 a sequence according to any one of SEQ ID NOs: 533, 558, 583, 608, 633, 658, or a fragment or fragment or variant thereof; D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein; E) optionally a histone stem loop, as defined herein; F) a poly(A) sequence comprising about 100 A nucleotides, e.g., representing the 3' end; G) Optionally, a chemically modified nucleotide, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0271] In some embodiments, the mRNA comprises the following elements in the 5'- to 3'-direction: A) the cap1 structure, as defined herein; B) the 5'-UTR, derived from the HSD17B4 gene as defined herein; C) at least one cds encoding an antigenic polypeptide which is or is derived from Escherichia coli FimH as defined herein, e.g. the coding sequence constituting at least one of the nucleic acid sequences 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 a sequence according to any one of SEQ ID NOs: 534, 559, 584, 609, 634, 659, or a fragment or fragment or variant thereof; D) a 3'-UTR derived from the 3'-UTR of the PSMB3 gene as defined herein; E) optionally a histone stem loop, as defined herein; F) a poly(A) sequence comprising about 100 A nucleotides, e.g., representing the 3' end; G) Optionally, a chemically modified nucleotide, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0272] The RNA sequences are provided in Table 2, where each row represents a particular preferred RNA construct of the present disclosure (compare Table 1), with a description of the construct shown in column A of Table 2 and the SEQ ID NO for the amino acid sequence of each construct provided in column B. The corresponding SEQ ID NO for the coding sequence encoding each construct is provided in Table 1.

[0273] The corresponding RNA sequences, in particular mRNA sequences, are provided in columns C and D, where column C provides RNA sequences having the UTR combination "HSD17B4 / PSMB3" and a 3'-terminal hSL-A100 tail, and column D provides nucleic acid sequences having the UTR combination "HSD17B4 / PSMB3" and a 3'-terminal A100 tail.

[0274] [Table 2-1] [Table 2-2] [Table 2-3]

[0275] In some embodiments, the coding RNA is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1150, 1152-1154, 1156-1158, 1158-1160, 1162-1170, 1174-1175, 1176-1180, 1182-1190, 1192-1204, 1194-1206, 1196-1208, 1206-1209, 1208-1300, 1208-1310, 1208-1320, 1208-1330, 1208-1340, 1208-1350, 1208-1360, 1208-1370, 1208-1380, 1208-1390, 1208-1391, 1208-1392, 1208-1393, 1208-1394, 1208-1395, 1208-1396, 1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, or a fragment or variant thereof, or comprising or consisting of a nucleic acid sequence that is identical to, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, a nucleic acid sequence according to any one of 1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, or a fragment or variant thereof.

[0276] In a further embodiment, the coding RNA is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270. and a coding RNA comprising or consisting of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence as set forth above, or a fragment or variant thereof, in which at least one, e.g. all, uracil nucleotides in said RNA sequence are replaced with pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides. In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 1271 to 1273, or a fragment or variant thereof, wherein all uracil nucleotides in the aforementioned RNA sequence are replaced by N1-methylpseudouridine (m1ψ) nucleotides. In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 1274-1276, or a fragment or variant thereof, wherein all uracil nucleotides in the aforementioned RNA sequence are replaced by pseudouridine (ψ) nucleotides.

[0277] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 679, 704, 729, 754, 779, 804, 829, 854, 879, 904, 929, 954, 979, 1004, 1029, 1054, 1079, 1104, 1129, 1154, 1179, 1204, 1229, 1254, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences are optionally free of chemically modified nucleotides.

[0278] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 683, 708, 733, 758, 783, 808, 833, 858, 883, 908, 933, 958, 983, 1008, 1033, 1058, 1083, 1108, 1133, 1158, 1183, 1208, 1233, 1258, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences are optionally free of chemically modified nucleotides.

[0279] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 684, 709, 734, 759, 784, 809, 834, 859, 884, 909, 934, 959, 984, 1009, 1034, 1059, 1084, 1109, 1134, 1159, 1184, 1209, 1234, 1259, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences are optionally free of chemically modified nucleotides.

[0280] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 679, 704, 729, 754, 779, 804, 829, 854, 879, 904, 929, 954, 979, 1004, 1029, 1054, 1079, 1104, 1129, 1154, 1179, 1204, 1229, 1254, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences optionally include pseudouridine (ψ) nucleotides.

[0281] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 683, 708, 733, 758, 783, 808, 833, 858, 883, 908, 933, 958, 983, 1008, 1033, 1058, 1083, 1108, 1133, 1158, 1183, 1208, 1233, 1258, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences optionally include pseudouridine (ψ) nucleotides.

[0282] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 684, 709, 734, 759, 784, 809, 834, 859, 884, 909, 934, 959, 984, 1009, 1034, 1059, 1084, 1109, 1134, 1159, 1184, 1209, 1234, 1259, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences optionally include pseudouridine (ψ) nucleotides.

[0283] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 679, 704, 729, 754, 779, 804, 829, 854, 879, 904, 929, 954, 979, 1004, 1029, 1054, 1079, 1104, 1129, 1154, 1179, 1204, 1229, 1254, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. Therein, the aforementioned RNA sequences optionally include N1-methylpseudouridine (m1ψ) nucleotides.

[0284] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 683, 708, 733, 758, 783, 808, 833, 858, 883, 908, 933, 958, 983, 1008, 1033, 1058, 1083, 1108, 1133, 1158, 1183, 1208, 1233, 1258, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences optionally include N1-methylpseudouridine (m1ψ) nucleotides.

[0285] In one embodiment, the coding RNA comprises or consists of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 684, 709, 734, 759, 784, 809, 834, 859, 884, 909, 934, 959, 984, 1009, 1034, 1059, 1084, 1109, 1134, 1159, 1184, 1209, 1234, 1259, or a fragment or variant thereof. In that embodiment, the aforementioned RNA sequence optionally comprises a 5'-terminal cap1 structure. In that embodiment, the aforementioned RNA sequences optionally include N1-methylpseudouridine (m1ψ) nucleotides.

[0286] In other embodiments, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), which is identical to an RNA sequence according to any one of SEQ ID NOs: 679, 829, 979, 1129, or a fragment or variant thereof.

[0287] In other embodiments, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), which is identical to an RNA sequence according to any one of SEQ ID NOs: 683, 833, 983, 1133, or a fragment or variant thereof.

[0288] In other embodiments, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C, U), which is identical to an RNA sequence according to any one of SEQ ID NOs: 684, 834, 984, 1134, or a fragment or variant thereof.

[0289] In one embodiment, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, which is a 5' capped (cap1) mRNA according to any one of SEQ ID NOs: 679, 829, 979, 1129, 1274, or a fragment or identical thereto.

[0290] In one embodiment, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, which is identical to an RNA sequence according to any one of SEQ ID NOs: 683, 833, 983, 1133, 1275, or a fragment or variant thereof.

[0291] In one embodiment, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides, which is identical to an RNA sequence according to any one of SEQ ID NOs: 684, 834, 984, 1134, 1276, or a fragment or variant thereof.

[0292] In one embodiment, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides, which is identical to an RNA sequence according to any one of SEQ ID NOs: 679, 829, 979, 1129, 1271, or a fragment or variant thereof. In one embodiment, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides, which is identical to an RNA sequence according to any one of SEQ ID NOs: 683, 833, 983, 1133, 1272, or a fragment or variant thereof.

[0293] In one embodiment, the coding RNA of the present disclosure is a 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides, which is identical to an RNA sequence according to any one of SEQ ID NOs: 684, 834, 984, 1134, 1273, or a fragment or variant thereof.

[0294] (2: Composition containing coding RNA encoding an antigenic polypeptide of Escherichia coli FimH) In a second aspect, there is provided a pharmaceutical composition comprising a coding RNA of the first aspect.

[0295] In particular, the embodiments relating to the pharmaceutical composition of the second aspect may be read and understood as preferred embodiments of the vaccine of the third aspect. Also, the embodiments relating to the vaccine of the third aspect may be read and understood as preferred embodiments of the pharmaceutical composition of the second aspect. Furthermore, the features and embodiments described in the context of the first aspect (RNA of the present disclosure) must be read and understood as preferred embodiments of the pharmaceutical composition of the second aspect.

[0296] In the context of this disclosure, a "composition" refers to any type of composition in which the specified components (e.g., (a) at least one untranslated region (UTR); and (b) a coding RNA comprising a coding sequence operably linked to the aforementioned UTR encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH) can be incorporated, optionally with any additional components, usually with at least one pharma- ceutically acceptable carrier or excipient. The composition may be a dry composition, such as a powder, granule, or solid lyophilized form. Alternatively, the composition may be in liquid form, and each component may be incorporated independently in dissolved or dispersed (e.g., suspended or emulsified) form.

[0297] In various embodiments, the coding RNA of the pharmaceutical composition is selected from the coding RNA defined in any of the embodiments of the first aspect.

[0298] In an embodiment, the coding RNA as comprised in the pharmaceutical composition is provided in an amount of at least about 100 ng up to about 500 μg, at least about 1 μg up to about 200 μg, at least about 1 μg up to about 100 μg, at least about 5 μg up to about 100 μg, for example at least about 10 μg up to about 50 μg, in particular about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg or 100 μg.

[0299] In one embodiment, the coding RNA of the composition is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-11 45, 1148-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, 1271-1276, or a fragment or variant thereof, or comprises or consists of an RNA sequence that is identical to, or is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of the sequences listed in the above table.

[0300] In some embodiments, the pharmaceutical composition comprises a plurality or at least more than one RNA species.

[0301] In one embodiment the pharmaceutical composition comprises a first coding RNA according to the first aspect and a second coding RNA encoding a polypeptide selected from or derived from Escherichia coli FimC.

[0302] In embodiments, the second coding sequence encodes a FimC amino acid sequence that is identical to, or 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 SEQ ID NOs: 317, 324, 496, 497, or a fragment or variant thereof. This is particularly preferred when the first coding sequence encodes a FimH amino acid sequence that is identical to or 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 SEQ ID NOs: 177-186, 247-256, in particular SEQ ID NOs: 498, 500, or is an immunogenic fragment or immunogenic variant thereof.

[0303] In an embodiment, the second coding RNA comprises a FimC coding sequence comprising at least one of the nucleic acid sequences identical or 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 SEQ ID NOs: 318-323, 325-330, 521, 522, 546, 547, 571, 572, 596, 597, 621, 622, 646, 647, or a fragment or variant thereof. This is particularly preferred when the first coding sequence comprises a FimH coding sequence comprising at least one of the nucleic acid sequences identical to or 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 SEQ ID NOs: 187-246, 257-316, 523, 525, 548, 550, 573, 575, 598, 600, 623, 625, 648, 650.

[0304] In an embodiment the second coding RNA is a nucleic acid sequence according to any one of SEQ ID NOs: 671, 672, 696, 697, 721, 722, 746, 747, 771, 772, 796, 797, 821, 822, 846, 847, 871, 872, 896, 897, 921, 922, 946, 947, 971, 972, 996, 997, 1021, 1022, 1046, 1047, 1071, 1072, 1096, 1097, 1121, 1122, 1146, 1147, 1171, 1172, 1196, 1197, 1221, 1222, 1246, 1247, or or comprises or consists of a FimC-encoding nucleic acid sequence that is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a fragment or variant of any of these sequences, optionally wherein at least one, e.g., all, uracil nucleotides in the aforementioned RNA sequence are replaced with pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0305] It is noteworthy that the preferred nucleic acid features (e.g., UTRs, cap structures, modifications, codon optimization) disclosed in the context of the FimH-encoding RNA sequence of the first embodiment are also applicable and may also be preferred for the FimC-encoding RNA sequence of the second embodiment.

[0306] Certain features and embodiments of the coding RNA of the first aspect provided herein can also be applied to the second coding RNA encoding FimC. Providing a pharmaceutical composition comprising a first coding RNA encoding FimH and a second coding RNA encoding FimC is particularly preferred because the FimH and FimC polypeptides encoded by the first and second coding RNAs can assemble in a non-covalent complex once translated. This is particularly suitable for stabilizing FimH when the coding sequence of the first RNA encoding FimH does not encode a donor strand peptide.

[0307] The E. coli FimC sequence and construct are recorded in Tables 1 and 2.

[0308] In various embodiments, the coding RNA of the pharmaceutical composition is formulated with a pharma- ceutically acceptable carrier or excipient.

[0309] For example, the term "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" as used herein includes liquid or non-liquid bases of compositions for administration. When the composition is provided in liquid form, the carrier can be water, e.g., pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g., phosphate, citrate, and other buffer solutions. Water or, e.g., buffers, e.g., aqueous buffers, including, e.g., sodium, calcium, or potassium salts, can be used. According to some embodiments, the sodium, calcium, or potassium salts can occur in the form of their halides, e.g., chlorides, iodides, or bromides, their hydroxides, carbonates, bicarbonates, or sulfates, and the like. Examples of sodium salts include NaCl, Na 2 HPO 4 , NaI, NaBr, Na 2 CO 3 , NaHCO 3 , Na 2 SO 4 Examples of optional potassium salts include KCl, KI, KBr, and K 2 CO 3 , K.H.C.O. 3 , K 2 SO 4 Examples of calcium salts include CaCl 2 , CaI 2 , CaBr 2 , CaCO 3 , CaSO 4 , Ca(OH) 2 Includes:

[0310] In addition, organic anions of the aforementioned cations may be present in the buffer. Thus, in embodiments, the pharmaceutical composition may include one or more pharma- ceutically acceptable carriers or excipients, such as to increase stability, increase cell transfection, allow sustained or delayed translation, increase translation of the encoded antigenic polypeptide in vivo, and / or change the release profile of the encoded antigenic peptide in vivo. In addition to conventional excipients, such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, the excipients of the present disclosure may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, and combinations thereof. In embodiments, one or more compatible solid or liquid fillers or diluents or encapsulating compounds suitable for administration to a subject may also be used. As used herein, the term "compatible" means that the components of the composition are capable of being mixed with at least one nucleic acid, and optionally multiple nucleic acids, of the composition under typical conditions of use (e.g., intramuscular or intradermal administration) in a manner such that there is no interaction that would substantially reduce the biological activity or pharmaceutical effectiveness of the composition. A pharmaceutically acceptable carrier or excipient must have a sufficiently high purity and sufficiently low toxicity to be suitable for administration to a subject to be treated.Compounds which may be used as pharma- ceutically acceptable carriers or excipients may be, for example, sugars such as lactose, glucose, trehalose, mannose, and sucrose; starches, for example, corn starch or potato starch; dextrose; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid lubricants, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, oil derived from theobroma; polyols, for example, polypropylene glycol, glycerol, sorbitol, mannitol, polyethylene glycol; alginic acid.

[0311] Pharmaceutical compositions of the present disclosure are preferably sterile and / or pyrogen-free.

[0312] <Formulation / Complexation> In some embodiments, the coding RNA of the pharmaceutical composition is complexed or accompanied with at least one additional compound to obtain a formulated composition.The formulation in this context can have the function of a transfection agent.The formulation in this context can also have the function of protecting RNA from degradation, for example to allow storage, shipping, etc.

[0313] In some embodiments, the coding RNA of the pharmaceutical composition is formulated with at least one compound, such as a peptide, protein, lipid, polysaccharide, and / or polymer.

[0314] In some embodiments, the coding RNA of the pharmaceutical composition is formulated with at least one cationic compound (cationic or, e.g., ionizable) or polycationic compound (cationic or, e.g., ionizable).

[0315] In some embodiments, the coding RNA of the pharmaceutical composition is complexed or associated, or at least partially complexed or associated, with one or more cationic (cationic or, e.g., ionizable) or polycationic compounds.

[0316] The term "cationic or polycationic compound" as used herein will be recognized and understood by those skilled in the art and is intended to refer to a charged molecule that is positively charged, for example, at a pH value in the range of about 1-9, a pH value in the range of about 3-8, a pH value in the range of about 4-8, a pH value in the range of about 5-8, for example, a pH value in the range of about 6-8, for example, a pH value in the range of about 7-8, for example, at physiological pH, for example, about 7.2-7.5. Thus, a cationic moiety, for example, a cationic peptide, cationic protein, cationic polymer, cationic polysaccharide, cationic lipid, can be any positively charged compound or polymer that is positively charged under physiological conditions. A "cationic or polycationic peptide or protein" can include at least one positively charged amino acid, or multiple positively charged amino acids, for example, amino acids selected from Arg, His, Lys, or Orn. ​​Thus, a "polycationic" moiety is also within the scope of exhibiting multiple positive charges under a given condition.

[0317] 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.

[0318] In some embodiments, the at least one cationic or polycationic compound is selected from a cationic or polycationic peptide or protein.

[0319] In some embodiments, the pharmaceutical composition comprises a coding RNA as defined herein and a polymeric carrier.

[0320] The term "polymeric carrier" as used herein will be recognized and understood by those skilled in the art and is intended to refer to, for example, a compound that facilitates the transport and / or complexation of another compound. A polymeric carrier is typically a carrier formed of a polymer. A polymeric carrier can entrain its cargo (e.g., RNA) by covalent or non-covalent interactions. A polymer can also be based on different subunits, such as copolymers. Suitable polymeric carriers in this context include, for example, polyethyleneimine (PEI).

[0321] In embodiments, the pharmaceutical composition comprises at least one RNA complexed or associated with a polymeric carrier and, optionally, at least one lipid component, as described in WO2012212008, WO2012212006, WO2012212007, and WO2012212009. In this context, the disclosures of WO2017212008, WO2017212006, WO2017212007, and WO2017212009 are incorporated herein by reference. In some embodiments, the lipidoid component of the polymeric carrier may be any one selected from the table of lipidoid structures in published PCT patent application WO20127212009A1 (pages 50-54).

[0322] <Formulation into lipid-based carriers> In some embodiments, the pharmaceutical composition comprises a lipid-based carrier.

[0323] In the context of the present disclosure, the term "lipid-based carrier" includes lipid-based delivery systems for RNA that include a lipid component. The lipid-based carrier may further include 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.

[0324] In the context of this disclosure, a typical "lipid-based carrier" is selected from liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes. The RNA of the pharmaceutical composition may be fully or partially incorporated or encapsulated in the lipid-based carrier, and the RNA may be located in the internal space of the lipid-based carrier, within the lipid layer / membrane of the lipid-based carrier, or may be associated with the outer surface of the lipid-based carrier. The incorporation of RNA into the lipid-based carrier may be referred to as "encapsulation". The "lipid-based carrier" is not limited to any form, and includes any form that is generated when, for example, an aggregation-reducing lipid and at least one additional lipid are combined in an aqueous environment, for example, in the presence of RNA. For example, LNPs, liposomes, lipid complexes, lipoplexes, etc. are within the scope of the term "lipid-based carrier". Lipid-based carriers can be of different sizes, including but not limited to multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments, small unilamellar vesicles (SUVs), which can be smaller than 50 nm in diameter, and large unilamellar vesicles (LUVs), which can be 50 nm to 500 nm in diameter. Liposomes are a specific type of lipid-based carrier, characterized as microscopic vesicles with an internal aqueous space isolated from the outside medium by a membrane consisting of one or more bilayers. In liposomes, at least one RNA is located in the internal aqueous space, typically wrapped in a partial or entire lipid portion of the liposome. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as synthetic or naturally derived lipids, which contain spatially separated hydrophilic and hydrophobic domains. Lipid nanoparticles (LNPs) are a specific type of lipid-based carrier, characterized as microscopic lipid particles with a solid or partially solid core. Typically, LNPs do not contain an internal aqueous space that is separated from the outside medium by a bilayer.In LNPs, at least one RNA is encapsulated or incorporated in the lipid portion of the LNP, surrounded by part or all of the lipid portion of the LNP.LNPs can include any lipid that can form a particle to which RNA can be attached or in which RNA can be encapsulated.For example, the lipid-based carriers described above are particularly suitable for intramuscular and / or intradermal administration.

[0325] In some embodiments, the lipid-based carrier of the pharmaceutical composition is selected from a liposome, a lipid nanoparticle, a lipoplex, and / or a nanoliposome.

[0326] 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 coding RNA of the present disclosure.

[0327] The term "encapsulated", such as incorporated, complexed, encapsulated, partially encapsulated, entrained, partially entrained, refers to an essentially stable combination of RNA and one or more lipids, such as in a lipid-based carrier (e.g., a larger complex or aggregate), without covalent binding of RNA. Lipid-based carrier-encapsulated RNA may be located completely or partially inside the lipid-based carrier (e.g., lipid portion and / or inner space) and / or lipid layer / membrane of the lipid-based carrier. Encapsulation of RNA in lipid-based carrier is also referred to as "embedding" herein, for example, because the RNA is contained inside the lipid-based carrier. Without wishing to be bound by assumption, the purpose of incorporating or encapsulating RNA in lipid-based carrier may be to protect the RNA from an environment that may include enzymes, chemicals, or conditions that degrade the RNA. Furthermore, incorporating RNA into lipid-based carriers may facilitate the uptake of RNA, thus enhancing the therapeutic effect of the RNA when administered to a cell or subject.

[0328] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises at least 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 a combination thereof.

[0329] 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 phospholipid, and a steroid or steroid analog.

[0330] <Cationic lipids> In some embodiments, the lipid-based carrier comprises a cationic or ionizable lipid.

[0331] The cationic or ionizable lipid of lipid-based carrier can be cationic or ionizable, that is, it is protonated when pH is lower than the pK of the ionizable group of lipid, but becomes gradually more neutral as pH value increases.At pH value below pK, lipid can be associated with negatively charged nucleic acid.In certain embodiments, cationic lipid comprises zwitterionic lipid, which becomes positively charged as pH decreases.

[0332] In some embodiments, the lipid-based carrier comprises a cationic or ionizable lipid that carries a net positive charge, e.g., at physiological pH, e.g., the cationic or ionizable lipid comprises a tertiary or quaternary nitrogen group. Thus, in some embodiments, the lipid-based carrier comprises a cationic or ionizable lipid selected from amino lipids.

[0333] In a further embodiment, the lipid formulation comprises a cationic or ionizable lipid as defined by formula I in paragraph

[0251] of WO2021222801, or a lipid selected from the disclosures in paragraphs

[0260] or

[0261] of WO2021222801. In another embodiment, the lipid formulation comprises a cationic or ionizable lipid selected from the group consisting of ATX-001 to ATX-132, such as ATX-0126, as disclosed in claim 90 of WO2021183563. The disclosures of WO2021222801 and WO2021183563, in particular the aforementioned lipids, are incorporated herein by reference.

[0334] In an embodiment, the cationic or ionizable lipid may be selected from the lipids disclosed in WO2018078053 (i.e., lipids derived from formulas I, II, and III of WO2018078053, or lipids defined in claims 1 to 12 of WO2018078053), the disclosure of which is incorporated herein by reference in its entirety. In this context, lipids disclosed in Table 7 of WO2018078053 (e.g., lipids derived from formulas I-1 to I-41) and lipids disclosed in Table 8 of WO2018078053 (e.g., lipids derived from formulas II-1 to II-36) may be suitably used in the context of the present disclosure. Accordingly, Formulae I-1 to I-41 and II-1 to II-36 of WO2018078053, and the specific disclosures thereof, are hereby incorporated by reference.

[0335] 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 in Table 9 of published PCT patent application WO2018078053. Accordingly, formulae III-1 to III-36 of WO2018078053, and the specific disclosures relating thereto, are incorporated herein by reference.

[0336] In various embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition (e.g., component B) of the present disclosure comprises a cationic lipid according to or derived from formula (III): [ka]

[0337] Formula (III) is further defined as follows: One of L1 and 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 and L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, or a direct bond; G1 and G2 are each independently an unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, C(=O)OR4, OC(=O)R4 or -NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; and x is 0, 1, or 2.

[0338] In one embodiment, the lipid-based carrier comprises a cationic lipid selected from or derived from formula III-3: [ka] The lipid of formula III-3 preferably used herein has the chemical term ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also called ALC-0315, i.e. CAS number 2036272-55-4.

[0339] Further suitable cationic lipids may be selected from or derived from cationic lipids according to PCT claims 1-14 of published patent application WO2021123332, or Table 1 of WO2021123332, the disclosures relating to claims 1-14 or Table 1 of WO2021123332 are incorporated herein by reference.

[0340] Thus, suitable cationic lipids may be selected from or derived from cationic lipids according to compounds 1 to 27 (C1-C27) in Table 1 of WO2021123332.

[0341] In other embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from (COATSOME® SS-EC) SS-33 / 4PE-15 (see C23 in Table 1 of WO2021123332).

[0342] 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 disclosed in Table 1 of WO2021123332: [ka]

[0343] In another embodiment, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a cationic lipid selected from or derived from 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, also referred to as SM-102. Other lipid-based carriers (e.g., LNP) of the pharmaceutical composition comprise a cationic lipid selected from the group consisting of squaramide ionizable amino lipids, such as those of formula (M1) and (M2): [ka] [ka] Here, the substituents (e.g., R 1 , R 2 , R 3 , R 5 , R 6 , R 7 , R 10 , M, M 1, m, n, o, l) are defined in claims 1 to 13 of US10392341B2; US10392341B2 is incorporated herein in its entirety.

[0344] Thus, 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.

[0345] In one embodiment, the lipid-based carrier of the pharmaceutical composition, such as an LNP, comprises a cationic lipid selected from or derived from ALC-0315.

[0346] In some embodiments, the lipid-based carriers of the present disclosure comprise two or more (different) cationic lipids as defined herein.

[0347] In certain embodiments, the cationic lipid as defined herein, such as the cationic lipid ALC-0315, is present in the lipid-based carrier in an amount of about 30 mol% to about 95 mol% based on the total lipid content of the lipid-based carrier. When multiple cationic lipids are included in the lipid-based carrier, the percentage applies to the combined cationic lipids.

[0348] In an embodiment, the cationic lipid is present in the lipid-based carrier in an amount of about 30 mol% to about 70 mol%. In one embodiment, the cationic lipid is present in the lipid-based carrier in an amount of about 40 mol% to about 60 mol%, for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol%. In an embodiment, the cationic lipid is present in the lipid-based carrier in an amount of about 47 mol% to about 48 mol%, for example, about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0 mol%, with 47.4 mol% being particularly preferred. In other embodiments, the cationic lipids are present in the lipid-based carrier in an amount of about 55 mol % to about 65 mol %, such as about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 mol %, with 59 mol % being particularly preferred.

[0349] In some embodiments, the cationic lipid is present in about 20 mol% to about 70 mol% or about 75 mol%, or about 45 mol% to about 65 mol%, or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol% of the total lipid present in the lipid-based carrier. In further embodiments, the LNP comprises about 25% to about 75% cationic lipid on a molar basis, e.g., 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% on a molar basis (based on 100% total molar lipid in the lipid nanoparticle).

[0350] In some embodiments, the ratio of cationic lipid to RNA is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11.

[0351] <Aggregation-reducing lipids> The term "aggregation-reducing lipid" refers to a molecule that includes both 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. It is therefore desirable to include lipid compounds that can reduce aggregation, for example by sterically stabilizing the lipid-based carrier. Such steric stabilization may occur when the compound has a sterically bulky but uncharged portion that shields or screens the charged portion of the lipid-based carrier from access to other lipid-based carriers in the composition. In the context of the present disclosure, stabilization of lipid-based carriers is achieved by including lipids that may include lipids with sterically bulky groups that are located, for example, on the exterior of the lipid-based carrier after formation of the lipid-based carrier. Suitable aggregation-reducing groups 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).

[0352] Lipids that include a polymer as an aggregation-reducing group are referred to herein as "polymer-conjugated lipids."

[0353] The term "polymer-conjugated lipid" refers to a molecule that includes both lipid and polymer moieties, and the polymer is suitable for reducing or preventing aggregation of lipid-based carriers that include RNA. A polymer should be understood as a substance or material that consists of a very large molecule or a macromolecule that is composed of many repeating subunits. In the context of this disclosure, suitable polymers are hydrophilic polymers. An example of a polymer-conjugated lipid is PEGylated or PEG-conjugated lipid.

[0354] In one embodiment, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid selected from polymer-conjugated lipids.

[0355] In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid (or PEGylated lipid, PEG lipid).

[0356] For example, the average molecular weight of the PEG moiety in the PEG-conjugated lipid is in the range of about 500 to about 8,000 daltons (eg, about 1,000 to about 4,000 daltons). In one embodiment, the average molecular weight of the PEG moiety is about 2,000 daltons.

[0357] 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, and 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)carbamyl]-1,2-dimyristyloxypropyl-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 LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a 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.

[0358] In some embodiments, the polymer-conjugated lipid, e.g., PEG-conjugated lipid, is selected from or derived from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000 DMG or DMG-PEG2000). As used in the art, "DMG-PEG 2000" is typically considered to be a mixture of 1,2-DMG PEG 2000 and 1,3-DMG PEG 2000 in a ratio of ∼97:3.

[0359] In other embodiments, the polymer-conjugated lipid, such as a PEG-conjugated lipid, is selected from or derived from C10-PEG2K or Cer8-PEG2K.

[0360] In one embodiment, the polymer-conjugated lipid, e.g., a PEG-conjugated lipid, is selected from or derived from formula (IVa): [ka] For example, n has an average value in the range of 30 to 60, such as 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 a further embodiment, the PEG lipid is of formula (IVa), where n is an integer selected such that the average molecular weight of the PEG lipid is about 2000 g / mol to about 3000 g / mol or about 2300 g / mol to about 2700 g / mol. In another embodiment, the PEG lipid is of formula (IVa), where n is an integer selected such that the average molecular weight of the PEG lipid is about 2000 g / mol.

[0361] The PEG-conjugated lipid of formula IVa suitable for use herein has the formula 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referred to as ALC-0159.

[0362] Thus, in some embodiments, the aggregation-reducing lipid is a PEG-conjugated lipid selected from or derived from DMG-PEG2000, C10-PEG2K, Cer8-PEG2K, or ALC-0159.

[0363] In one embodiment, the lipid-based carrier, e.g., the LNP of the pharmaceutical composition, comprises an aggregation-reducing lipid selected from or derived from ALC-0159.

[0364] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises an aggregation-reducing lipid, and the aggregation-reducing lipid is not a PEG-conjugated lipid.

[0365] In some embodiments, the lipid-based carrier comprises less than about 3 mol%, less than about 2 mol%, or less than about 1 mol% of aggregation-reducing lipid based on the total moles of lipid in the lipid-based carrier. In further embodiments, the lipid-based carrier comprises from about 0.1% to about 10% aggregation-reducing lipid on a molar basis, such as from about 0.5% to about 10%, from 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% on a molar basis (based on 100% total moles of lipid in the lipid-based carrier). In other embodiments, the lipid-based carrier comprises about 1.0% to about 2.0% aggregation-reducing lipid on a molar basis, such as 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%, in particular about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, for example 1.7% (based on 100% total moles of lipid in the lipid-based carrier). In other embodiments, the lipid-based carrier comprises about 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, for example 2.5%, aggregation-reducing lipid on a molar basis (based on 100% total moles of lipid in the lipid-based carrier). In various embodiments, the molar ratio of cationic lipid to aggregation-reducing lipid ranges from about 100:1 to about 25:1.

[0366] <Neutral lipids> In some embodiments, the lipid-based carrier (eg, LNP) comprises a neutral lipid or a phospholipid.

[0367] The term "neutral lipid" refers to any one of a number of lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Suitable neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of a neutral lipid for use in the particles described herein is generally guided by considerations, for example, of the size of the lipid particle and the stability of the lipid particle in the bloodstream. For example, the neutral lipid is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In one embodiment, the neutral lipid comprises a saturated fatty acid having a carbon chain length in the range of C10-C20. In another embodiment, neutral lipids having mono- or di-unsaturated fatty acids having a carbon chain length in the range of C10-C20 are used. Additionally, neutral lipids having a mixture of saturated and unsaturated fatty acid chains may also be used.

[0368] In some embodiments, the lipid-based carrier comprises one or more neutral lipids, wherein the neutral lipids are distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexylamine (DOPE). The phosphatidylethanolamine is selected from the group consisting of 1,2-diphenylphosphatidylethanolamine (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-diereidoyl-sn-glycero-3-phosphoethanolamine (transDOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), or a mixture thereof.

[0369] In some embodiments, the neutral lipid of the lipid-based carrier (eg, LNP) of the pharmaceutical composition is selected from or derived from 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC).

[0370] In other embodiments, the neutral lipid of the lipid-based carrier (eg, LNP) of the pharmaceutical composition is selected from or derived from 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE).

[0371] Thus, in some embodiments, the lipid-based carrier (e.g., LNP) of the pharmaceutical composition comprises a neutral lipid selected from or derived from DSPC, DHPC, or DPhyPE.

[0372] In some embodiments, the lipid-based carrier, eg, the LNP of the pharmaceutical composition, comprises a neutral lipid selected from or derived from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0373] In various embodiments, the molar ratio of cationic lipid to neutral lipid in the lipid-based carrier ranges from about 2:1 to about 8:1.

[0374] The neutral lipid is, for example, about 5 mol% to about 90 mol%, about 5 mol% to about 10 mol%, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 mol% of the total lipid present in the lipid-based carrier. In one embodiment, the lipid-based carrier contains about 0% to about 15% or 45% neutral lipid on a molar basis, for example, about 3% to about 12% or about 5% to about 10% neutral lipid. For example, the lipid-based carrier may contain about 15%, about 10%, about 7.5%, or about 7.1% neutral lipid on a molar basis (based on 100% total moles of lipid in the lipid-based carrier).

[0375] <Steroids, steroid analogs or sterols> In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, a steroid analog, or a sterol.

[0376] Suitably, the steroid, steroid analog or sterol may be derived from or selected from cholesterol, cholesteryl hemisuccinate (CHEMS) and their derivatives. In another embodiment, 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), such as a compound having the structure of formula I as disclosed in claim 1 of WO2020061332; the disclosure of WO2020061332, particularly the disclosure of formula I and plant sterols, is incorporated herein by reference. In a further embodiment, the steroid is an imidazole cholesterol ester or "ICE", as disclosed in paragraphs

[0320] and

[0339] to

[0340] of WO2019226925; WO2019226925 is incorporated herein by reference in its entirety.

[0377] In some embodiments, the lipid-based carrier of the pharmaceutical composition comprises a steroid, a steroid analog, or a sterol selected from, derived from, or including cholesterol.

[0378] The molar ratio of cationic lipid to cholesterol in the lipid-based carrier can range from about 2: 1 to about 1: 1. In some embodiments, the cholesterol can be PEGylated.

[0379] In some embodiments, the lipid-based carrier comprises about 10 mol% to about 60 mol% or about 25 mol% to about 40 mol% of sterol (based on 100% of the total moles of lipids in the lipid-based carrier). In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol% of the total lipids present in the lipid-based carrier. In another embodiment, the lipid-based carrier comprises about 5% to about 50% of sterol on a molar basis, for example 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% on a molar basis (based on 100% of the total moles of lipids in the lipid-based carrier). In some embodiments, the lipid-based carrier comprises about 28%, about 29%, or about 30% sterol (based on 100% total moles of lipid in the lipid-based carrier). In some embodiments, the lipid-based carrier comprises about 40.9% sterol (based on 100% total moles of lipid in the lipid-based carrier).

[0380] References to other suitable cationic or ionizable, neutral, steroid / sterol or aggregation-reducing lipids: Other suitable cationic or ionizable, neutral, steroid / sterol or aggregation reducing lipids can be found in WO2010053572, WO2011068810, WO2012170889, WO2012170930, WO2013052523, WO2013090648, WO2013149140, WO2013149141, WO2013151663, WO2013151664, WO2013151665, WO2013151666, WO2013151667, WO2013151668, WO2013151669, WO20131 51670, WO2013151671WO2013151672, WO2013151736, WO2013185069, WO2014081507, WO2014089486, WO2014093924, WO2014144196, WO2014152211, WO 2014152774, WO2014152940, WO2014159813, WO2014164253, WO2015061461, WO2015061467, WO2015061500, WO2015074085, WO2015105926, WO20151482 47, WO2015164674, WO2015184256, WO2015199952, WO2015200465, WO2016004318, WO2016022914, WO2016036902, WO2016081029, WO2016118724, WO20 16118725, WO2016176330, WO2017004143, WO2017019935, WO2017023817, WO2017031232, WO2017049074, WO2017049245, WO2017070601, WO201707061 3, WO2017070616, WO2017070618, WO2017070620, WO2017070622, WO2017070623, WO2017070624, WO2017070626, WO2017075038, WO2017075531, WO201 7099823, WO2017106799, WO2017112865, WO2017117528, WO2017117530, WO2017180917, WO2017201325, 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, WO2010054406, WO2010080724, WO2010088537, WO2010129709, WO201021865, WO2011022460, W O2011043913, WO2011090965, WO2011149733, WO2011153120, WO2011153493, WO2012040184, W O2012044638, WO2012054365, WO2012061259, WO2013063468, WO2013086354, WO2013086373, US 7893302B2, US7404969B2, US8158601B2, US8283333B2, US8466122B2, US8569256B2, US201000 36115, US20110256175, US20120202871, US20120027803, US20120128760, US20130064894, US2 US20130129785, US20130150625, US20130178541, US20130225836, and US20140039032; the specific disclosures of the above publications regarding cationic or ionizable, neutral, sterol, or aggregation-reducing lipids suitable for lipid-based carriers are incorporated herein by reference.

[0381] For example, suitable cationic lipids or cationizable or ionizable lipids include DSDMA, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salts), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12 (WO2015200465), 1,2-dioleoyloxy-3-trimethylaminopropane chloride (DOTAP ... 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Di-y-linoleyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5, 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), ICE (imidazole based), HGT500 0, 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-Dilinoleyloxo-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)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3, US20100324120), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N,N 16-Diundecyl-4,7,10,13-tetraazahexadecane-l,16-diamide), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(Dimethylamino)butanoic acid (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z.31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3 phosphoethanolamine (DOPE), GIBCO / BRL, Grand Rapids, MD). Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), manufactured by GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, manufactured by Promega Corp., Madison, Wis.Further suitable cationic or ionizable lipids include, but are not limited to, those described in International Patent Publication WO2010053572 (and in particular, 1,1'-(2-(4-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200) described in paragraph

[0225] of WO20153572) and WO201217093 0, both of which 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 (DLinDAP), 1,2 -Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLin AP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-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).

[0382] Lipid-Based Carrier Compositions In some embodiments, the lipid-based carrier of the pharmaceutical composition, such as an LNP, comprises a coding RNA as defined in the first aspect, a cationic lipid as defined herein, an aggregation-reducing lipid as defined herein, optionally a neutral lipid as defined herein, and optionally a steroid or steroid analogue as defined herein.

[0383] In some embodiments, the lipid-based carrier comprising the coding RNA of the first aspect comprises: (i) at least one cationic or ionizable lipid, e.g., as defined herein; (ii) at least one neutral lipid or phospholipid, e.g., as defined herein; (iii) at least one steroid or steroid analog, e.g., as defined herein; and (iv) at least one aggregation-reducing lipid, e.g., as defined herein.

[0384] In some embodiments, the lipid-based carrier comprising the coding RNA of the first aspect comprises: (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); (ii) at least one neutral lipid selected from or derived from DSPC, DHPC, or DPhyPE; (iii) at least one steroid or steroid analog selected from or derived from cholesterol; and (iv) at least one aggregation-reducing lipid selected from or derived from DMG-PEG2000, C10-PEG2K, Cer8-PEG2K, or ALC-0159; and Here, the lipid-based carrier encapsulates the RNA.

[0385] In some embodiments, the cationic lipid (as defined herein), neutral lipid (as defined herein), steroid or steroid analog (as defined herein), and / or aggregation-reducing lipid (as defined herein) may be combined in various relative ratios.

[0386] In some embodiments, the lipid-based carrier comprises (i)-(iv) in a molar ratio of about 20-60% cationic or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analog, and about 0.5-15% aggregation-reducing lipid, e.g., a polymer-conjugated lipid, e.g., such that the lipid-based carrier encapsulates RNA.

[0387] For example, the ratio of cationic or ionizable lipids to neutral lipids to steroids, or the ratio of steroid analogs to aggregation-reducing lipids can be between about 30-60:20-35:20-30:1-15, 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.

[0388] In some embodiments, the lipid-based carrier, such as a LNP comprising the coding RNA of the first aspect, comprises: (i) at least one cationic lipid selected from SM-102; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from DMG-PEG2000; and wherein the lipid-based carrier encapsulates RNA, e.g., i) through (iv) are in a weight ratio of about 50% cationic lipid, about 10% neutral lipid, about 38.5% steroid or steroid analog, and about 1.5% aggregation-reducing lipid; For example, here, a lipid-based carrier encapsulates RNA.

[0389] In some embodiments, the lipid-based carrier, such as a LNP comprising the coding RNA of the first aspect, comprises: (i) at least one cationic lipid selected from SM-102; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from DMG-PEG2000; and wherein the lipid-based carrier encapsulates RNA, e.g., i) through (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; For example, where a lipid-based carrier encapsulates RNA, a suitable N / P ratio for this formulation is about 4.85 (molar ratio of lipid to RNA).

[0390] In some embodiments, the lipid-based carrier, e.g., the LNP comprising the coding RNA of the first aspect, comprises: (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); (ii) at least one neutral lipid selected from DPhyPE; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from DMG-PEG2000; and Here, the lipid-based carrier encapsulates RNA, and such LNPs are referred to herein as GN-LNPs.

[0391] In one embodiment, the lipid-based carrier, e.g., LNP, comprising the coding RNA of the first aspect, comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159.

[0392] In one embodiment, the lipid based carrier, preferably a LNP comprising the coding RNA of the first aspect, comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159; and Here, the lipid-based carrier encapsulates RNA, e.g., i)-(iv) are in 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. Such LNPs are referred to herein as 315-LNPs.

[0393] In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNPs) having a molar ratio of about 50:10:38.5:1.5, such as 47.5:10:40.8:1.7, or such as 47.4:10:40.9:1.7 (i.e., mol % of cationic lipid (e.g., lipid III-3 (ALC-0315) above), DSPC, cholesterol, and PEG-lipid (e.g., PEG-lipid of formula (IVa) above where n=49, e.g., PEG-lipid of formula (IVa) above where n=45 (ALC-0159) above); solubilized in ethanol).

[0394] In one embodiment, the lipid-based carrier, e.g., LNP, comprising the coding RNA of the first aspect, comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159; and wherein the lipid-based carrier encapsulates RNA, for example, (i)-(iv) are about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, for example, wherein the lipid-based carrier encapsulates RNA, wherein the RNA is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973 The RNA sequence may be identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, any one of, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1220, 1223-1245, or 1248-1270, or a fragment or variant thereof, or may consist of the RNA sequence. In such an embodiment, the RNA is, for example, an mRNA comprising a cap1 structure and an RNA sequence that is not chemically modified (e.g., is composed of unmodified ribonucleotides).

[0395] An mRNA sequence in that context is, for example, SEQ ID NO: 829, 679, or any fragment or variant thereof. Other mRNA sequences in that context are SEQ ID NO: 834, 684, or any fragment or variant thereof. Other mRNA sequences in that context are SEQ ID NO: 833, 683, or any fragment or variant thereof.

[0396] In some embodiments, the lipid-based carrier, e.g., the LNP comprising the coding RNA of the first aspect, comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159; and wherein the lipid-based carrier encapsulates RNA, for example, (i)-(iv) are about 47.4% cationic lipid, about 10% neutral lipid, about 40.9% steroid or steroid analog, and about 1.7% aggregation-reducing lipid, for example, wherein the lipid-based carrier encapsulates RNA, wherein the RNA is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, 1271-1276, or a fragment or variant thereof, or comprises or consists of an RNA sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the RNA sequences. In such an embodiment, the RNA is, for example, an mRNA comprising a cap1 structure and an RNA sequence in which all uracils are replaced with pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0397] An mRNA sequence in this context is, for example, SEQ ID NO: 829, 679, 1271, 1274, or any fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 834, 684, 1273, 1276, or any fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 833, 683, 1272, 1275, or any fragment or variant thereof.

[0398] In some embodiments, the wt / wt ratio of lipid to RNA in the lipid-based carrier is about 10:1 to about 60:1, e.g., about 40:1. In some embodiments, the wt / wt ratio of lipid to RNA is about 20:1 to about 30:1, e.g., about 25:1. In other embodiments, the wt / wt ratio of lipid to RNA is in the range of 20 to 60, e.g., about 3 to about 15, about 5 to about 13, about 4 to about 8, or about 7 to about 11.

[0399] The amount of lipids comprised in the lipid-based carrier may be selected taking into account the amount of RNA cargo. In one embodiment, these amounts are selected so that the lipid-based carrier encapsulating the RNA results in an N / P ratio in the range of 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 may be calculated, for example, on the basis that 1 μg of RNA typically contains about 3 nmol of phosphate residues, when the RNA exhibits a statistical distribution of bases. The "N" value of a lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic groups and - if present - cationizable groups.

[0400] In embodiments, the N / P ratio can range from about 1 to about 50. In other embodiments, the range is from about 1 to about 20, e.g., from about 1 to about 15, from about 1 to about 10, or from about 5 to about 7. For "GN-LNP", a preferred N / P (molar ratio of lipid to RNA) is about 14 or about 17. For "315-LNP", a preferred N / P (molar ratio of lipid to RNA) is about 6. Another preferred N / P ratio is about 4.85 or 5 (molar ratio of lipid to RNA).

[0401] In various embodiments, the pharmaceutical composition comprises a lipid-based carrier (that encapsulates the RNA) having a defined size (particle size, uniform size distribution).

[0402] In this specification, the size of lipid-based carrier of pharmaceutical composition is typically described as Z-average size. The term "average diameter", "mean diameter", "diameter" or "size" of particles (e.g. lipid-based carrier) is used synonymously with the value of Z-average. The term "Z-average size" refers to the average diameter of particles measured by dynamic light scattering (DLS) by data analysis using the so-called cumulant algorithm, which results in the so-called Z-average having the dimension of length and the polydispersity index (PI) being dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321).

[0403] The term "dynamic light scattering" or "DLS" refers to a method for analyzing particles in a liquid, where the liquid is typically illuminated with a monochromatic light source and the light scattered by the particles in the liquid is detected. Thus, DLS can be used to measure particle size in a liquid. Suitable DLS protocols are known in the art. DLS instruments are commercially available (e.g., Zetasizer Nano Series, Malvern Instruments, Worcestershire, UK, etc.). DLS instruments employ either a 90° detector (e.g., DynaPro® NanoStar® from Wyatt Technology or Zetasizer Nano S90® from Malvern Instruments) or a backscattering detection system with 173° (e.g., Zetasizer Nano S® from Malvern Instruments) and 158° (DynaPro Plate Reader® from Malvern Instruments), which are close to 180° incident light. Typically, DLS measurements are performed at a temperature of about 25°C. DLS is also used in the context of the present disclosure to measure the polydispersity index (PDI) and / or main peak diameter of lipid-based carriers incorporating RNA.

[0404] In various embodiments, the lipid-based carrier of the pharmaceutical composition in which RNA is encapsulated has a diameter of about 50 nm to 200 nm, about 50 nm to 190 nm, about 50 nm to 180 nm, about 50 nm to 170 nm, about 50 nm to 160 nm, about 50 nm to 150 nm, about 50 nm to 140 nm, about 50 nm to 130 nm, about 50 nm to 120 nm, about 50 nm to 110 nm, about 50 nm to 100 nm, about 50 nm to 90 nm, about 50 nm to 80 nm, about 50 nm to 70 nm, about 50 nm to 60 nm, about 60 nm to 200 nm, about 60 nm to 190 nm, about 60 nm to 180 nm, about 60 nm to 170 nm, about 60 nm to 160 nm, and having a Z-average size in the range of about 60 nm to 150 nm, about 60 nm to 140 nm, about 60 nm to 130 nm, about 60 nm to 120 nm, about 60 nm to 110 nm, about 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, or 60 nm to 70 nm, for example, 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.

[0405] In one embodiment, the lipid-based carrier of the pharmaceutical composition encapsulating the RNA has a Z-average size in the range of about 50 nm to 200 nm, such as in the range of about 50 nm to 150 nm, such as in the range of about 50 nm to 120 nm.

[0406] Suitably, the pharmaceutical composition contains less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of lipid-based carriers having a particle size greater than about 500 nm.

[0407] Preferably, the pharmaceutical composition contains less than about 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of LNPs having a particle size smaller than about 20 nm.

[0408] Preferably, at least about 80%, 85%, 90%, 95% of the lipid-based carrier of the composition has a spherical form.

[0409] In embodiments, the polydispersity index (PDI) of the lipid-based carrier is typically in the range of 0.1 to 0.5. In certain embodiments, the PDI is less than 0.2. Typically, the PDI is determined by dynamic light scattering.

[0410] In some embodiments, 80% of the RNA contained in the pharmaceutical composition is encapsulated in the lipid-based carrier, for example 85% of the RNA contained in the pharmaceutical composition is encapsulated in the lipid-based carrier, for example 90% of the RNA contained in the pharmaceutical composition is encapsulated in the lipid-based carrier, for example 95% of the RNA contained in the pharmaceutical composition is encapsulated in the lipid-based carrier. The percentage of encapsulation can be determined by the RiboGreen assay, as is known in the art.

[0411] According to some embodiments, the lipid-based carrier, for example encapsulating or containing RNA, is purified by at least one purification step, for example at least one TFF step, and / or at least one clarification step, and / or at least one filtration step.

[0412] <Antagonist of RNA-sensing pattern recognition receptor> In some embodiments, the pharmaceutical composition comprises at least one antagonist of at least one RNA-sensing pattern recognition receptor. Such antagonists can be co-formulated, for example, in a lipid-based carrier as defined herein.

[0413] 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.

[0414] In that context, in some embodiments, 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.

[0415] In embodiments in that context, the at least one antagonist of the at least one RNA sensing pattern recognition receptor is selected from a nucleotide, a nucleotide analog, a nucleic acid, a peptide, a protein, a small molecule, a lipid, or a fragment, variant or derivative of any of these.

[0416] In that context, in some embodiments, 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.

[0417] In an embodiment in 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 selected from the group consisting of SEQ ID NOs: 85-212 of WO2021028439, or a fragment of any of these sequences, 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 said nucleic acid sequence.

[0418] 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 selected from the group consisting of SEQ ID NOs: 85-87, 149-212 of WO2021028439, or a fragment of any of these sequences, 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 said nucleic acid sequence.

[0419] A suitable antagonist of at least one RNA-sensing pattern recognition receptor in the context of the present disclosure is 5'-GAG CGmG CCA-3' (sequence number 85 of WO2021028439), or a fragment thereof.

[0420] In that context, in some embodiments, 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 preferably in the range of about 1:1, to about 100:1, or in the range of about 20:1, to about 80:1.

[0421] In that context, in some embodiments, the weight to weight ratio of at least one RNA encoding an antigenic peptide or protein, as defined herein, to at least one antagonist of at least one RNA sensing pattern recognition receptor, as defined herein, is preferably in the range of about 1:1, to about 1:30, or in the range of about 1:2, to about 1:10.

[0422] In an embodiment in that 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, e.g., formulated separately in a lipid-based carrier, as defined herein.

[0423] In some embodiments in that 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, e.g., co-formulated in a lipid-based carrier, as defined herein.

[0424] In embodiments of compositions comprising at least one antagonist of at least one RNA sensing pattern recognition receptor, at least one RNA encoding an antigenic peptide or protein, as defined herein, does not contain chemically modified nucleotides, e.g., pseudouridine (ψ) or N1-methylpseudouridine (m1ψ), as defined herein.

[0425] <Presentation> In some embodiments, the pharmaceutical composition is freeze dried, spray dried or spray freeze dried.

[0426] Thus, the pharmaceutical composition can be freeze-dried (e.g., according to WO2016165831 or WO2011069586) to obtain a temperature-stable composition. The pharmaceutical composition can also be dried using spray drying or spray freeze drying (e.g., according to WO2016184575 or WO2016184576) to obtain a temperature-stable composition (powder) as defined herein.

[0427] The cryoprotectant for freeze-drying and / or spray-drying can be selected from trehalose, sucrose, mannose, dextran and inulin.A preferred cryoprotectant is sucrose, optionally including a further cryoprotectant.Another preferred cryoprotectant is trehalose, optionally including a further cryoprotectant.Thus, the pharmaceutical composition may include at least one cryoprotectant.

[0428] In some embodiments, the pharmaceutical composition is a liquid composition or a dry composition, such as a lyophilized / freeze-dried composition, that can be reconstituted in a liquid carrier.

[0429] In some embodiments, the pharmaceutical composition (or liquid carrier) comprises a sugar at a concentration of about 50 mM to about 300 mM, for example, sucrose at a concentration of about 150 mM.

[0430] In some embodiments, the pharmaceutical composition (or liquid carrier) comprises a salt at a concentration of about 10 mM to about 200 mM, for example, NaCl at a concentration of about 75 mM.

[0431] In some embodiments, the pharmaceutical composition (or liquid carrier) contains a buffer, such as Na, at a concentration of 1 mM to about 100 mM. 2 HPO 4 , Na 3 PO 4 Or Tris(Trometamol). In another embodiment, the pharmaceutical composition (or liquid carrier) comprises about 2.4 mM Tris(Trometamol), about 1.4 mM glacial acetic acid, about 3.9 mM acetic acid, and about 254 mM sugar.

[0432] In some embodiments, the pharmaceutical composition (or liquid carrier) has a pH in the range of about pH 7.0 to about pH 8.0, for example about pH 7.4.

[0433] (3: Vaccine containing coding RNA encoding an antigenic polypeptide that is or is derived from E. coli FimH) In a third aspect, there is provided a vaccine against E. coli.

[0434] In particular, the embodiments relating to the composition of the second aspect may be read and understood as preferred embodiments of the vaccine of the third aspect as well. Also, the embodiments relating to the vaccine of the third aspect may be read and understood as preferred embodiments of the composition of the second aspect as well. Furthermore, the features and embodiments described in the context of the first aspect (the coding RNA of the present disclosure) must be read and understood as preferred embodiments of the vaccine of the third aspect.

[0435] In some embodiments, the vaccine comprises a coding RNA of the first aspect or at least one composition of the second aspect.

[0436] The term "vaccine" will be recognized and understood by those skilled in the art and contemplates, for example, a prophylactic or therapeutic material that provides at least one epitope or antigen, e.g., 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 encoded RNA described above, which comprises 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 encoded RNA of the first aspect).

[0437] 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.

[0438] In some embodiments, administration of the vaccine, e.g., the vaccine to a subject, induces a humoral immune response against E. coli. In one embodiment, the aforementioned humoral immune response is against E. coli FimH. In one embodiment, administration of the vaccine, e.g., the vaccine to a subject, induces a humoral immune response against E. coli, e.g., a humoral immune response against E. coli FimH, in the urine of the subject upon administration of the vaccine.

[0439] In one embodiment, administration of the vaccine, e.g., the vaccine to a subject, induces a neutralizing antibody titer against E. coli. In one embodiment, the aforementioned antibody is an IgG antibody. In one embodiment, the aforementioned antibody is against E. coli FimH. In one embodiment, administration of the vaccine, e.g., the vaccine to a subject, induces a neutralizing antibody titer against E. coli, e.g., a neutralizing antibody titer against E. coli FimH, in the urine of the subject upon administration of the vaccine.

[0440] In one embodiment, the vaccine of the present disclosure induces antibodies capable of inhibiting bacterial adhesion to urothelial cells. Suitable methods for measuring inhibition of bacterial adhesion are described herein and in the Examples.

[0441] Methods for measuring bacterial adhesion are known in the art.Suitably, the methods described in Thomas WE, et al. Cell. 2002 Jun 28;109(7):913-23 (hereby incorporated by reference); or Hartmann M, et al. FEBS Lett. 2012 May 21;586(10):1459-65 (hereby incorporated by reference); or Falk P, et sl. Methods Cell Biol. 1994;45:165-92 (hereby incorporated by reference); or Garcia Mendez KB, et al. Int J Exp Pathol. 2016 Apr;97(2):194-201 (hereby incorporated by reference) can be used. In one embodiment, bacterial adhesion is measured (simply) using the BAI assay as follows and described in the Examples: UPEC strains engineered to express the mCherry fluorescent marker are incubated with a monolayer of SV-HUC-1(ATTCC) in a 96-well plate for 30 minutes in the presence of specific serum against FimH derivatives or positive / negative controls. After adhesion, cells are washed extensively to remove unbound bacteria and fixed with formaldehyde. Finally, the specific fluorescent signal associated with the attached bacteria is recorded using an automated high-content screening microscope (Opera Phenix) and quantified with Harmony software.

[0442] In some embodiments, the immune response is effective in preventing or treating one or more symptoms associated with UTI in a subject in need thereof.In certain embodiments, the immune response is effective in preventing or reducing symptoms of UTI, for example, in at least 30%, for example at least 40%, for example at least 50% of subjects administered the vaccine.Symptoms of UTI may vary depending on the nature of infection and may include, but are not limited to, difficulty in urinating, increased frequency or urgency, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea.

[0443] In certain embodiments, immune response is effective for preventing or reducing organ failure caused by UTI.In certain embodiments, immune response is effective for reducing the possibility of hospitalization of the subject suffering from UTI.In some embodiments, immune response is effective for shortening the hospitalization period of the subject suffering from UTI.

[0444] The vaccines may be used in accordance with the present disclosure for veterinary medical purposes (mammalian, vertebrate, or avian) as well as human medical purposes.

[0445] Suitable routes of administration of the vaccine include nasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal, or subcutaneous. Thus, in some embodiments, the vaccine is suitable for nasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal, or subcutaneous administration.

[0446] In one embodiment, the vaccine is suitable for intramuscular administration.

[0447] In one embodiment, the vaccine comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159; and wherein the lipid-based carrier encapsulates the RNA of the present disclosure, e.g., wherein (i)-(iv) comprise 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 is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 99 The RNA sequence may be identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, any one of SEQ ID NOs: 8-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, or a fragment or variant thereof, or may consist of the RNA sequence. In such an embodiment, the RNA is, for example, an mRNA comprising a cap1 structure, and the RNA sequence is not chemically modified (e.g., consists of unmodified ribonucleotides). The mRNA sequence in that context is, for example, SEQ ID NO: 829, 679, or a fragment or variant of any of these. Other mRNA sequences in that context are SEQ ID NO: 834 684, or any fragment or variant thereof. Other mRNA sequences in that context are SEQ ID NO: 833, 683, or any fragment or variant thereof.

[0448] In one embodiment, the vaccine comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159; and wherein the lipid-based carrier encapsulates the RNA of the present disclosure, e.g., wherein i)-(iv) comprise 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 is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020 , 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, 1274-1276, or a fragment or variant thereof, or comprises or consists of an RNA sequence that is identical to or is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the following: An mRNA sequence in this context is, for example, SEQ ID NO: 829, 679, 1274, or any fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 834, 684, 1276, or any fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 833, 683, 1275, or any fragment or variant thereof.

[0449] In one embodiment, the vaccine comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid or steroid analog selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159; and wherein the lipid-based carrier encapsulates the RNA of the present disclosure, e.g., wherein i)-(iv) comprise 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 is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020 , 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1220, 1223-1245, 1248-1270, 1271-1273, or a fragment or variant thereof, or comprises or consists of an RNA sequence that is identical to or is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the following: An mRNA sequence in this context is, for example, SEQ ID NO: 829, 679, 1271, or any fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 834, 684, 1273, or any fragment or variant thereof. Other mRNA sequences in this context are SEQ ID NO: 833, 683, 1272, or any fragment or variant thereof.

[0450] (4: Kit or parts kit) In a fourth aspect, there is provided a kit or kit of parts suitable for treating or preventing an infection caused by Escherichia coli. Of note, the RNA-related embodiments of the first aspect may be read and understood as preferred embodiments of the kit or kit of parts of the fourth aspect as well. Also, the pharmaceutical composition of the second aspect or the vaccine of the third aspect may be read and understood as preferred embodiments of the kit or kit of parts of the fourth aspect as well. In some embodiments, the kit or kit of parts comprises at least one coding RNA of the first aspect, at least one composition of the second aspect, and / or at least one vaccine of the third aspect.

[0451] In addition, the kit or kit of parts may include a liquid vehicle for solubilization, and / or technical instructions providing information regarding administration and dosage of the components.

[0452] The kit may further comprise additional components as described in the context of the composition of the second aspect, and / or the vaccine of the third aspect.

[0453] The technical instructions of the aforementioned kits may include information regarding administration and dosage, as well as patient groups. Such kits, e.g. kits of parts, may be applied for example for any application or use mentioned herein, such as for use of the RNA of the first aspect, the composition of the second aspect, the vaccine of the third aspect, for the treatment or prevention of an infection or disease caused by Escherichia coli, or a disorder related thereto.

[0454] Suitably, the coding RNA, the composition or the vaccine are provided in separate parts of the kit.

[0455] In some embodiments, the coding RNA, composition, or vaccine is lyophilized or spray (freeze) dried.

[0456] In embodiments in which the pharmaceutical composition is provided as a freeze-dried or spray-freeze-dried or spray-dried composition, the kit or kit-of-parts may suitably include a buffer for reconstitution of the freeze-dried or spray-freeze-dried or spray-dried composition.

[0457] Thus, the kit or kit of parts may further comprise buffers for the reconstitution and / or dilution of the RNA, composition or vaccine.

[0458] In some embodiments, the buffer for reconstitution and / or dilution is a sterile buffer. In some embodiments, the buffer contains a salt, such as NaCl, optionally at a concentration of about 0.9%. Such a buffer can optionally contain a preservative.

[0459] In some embodiments, a kit or kit-of-parts as defined herein comprises at least one syringe.

[0460] In some embodiments, the kit or kit of parts comprises the following components: a) At least one container or vial containing a composition or vaccine as defined herein. b) optionally at least one dilution container or vial containing a sterile dilution buffer, preferably a buffer containing NaCl, and optionally containing a preservative; c) optionally, at least one means for transferring the composition or vaccine from the container to a dilution container; and d) At least one syringe for administering the composition or vaccine to a subject, e.g., a syringe configured for intramuscular administration to a human subject.

[0461] (5: Medical Use) In a further aspect there is provided a medical use of a coding RNA as defined herein, a composition as defined herein, a vaccine as defined herein, or a kit or kit-of-parts as defined herein.

[0462] It should be noted that the embodiments related to the previous aspects may likewise be read and understood as preferred embodiments for medical applications of the present invention (and vice versa).

[0463] Thus, there is provided a coding RNA of the present disclosure, and / or a composition of the present disclosure, and / or a vaccine of the present disclosure, and / or a kit or kit of parts of the present disclosure, for use as a medicament. In a further aspect, there is provided a secondary medical use of a coding RNA as defined herein, a composition as defined herein, a vaccine as defined herein, or a kit or kit of parts as defined herein.

[0464] Thus, there is provided a coding RNA of the present disclosure, and / or a composition of the present disclosure, and / or a vaccine of the present disclosure, and / or a kit or kit of parts of the present disclosure for use in treating or preventing a disease caused by 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.

[0465] In some embodiments, the coding 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 parts of the present disclosure are provided for use in 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 symptoms of UTI, for example, in at least 30%, such as at least 40%, such as at least 50% of subjects administered the vaccine.Symptoms of UTI may vary depending on the nature of infection and may include, but are not limited to, difficulty in urinating, increased frequency or urgency, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea.

[0466] In certain embodiments, the use is for preventing or reducing organ failure caused by UTI.In certain embodiments, the use is for reducing the possibility of the subject suffering from UTI being hospitalized.In some embodiments, the use is for shortening the duration of hospitalization of the subject suffering from UTI.

[0467] In the context of medical applications, the coding 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 parts of the present disclosure may, for example, be administered locally.

[0468] In that context, administration can be intranasal, oral, sublingual, intravenous, intramuscular, intradermal, transdermal, or subcutaneous, such as intramuscular.

[0469] In one embodiment, administration can be by conventional needle injection, for example intramuscular injection.

[0470] In some embodiments, the use may be for human medical purposes or for veterinary medical purposes, hi one embodiment, the use may be for human medical purposes.

[0471] In some embodiments, the use is for naive subjects, i.e. subjects who do not have E. coli infection or have never had UTI before.In some embodiments, the use is for subjects who are at risk of acquiring or developing UTI before symptoms appear or symptoms become severe, such as immunocompromised or immunodeficient individuals.In certain embodiments, the use is for subjects who have UTI or have previously been diagnosed with UTI.

[0472] As used herein, the term "at risk subject" refers to a person who is more susceptible to a condition than the average human adult population. Examples of "at risk subjects" include those who have one or more risk factors for UTI, including but not limited to elderly people, people with immune disorders, 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 urinary system procedures such as urinary tract surgery, etc.

[0473] In certain embodiments, the use is for subjects with UPEC infection or previously diagnosed with UPEC infection. In some embodiments, the use is for subjects suffering from recurrent UTI. In some embodiments, the use is for subjects suffering from recurrent UTI but healthy at the time of treatment. In some embodiments, the use is for subjects with or at risk of E. coliemia or sepsis. In some embodiments, the subject to which the composition or method of the present disclosure is administered or applied has a condition that requires the use of a catheter, such as a urinary catheter, which leads to the risk of CAUTI, i.e., catheter-associated UTI. In some embodiments, the use is for subjects undergoing a pre-planned surgery.

[0474] In certain embodiments, the use is in human adults over 50 years of age. In certain embodiments, the use is in human adults over 55 years of age, over 60 years of age, or over 65 years of age. In certain embodiments, the use is in females between about 16 and 50 years of age, such as females between about 16 and 35 years of age. In certain embodiments, the use is in subjects with diabetes.

[0475] (6:Treatment method) In a further aspect, a method for treating or preventing a disease caused by E. coli is provided. The method comprises administering to a subject in need thereof an effective amount of a coding 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 of parts according to the fourth aspect. Also provided is a method for inducing an immune response in a subject in need thereof. Advantageously, the immune response is effective to prevent or treat one or more symptoms associated with UTI in a subject in need thereof.

[0476] There is also provided the use of a coding 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 of parts according to the fourth aspect for enhancing an immune response in a mammal, e.g. for treating and / or preventing a disease. There is also provided the use of a coding 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 of parts according to the fourth aspect for the manufacture of a medicament for enhancing an immune response in a mammal, e.g. for treating and / or preventing a disease, e.g. E. coli infection.

[0477] It should be noted that the embodiments related to the above-mentioned aspects may also be read and understood as preferred embodiments for medical applications of the present disclosure.

[0478] Additionally, certain features and embodiments relating to the treatment methods provided herein may also be applied to the medical applications of the present disclosure, and vice versa.

[0479] Preventing (suppressing) or treating a disease, particularly an infection caused by E. coli, refers to suppressing the full development of a disease or condition in a subject at risk of a disease, such as an infection. "Treatment" refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "amelioration" in relation to a disease or pathological condition refers to an observable beneficial effect of treatment. Suppressing a disease can include preventing a disease or reducing the risk of a disease, such as preventing E. coli infection or reducing the risk of infection. A beneficial effect can be evidenced, for example, by a delay in the onset of clinical symptoms of a disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of a disease, a delay in the progression of a disease, a reduction in viral load, an improvement in the overall health or well-being of the subject, or other parameters specific to a particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not show signs of a disease or who shows only early signs, with the aim of reducing the risk of developing pathology.

[0480] In some embodiments, a method of treating or preventing a disease, disorder or condition is provided, the method comprising administering or administering a coding RNA of the present disclosure, and / or a composition of the present disclosure, and / or a vaccine of the present disclosure, and / or a kit or kit of parts of the present disclosure to a subject in need thereof.

[0481] In some embodiments, the disease, disorder or condition is an infectious disease caused by E. coli or a disorder associated with such an infectious disease.

[0482] In certain embodiments, the method of inducing an immune response in a subject of the present disclosure provides for vaccination of the subject to induce protective immunity against infection with an E. coli strain expressing FimH.

[0483] In one embodiment, the method induces a humoral immune response against E. coli.

[0484] In one embodiment, the aforementioned humoral immune response is against E. coli FimH. In one embodiment, the method induces a humoral immune response against E. coli, for example against E. coli FimH, in the urine of the subject.

[0485] In one embodiment, the method induces neutralizing antibody titers against E. coli. In one embodiment, the aforementioned antibody is an IgG antibody. In one embodiment, the aforementioned antibody is against E. coli FimH. In one embodiment, the method induces neutralizing antibody titers against E. coli, e.g., E. coli FimH, in the urine of the subject.

[0486] In one embodiment, the disclosed method elicits antibodies capable of inhibiting bacterial adhesion to urothelial cells. Suitable methods for measuring inhibition of bacterial adhesion are described herein and in the Examples.

[0487] In certain embodiments, the immune response induced in a subject following administration of a coding RNA, pharmaceutical composition or vaccine according to the present disclosure is effective in resolving a UTI.

[0488] In certain embodiments, the immune response induced in a subject after administration of a coding RNA, pharmaceutical composition or vaccine according to the present disclosure is effective in preventing or reducing symptoms of UTI, for example, in at least 30%, such as at least 40%, such as at least 50% of subjects administered the composition. Symptoms of UTI may vary depending on the nature of the infection and may include, but are not limited to, difficulty in urinating, increased frequency or urgency, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea.

[0489] In certain embodiments, the immune response induced in a subject after administration of the coding RNA, pharmaceutical composition or vaccine of the present disclosure is effective for preventing or reducing organ failure caused by UTI.In certain embodiments, the immune response induced in a subject after administration of the coding RNA, pharmaceutical composition or vaccine of the present disclosure is effective for reducing the possibility of hospitalization of a subject suffering from UTI.In some embodiments, the immune response induced in a subject after administration of the composition of the present disclosure is effective for shortening the hospitalization period of a subject suffering from UTI.

[0490] In some embodiments, application or administration is via intranasal administration, oral administration, sublingual administration, intramuscular injection, intravenous injection, transdermal injection, or intradermal injection. In one embodiment, application or administration is via intramuscular injection.

[0491] As used herein in the context of the present disclosure, the term "effective amount" refers to an amount sufficient to induce desired immune effect or immune response in a subject.In certain embodiments, "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) prevent the progression or onset of UTI or symptoms associated therewith; (ii) prevent or reduce the recurrence of UTI or symptoms associated therewith; (iii) prevent, reduce or improve the severity of UTI or symptoms associated therewith; (iv) reduce the duration of infectious UTI or symptoms associated therewith; (v) prevent the clinical progression of UTI or symptoms associated therewith; (vi) cause the regression of UTI or symptoms associated therewith; (vii) prevent or reduce organ failure caused by UTI; (viii) reduce the chance or frequency of hospitalization of a subject with UTI; (ix) reduce the duration of hospitalization of a subject with UTI; (x) eliminate UTI; and / or (xi) enhance or improve the preventive or therapeutic effect of another therapy.

[0492] The selection of a specific effective dose can be determined by a person skilled in the art (e.g., through clinical trials) based on the consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the medical history of the subject, the subject's physical condition, such as the subject's age, weight and / or immune status, the composition to be administered, and other factors known to those skilled in the art. The exact dose to be employed in the formulation also depends on the route of administration, the severity of the disease, and should be determined according to the judgment of the person skilled in the art and the circumstances of each patient. Effective doses can be estimated from dose-response curves obtained from in vitro or animal model test systems.

[0493] In one embodiment, the subject in need is a mammalian subject, such as a human subject.

[0494] In certain embodiments, the disclosed method is administered or applied to a naive subject, i.e., a subject who does not have an E. coli infection or has never had a UTI before. In one embodiment, the disclosed composition or method is administered or applied to a subject who is at risk of acquiring or developing a UTI, such as an immunocompromised or immunodeficient individual, before symptoms appear or become severe. In certain embodiments, the disclosed method is administered or applied to a subject who has a UTI or has previously been diagnosed with a UTI.

[0495] In certain embodiments, the disclosed method is administered or applied to a subject with or previously diagnosed with a UPEC infection. In some embodiments, the disclosed composition or method is administered or applied to a subject suffering from recurrent UTI. In some embodiments, the disclosed method is administered or applied to a subject suffering from recurrent UTI but healthy at the time of treatment. In some embodiments, the disclosed method is administered or applied to a subject with or at risk of E. coliemia or sepsis. In some embodiments, the subject to which the disclosed method is applied has a condition that requires the use of a catheter, such as a urinary catheter, which leads to the risk of CAUTI, i.e., catheter-associated UTI. In some embodiments, the disclosed method is applied to a subject undergoing a pre-planned surgery.

[0496] In certain embodiments, the subject that the method of the present disclosure is applied to is a human subject, for example, the human subject that is at risk of having disease UTI.In certain embodiments, the subject that the composition or method of the present disclosure is applied to is a human adult over 50 years old.In certain embodiments, the subject that the method of the present disclosure is applied to is a human adult over 55 years old, over 60 years old, or over 65 years old.

[0497] In certain embodiments, the subject to which the method of the present disclosure is applied is a female between about 16 and 50 years of age, for example, a female between about 16 and 35 years of age. In certain embodiments, the subject to which the method of the present disclosure is applied has diabetes.

[0498] <Brief explanation of the table> Table 1: Sequences (amino acid sequences and coding sequences) Table 2: RNA constructs Table 3: RNA constructs encoding antigen designs used in the examples Table 4: Lipid-based carrier composition in the examples Table 5: RNA constructs used for Western blot analysis (Example 2.1) Table 6: Vaccination regimen (Example 2.2) Table 7: BAI titers of serum antibody responses to E. coli FimH antigen designs (Example 2.3) Table 8: Vaccination regimen (Example 3.1) Table 9: BAI titers of serum antibody responses to E. coli FimH antigen designs (Example 3.2) Table 10: mRNA constructs used for Western blot analysis (Example 4.1) Table 11: Vaccination regimen (Example 4.2) Table 12: BAI titers of serum antibody responses to E. coli FimH anti-antigen designs (Example 4.3) Table 13: Additional sequences of less than 10 specific nucleotides or less than 4 specific amino acids.

[0499] <Numbered embodiment> Below, embodiments of the present invention are presented as a list of numbered embodiments (embodiment 1 to embodiment 110).

[0500] Embodiment 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 fimbria D-mannose specific adhesin (FimH).

[0501] Embodiment 2. The coding RNA of embodiment 1, wherein the E. coli FimH comprises an amino acid sequence identical to or 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 SEQ ID NOs: 177-186, 247-256, or is an immunogenic fragment or immunogenic variant thereof.

[0502] Embodiment 3. The coding RNA of embodiment 1 or 2, wherein the coding sequence further encodes one or more additional peptide or protein elements selected from a donor chain peptide, a signal peptide, an antigen clustering domain, or a transmembrane domain.

[0503] Embodiment 4. The coding RNA of embodiment 3, wherein the one or more further peptide or protein elements are donor chain peptides, and optionally 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 chain peptide.

[0504] Embodiment 5. The coding RNA of embodiment 4, wherein the donor strand peptide comprises or consists of the 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, such as 1, 2, 3 or 4, single amino acid mutations compared to SEQ ID NO: 338 or SEQ ID NO: 339.

[0505] Embodiment 6. The coding RNA of embodiment 4, wherein the donor strand peptide comprises or consists of the amino acid sequence of SEQ ID NO: 338.

[0506] Embodiment 7. The coding RNA according to any one of embodiments 1 to 6, wherein the coding sequence further codes for a peptide linker.

[0507] Embodiment 8. The coding RNA of embodiments 1 to 7, wherein 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 element; and a donor chain peptide.

[0508] Embodiment 9. The coding RNA according to embodiment 7 or 8, wherein the peptide linker comprises any one of SEQ ID NOs: 352 to 358 or consists of said SEQ ID NOs.

[0509] Embodiment 10. The coding RNA according to embodiments 7 to 9, wherein the peptide linker comprises or consists of SEQ ID NO:352.

[0510] Embodiment 11. The coding RNA of any one of embodiments 1 to 10, wherein the antigenic polypeptide is in a low mannose binding affinity conformation.

[0511] Embodiment 12. A coding RNA according to any one of embodiments 1 to 11, wherein the coding sequence further encodes an antigen clustering domain.

[0512] Embodiment 13. The coding RNA of embodiment 12, wherein the antigen clustering domain is selected from or derived from ferritin or lumazine synthase.

[0513] Embodiment 14. The coding RNA according to any one of embodiments 12 or 13, wherein the amino acid sequence of the antigen clustering domain 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 any one of the amino acid sequences SEQ ID NOs: 457-459, 443, 444, or a fragment or variant thereof.

[0514] Embodiment 15. A coding RNA according to any one of embodiments 1 to 11, wherein the coding sequence further codes for a transmembrane domain.

[0515] Embodiment 16. The coding RNA of claim 15, wherein the transmembrane domain is heterologous and optionally is selected or derived from an influenza HA transmembrane domain, such as SEQ ID NO: 478.

[0516] Embodiment 17. A coding RNA according to any one of embodiments 1 to 16, wherein the coding sequence further codes for a signal peptide.

[0517] Embodiment 18. The coding 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), and optionally the amino acid sequence of the signal peptide 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 any one of the amino acid sequences SEQ ID NOs: 394-400, or a fragment or variant thereof.

[0518] Embodiment 19. The coding RNA according to embodiment 17 or 18, wherein the signal peptide is selected from or derived from IgE or IgK, and optionally the amino acid sequence of the signal peptide 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 any one of the amino acid sequences SEQ ID NO: 394, 395, or a fragment or variant thereof.

[0519] Embodiment 20(a). A coding RNA according to any one of embodiments 1 to 19, wherein the coding sequence codes for the following elements, for example from N-terminus to C-terminus: a) signal peptide, antigenic polypeptide; b) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide; c) antigen clustering domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide; d) signal peptides, antigen clustering domains, peptide linkers, antigenic polypeptides, peptide linkers, donor chain peptides; e) a signal peptide, an antigenic polypeptide, a peptide linker, a donor chain peptide, a peptide linker, an antigen clustering domain; or f) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, transmembrane domain.

[0520] Embodiment 20(b). The coding sequence encodes the following elements, for example from N-terminus to C-terminus: a signal peptide, an antigenic polypeptide, a peptide linker, and a donor chain peptide; optionally, the signal peptide is selected from SEQ ID NOs: 394-400, and optionally, the signal peptide is SEQ ID NO: 395; the antigenic polypeptide is selected from SEQ ID NOs: 247-256, and optionally, the antigenic polypeptide is SEQ ID NO: 247; the peptide linker is selected from SEQ ID NOs: 352-354, and optionally, the peptide linker is SEQ ID NO: 352; the donor chain peptide is selected from SEQ ID NOs: 338, 339, and optionally, the donor chain peptide is SEQ ID NO: 338.

[0521] Embodiment 21. The coding sequence encodes, e.g., from N-terminal to C-terminal, the following elements: a signal peptide, an antigenic polypeptide as defined herein, a (first) peptide linker, a donor chain peptide, a (second) peptide linker; and an antigen clustering domain; optionally, the signal peptide is selected from SEQ ID NOs: 394-400, optionally the signal peptide is SEQ ID NO: 394; the antigenic polypeptide is selected from SEQ ID NOs: 247-256, optionally the antigenic polypeptide is SEQ ID NO: 247; the (first) peptide linker is selected from SEQ ID NO: The coding RNA of any one of embodiments 1 to 19, wherein the antigen clustering domain is selected from SEQ ID NOs: 352 to 354, and optionally the (first) peptide linker is SEQ ID NO: 352; the donor chain peptide is selected from SEQ ID NOs: 338, 339, and optionally the donor chain peptide is SEQ ID NO: 338; the (second) peptide linker is selected from SEQ ID NOs: 355 to 358, and optionally the (second) peptide linker is SEQ ID NO: 355; the antigen clustering domain is selected from SEQ ID NOs: 443, 444, 457 to 459, and optionally the peptide linker is SEQ ID NO: 444 or 459.

[0522] Embodiment 22. A coding RNA according to any one of embodiments 1 to 21, wherein the coding sequence encodes an amino acid sequence identical or 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 SEQ ID NOs: 177-186, 247-256, 498-520, 1277, or an immunogenic fragment or immunogenic variant thereof.

[0523] Embodiment 23(a) A coding RNA according to any one of embodiments 1 to 22, wherein the coding sequence encodes an amino acid sequence identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 504, 508, and 509, or an immunogenic fragment or immunogenic variant thereof.

[0524] Embodiment 23(b). A coding RNA according to any one of embodiments 1 to 22 and 23(a), wherein the coding sequence encodes an amino acid sequence identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 504, or an immunogenic fragment or immunogenic variant thereof.

[0525] Embodiment 23(c). The coding RNA of any one of embodiments 1 to 22, 23(a) and 23(b), wherein the coding sequence encodes an amino acid sequence identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 508, or an immunogenic fragment or immunogenic variant thereof.

[0526] Embodiment 23(d). The coding RNA of any one of embodiments 1 to 22 and 23(a) to 23(c), wherein the coding sequence encodes an amino acid sequence identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 509, or an immunogenic fragment or immunogenic variant thereof.

[0527] Embodiment 24. A coding RNA according to any one of embodiments 1 to 22, wherein the coding sequence comprises a nucleic acid sequence which 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 a sequence according to any one of SEQ ID NOs: 187 to 246, 257 to 316, 523 to 545, 548 to 570, 573 to 595, 598 to 620, 623 to 645, 648 to 670, or a fragment or variant thereof.

[0528] Embodiment 25. The coding RNA according to any one of embodiments 1 to 24, wherein the coding sequence is a codon-modified coding sequence, and 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, and optionally, the at least one codon-modified coding sequence is selected from a C-maximized coding sequence, a CAI-maximized coding sequence, a human codon usage adapted coding sequence, a G / C content modified coding sequence, and a G / C optimized coding sequence, or any combination thereof.

[0529] Embodiment 26(a) The coding RNA according to embodiment 25, wherein the coding sequence comprises at least one nucleic acid sequence identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or variant thereof.

[0530] Embodiment 26(b). A coding RNA according to any one of embodiments 1 to 25 and 26(a), wherein the coding sequence comprises at least one nucleic acid sequence identical or 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 SEQ ID NOs: 529, 533, 534, 554, 558, 559, 579, 583, 584, 604, 608, 609, 629, 633, 634, 654, 658, 659, or a fragment or variant thereof.

[0531] Embodiment 27. A coding RNA according to any one of embodiments 1 to 25, 26(a) and 26(b), wherein the coding sequence is a G / C-optimized coding sequence.

[0532] Embodiment 28. A coding RNA according to embodiment 27, wherein the coding sequence comprises at least one nucleic acid sequence identical or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 523-545, 548-570, 648-670, or a fragment or variant thereof.

[0533] Embodiment 29. A coding RNA according to embodiment 27, wherein the coding sequence comprises at least one nucleic acid sequence identical or 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 SEQ ID NOs: 529, 533, 534, 554, 558, 559, 654, 658, 659, or a fragment or variant thereof.

[0534] Embodiment 30. The coding RNA according to any one of embodiments 1 to 29, wherein at least one UTR is selected from at least one 5'-UTR and / or at least one 3'-UTR, and optionally at least one UTR is selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR.

[0535] Embodiment 31. The coding RNA according to embodiment 30, comprising at least one 3'-UTR, wherein at least one 3'-UTR comprises or consists of a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or a homolog, fragment or mutant of any one of these genes.

[0536] Embodiment 32. A coding RNA according to embodiment 31, wherein at least one heterologous 3'-UTR comprises or consists of a nucleic acid sequence identical to 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: 67-90, 109-120, or a fragment or variant thereof.

[0537] Embodiment 33. The coding RNA of embodiment 31, wherein the coding RNA comprises a 3'-UTR derived from or selected from the PSMB3 gene.

[0538] Embodiment 34. A coding 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 identical to, or 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 SEQ ID NOs: 67, 68, 109-120, or a fragment or variant thereof.

[0539] Embodiment 35. A coding RNA according to any one of embodiments 30 to 34, comprising at least one 5'-UTR, wherein at least one (heterologous) 5'-UTR comprises or consists of a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or a homologue, fragment or variant thereof.

[0540] Embodiment 36. A coding 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 identical or 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 SEQ ID NOs: 1 to 32, 65, 66, or a fragment or variant thereof.

[0541] Embodiment 37. A coding RNA according to embodiment 35 or 36, wherein at least one (heterologous) 5'-UTR is selected from HSD17B4, and optionally the 5'-UTR derived from or selected from HSD17B4 comprises or consists of a nucleic acid sequence identical or 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 SEQ ID NOs: 1, 2, 65, 66, or a fragment or variant thereof.

[0542] Embodiment 38. A coding 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.

[0543] Embodiment 39. A coding RNA according to any one of embodiments 1 to 38, wherein the coding RNA of the present invention is monocistronic.

[0544] Embodiment 40. A coding RNA according to any one of embodiments 1 to 39, comprising at least one poly(A) sequence, optionally comprising at least one poly(A) sequence comprising from about 40 to about 500 adenosine nucleotides, such as from about 60 to about 250 adenosine nucleotides, such as from about 60 to about 150 adenosine nucleotides.

[0545] Embodiment 41. The coding RNA of embodiment 40, wherein at least one poly(A) sequence comprises about 100 adenosine nucleotides.

[0546] Embodiment 42. A coding RNA according to embodiment 40 or 41, in which at least one poly(A) sequence is located directly at the 3'-end, and optionally, the 3'-terminal nucleotide is an adenosine.

[0547] Embodiment 43. A coding RNA according to any one of embodiments 1 to 42, comprising at least one poly(C) sequence and / or at least one miRNA binding site and / or a histone stem-loop sequence.

[0548] Embodiment 44. The coding RNA of embodiment 43, comprising at least one histone stem loop.

[0549] Embodiment 45. The coding RNA of embodiment 44, wherein the histone stem loop sequence comprises or consists of a nucleic acid sequence identical to or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 136, 137, or a fragment or variant thereof.

[0550] Embodiment 46. A coding RNA according to any one of the preceding embodiments, comprising or consisting of an RNA sequence which comprises at least one 3'-terminal sequence element, and optionally which 3'-terminal sequence element is identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 138 to 172, or a fragment or variant thereof.

[0551] Embodiment 47. The coding RNA of embodiment 47, comprising a 3'-terminal sequence element that comprises or consists of an RNA sequence identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO: 144, or a fragment or variant thereof.

[0552] Embodiment 48. A coding RNA according to any one of the preceding embodiments, comprising a 5'-terminal sequence element that comprises or consists of an RNA sequence that is identical or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 121 to 127, or a fragment or variant of said sequence.

[0553] Embodiment 49. The coding RNA according to embodiment 48, comprising a 5'-terminal sequence element consisting of or consisting of an RNA sequence identical to, or at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO: 122, or a fragment or variant thereof.

[0554] Embodiment 50. A coding RNA according to any one of embodiments 1 to 49, comprising a 5'-cap structure.

[0555] Embodiment 51. A coding RNA according to embodiment 50, wherein the 5'-cap structure is selected from a cap1 structure or a modified cap1 structure.

[0556] Embodiment 52. A coding RNA according to embodiment 50 or 51, wherein the 5'-cap structure is co-transcriptionally added using a trinucleotide cap analog, in particular in RNA in vitro transcription.

[0557] Embodiment 53. A coding RNA according to any one of embodiments 50 to 52, comprising a cap1 structure.

[0558] Embodiment 54. A coding RNA according to embodiment 53, wherein the cap1 structure is formed via co-transcriptional capping using the trinucleotide cap analogue m7G(5')ppp(5')(2'OMeA)pG or m7G(5')ppp(5')(2'OMeG)pG.

[0559] Embodiment 55. The coding RNA of embodiment 54, wherein the cap1 analog is m7G(5')ppp(5')(2'OMeA)pG.

[0560] Embodiment 56. A coding RNA according to any one of embodiments 1 to 55, optionally comprising at least one modified nucleotide selected from pseudouridine (ψ) or N1-methylpseudouridine (m1ψ).

[0561] Embodiment 57. The coding RNA of embodiment 56, wherein the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), and optionally, essentially all uracil nucleotides are replaced with pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0562] Embodiment 58. A coding RNA according to any one of embodiments 1 to 57, wherein the nucleic acid comprises at least one modified nucleotide which is N1-methylpseudouridine (m1ψ).

[0563] Embodiment 59. The coding RNA of embodiment 57 or 58, wherein essentially all uracil nucleotides are replaced by N1-methylpseudouridine (m1ψ) nucleotides.

[0564] Embodiment 60. The coding RNA according to any one of embodiments 1 to 59, wherein the coding RNA is selected from mRNA, a coding self-replicating RNA, a coding circular RNA, a coding viral RNA, or a coding replicon RNA.

[0565] Embodiment 61. The coding RNA of embodiment 60, wherein the coding RNA is an mRNA.

[0566] Embodiment 62. A coding RNA according to any one of embodiments 1 to 61, which is an in vitro transcribed RNA, optionally wherein the RNA in vitro transcription is carried out in the presence of a sequence-optimized nucleotide mixture.

[0567] Embodiment 63. The coding RNA according to any one of embodiments 1 to 62, which is a purified RNA, optionally wherein the RNA is 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.

[0568] Embodiment 64. The coding RNA of embodiment 63, wherein the RNA is purified by RP-HPLC and / or TFF.

[0569] Embodiment 65. A coding RNA according to any one of embodiments 1 to 64, having 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%.

[0570] Embodiment 66. A coding RNA according to any one of embodiments 1 to 65, comprising, for example in the 5' to 3' direction, the following elements: A) 5'-cap structure; B) a 5'-UTR, for example selected from or derived from the 5'-UTR of the HSD17B4 gene; C) at least one coding sequence encoding an antigenic polypeptide selected from or derived from Escherichia coli FimH; D) a 3'-UTR, for example selected from or derived from the 3'-UTR of the PSMB3 gene; E) optionally, a histone stem loop; and F) A poly(A) sequence, for example comprising about 100 A nucleotides.

[0571] Embodiment 67. SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195, 1198-1 67. The coding RNA according to any one of the preceding claims, comprising or consisting of a nucleic acid sequence identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of the preceding claims, or a fragment or variant thereof.

[0572] Embodiment 68. The coding RNA of embodiment 67, wherein at least one, e.g. all, uracil nucleotides in the aforementioned RNA sequence are replaced by pseudouridine (ψ) nucleotides and / or N1-methylpseudouridine (m1ψ) nucleotides.

[0573] Embodiment 69. A coding RNA according to any one of embodiments 1 to 68, comprising or consisting of a nucleic acid sequence identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 679, 704, 729, 754, 779, 804, 829, 854, 879, 904, 929, 954, 979, 1004, 1029, 1054, 1079, 1104, 1129, 1154, 1179, 1204, 1229, 1254, or a fragment or variant thereof, optionally comprising a 5'-terminal cap1 structure.

[0574] Embodiment 70. A coding RNA according to any one of embodiments 1 to 69, comprising or consisting of a nucleic acid sequence identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 683, 708, 733, 758, 783, 808, 833, 858, 883, 908, 933, 958, 983, 1008, 1033, 1058, 1083, 1108, 1133, 1158, 1183, 1208, 1233, 1258, or a fragment or variant thereof, and optionally comprising a 5'-terminal cap1 structure.

[0575] Embodiment 71. In one embodiment, the coding RNA according to any one of embodiments 1 to 70, comprising or consisting of a nucleic acid sequence identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence according to any one of SEQ ID NOs: 684, 709, 734, 759, 784, 809, 834, 859, 884, 909, 934, 959, 984, 1009, 1034, 1059, 1084, 1109, 1134, 1159, 1184, 1209, 1234, 1259, or a fragment or variant thereof, optionally comprising a 5'-terminal cap1 structure.

[0576] Embodiment 72(a). A 5' capped (cap1) 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; The coding RNA of embodiment 69, which is identical to an RNA sequence according to any one of SEQ ID NOs: 679, 829, 979, 1129, 1271, 1274, or a fragment or variant thereof.

[0577] Embodiment 72(b). A 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides; The coding RNA of embodiment 69, which is identical to the RNA sequence of SEQ ID NO: 1271, or a fragment or variant thereof.

[0578] Embodiment 72(c). A 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides; The coding RNA of embodiment 69, which is identical to the RNA sequence according to SEQ ID NO: 1274, or a fragment or variant thereof.

[0579] Embodiment 73(a). A 5' capped (cap1) 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; The coding RNA of embodiment 70, which is identical to the RNA sequence according to any one of SEQ ID NOs: 683, 833, 983, 1133, 1272, 1275, or a fragment or variant thereof.

[0580] Embodiment 73(b). A 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides; The coding RNA according to embodiment 70, which is identical to the RNA sequence according to SEQ ID NO: 1272, or a fragment or variant thereof.

[0581] Embodiment 73(c). A 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides; The coding RNA according to embodiment 70, which is identical to the RNA sequence according to SEQ ID NO: 1275, or a fragment or variant thereof.

[0582] Embodiment 74(a). A 5' capped (cap1) 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; The coding RNA of embodiment 71, which is identical to an RNA sequence according to any one of SEQ ID NOs: 684, 834, 984, 1134, 1273, 1276, or a fragment or variant thereof.

[0583] Embodiment 74(b). A 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified N1-methylpseudouridine (m1ψ) ribonucleotides; The coding RNA of embodiment 69, which is identical to the RNA sequence according to SEQ ID NO: 1273, or a fragment or variant thereof.

[0584] Embodiment 74(c). A 5' capped (cap1) mRNA comprising or consisting of an RNA sequence consisting of unmodified ribonucleotides (A, G, C) and chemically modified pseudouridine (ψ) ribonucleotides; The coding RNA of embodiment 69, which is identical to the RNA sequence according to SEQ ID NO: 1276, or a fragment or variant thereof.

[0585] Embodiment 75. A pharmaceutical composition comprising a coding RNA according to any one of embodiments 1 to 74.

[0586] Embodiment 76. A pharmaceutical composition according to embodiment 75, comprising at least one pharma- ceutically acceptable carrier or excipient.

[0587] Embodiment 77. A pharmaceutical composition according to embodiment 75 or 76, comprising a lipid-based carrier, optionally wherein the coding RNA is formulated in the lipid-based carrier.

[0588] Embodiment 78. The pharmaceutical composition according to embodiment 77, wherein the lipid-based carrier is selected from liposomes, lipid nanoparticles, lipoplexes, solid lipid nanoparticles, lipopolyplexes, and / or nanoliposomes.

[0589] Embodiment 79. The pharmaceutical composition of embodiment 78, wherein the lipid-based carrier is a lipid nanoparticle, and optionally the lipid nanoparticle encapsulates the coding RNA.

[0590] Embodiment 80. A pharmaceutical composition according to any one of embodiments 75 to 79, wherein the coding RNA is formulated in at least one cationic or polycationic compound.

[0591] Embodiment 81. The pharmaceutical composition according to embodiment 80, wherein 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 a combination thereof.

[0592] Embodiment 82. The pharmaceutical composition of any one of embodiments 77 to 81, wherein the lipid-based carrier comprises at least one lipid selected from an aggregation-reducing lipid, a cationic lipid or an ionizable lipid, a neutral lipid or a phospholipid, or a steroid, a steroid analog or a sterol, or a combination thereof.

[0593] Embodiment 83. The pharmaceutical composition of embodiment 82, wherein 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, steroid analog or sterol.

[0594] Embodiment 84. The pharmaceutical composition of 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:

[0595] Embodiment 85. The pharmaceutical composition of any one of embodiments 77 to 84, wherein the lipid-based carrier comprises a cationic lipid selected from or derived from ALC-0315.

[0596] Embodiment 86. The pharmaceutical composition of any one of embodiments 77 to 85, wherein the lipid-based carrier comprises an aggregation-reducing lipid selected from a polymer-conjugated lipid, and optionally the polymer-conjugated lipid is a PEG-conjugated lipid selected from or derived from formula IVa, such as ALC-0159 or a PEG-conjugated lipid selected from or derived from formula IVa.

[0597] Embodiment 87. A pharmaceutical composition according to any one of embodiments 77 to 86, wherein the lipid-based carrier comprises a neutral lipid selected from or derived from DSPC.

[0598] Embodiment 88. The pharmaceutical composition of any one of embodiments 77 to 87, wherein the lipid-based carrier comprises a steroid, a steroid analogue or a sterol, optionally selected from or derived from cholesterol.

[0599] Embodiment 89. The pharmaceutical composition of any one of embodiments 77 to 88, wherein the lipid-based carrier comprises: (i) at least one cationic lipid, e.g., as described in embodiment 84 or 85; (ii) at least one neutral lipid, e.g., as described in embodiment 87; (iii) at least one steroid, steroid analog, or sterol, e.g., as described in embodiment 88; and (iv) at least one aggregation-reducing lipid, e.g., as described in embodiment 86.

[0600] Embodiment 90. The pharmaceutical composition of any one of embodiments 77 to 89, wherein the lipid-based carrier comprises: (i) at least one cationic lipid selected from ALC-0315; (ii) at least one neutral lipid selected from DSPC; (iii) at least one steroid, steroid analog, or sterol selected from cholesterol; and (iv) at least one aggregation-reducing lipid selected from ALC-0159.

[0601] Embodiment 91. The pharmaceutical composition of any one of embodiments 89-90, wherein the lipid-based carrier comprises (i)-(iv) in a molar ratio of about 20-60% cationic lipid or ionizable lipid, about 5-25% neutral lipid, about 25-55% steroid or steroid analog, and about 0.5-15% aggregation-reducing lipid.

[0602] Embodiment 92. A pharmaceutical composition according to any one of embodiments 77 to 91, wherein the wt / wt ratio of lipid to coding RNA in the lipid-based carrier is from about 10:1 to about 60:1, for example from about 20:1 to about 30:1.

[0603] Embodiment 93. A pharmaceutical composition according to any one of embodiments 77 to 92, wherein the N / P ratio of the lipid-based carrier in which the nucleic acid is encapsulated is in the range of about 1 to about 10, for example in the range of about 5 to about 7.

[0604] Embodiment 94. A pharmaceutical composition according to any one of embodiments 77 to 93, wherein the lipid-based carrier has a Z-average size in the range of about 50 nm to about 120 nm.

[0605] Embodiment 95. A pharmaceutical composition according to any one of embodiments 75 to 94, further comprising at least one antagonist of at least one RNA-sensing pattern recognition receptor selected from Toll-like receptors, for example a TLR7 antagonist and / or a TLR8 antagonist.

[0606] Embodiment 96. A pharmaceutical composition according to any one of embodiments 75 to 95, wherein the composition is a liquid composition or a dry composition.

[0607] Embodiment 97. A vaccine comprising a coding RNA according to any one of embodiments 1 to 74 or a pharmaceutical composition according to any one of embodiments 75 to 96.

[0608] Embodiment 98. The vaccine of embodiment 97, for example, wherein administration of the vaccine to a subject induces a humoral immune response against E. coli, FimH.

[0609] Embodiment 99. The vaccine of embodiment 97 or 98, e.g., wherein administration of the vaccine to a subject induces a neutralizing antibody titer against E. coli, and optionally wherein said antibody is an IgG antibody. In one embodiment, said antibody is against E. coli FimH. In one embodiment, e.g., administration of the vaccine to a subject induces a neutralizing antibody titer against E. coli, e.g., a neutralizing antibody titer against E. coli FimH, in the urine of the subject at the time of administration of the vaccine.

[0610] Embodiment 100. A kit or kit of parts comprising a coding RNA according to any one of embodiments 1 to 74, a pharmaceutical composition according to any one of embodiments 75 to 96 and / or a vaccine according to any one of embodiments 97 to 99, optionally comprising a liquid vehicle for solubilization and optionally comprising technical instructions providing information on the administration and dosing of the components.

[0611] Embodiment 101. A coding RNA according to any one of embodiments 1 to 74, a pharmaceutical composition according to any one of embodiments 75 to 96, a vaccine according to any one of embodiments 97 to 99, or a kit or kit of parts according to embodiment 100, for use as a medicament.

[0612] Embodiment 102. A coding RNA according to any one of embodiments 1 to 74, a pharmaceutical composition according to any one of embodiments 75 to 96, a vaccine according to any one of embodiments 97 to 99, or a kit or kit of parts according to embodiment 100, for use in the treatment or prevention of one or more symptoms associated with a urinary tract infection (UTI) in a subject in need thereof.

[0613] Embodiment 103. A coding RNA according to any one of embodiments 1 to 74, a pharmaceutical composition according to any one of embodiments 75 to 96, a vaccine according to any one of embodiments 97 to 99 or a kit or kit of parts according to embodiment 100 for use in the treatment or prevention of a disease caused by E. coli.

[0614] Embodiment 104. A method for treating or preventing a disorder, comprising administering to a subject in need thereof an effective amount of a coding RNA according to any one of embodiments 1 to 74, a pharmaceutical composition according to any one of embodiments 75 to 96, a vaccine according to any one of embodiments 97 to 99, or a kit or kit of parts according to embodiment 100.

[0615] Embodiment 105 The method of embodiment 104, eliciting a humoral immune response against E. coli FimH, and optionally eliciting a humoral immune response in the urine of the subject.

[0616] Embodiment 106. The method of embodiment 104 or 105, which induces neutralizing antibody titers against E. coli, and optionally, said antibody is an IgG antibody.

[0617] Embodiment 107. The method of embodiment 106, which induces neutralizing antibody titers against E. coli, e.g., E. coli FimH, in the subject's urine.

[0618] Embodiment 108. The method of any one of embodiments 104 to 107, which induces antibodies capable of inhibiting bacterial adhesion.

[0619] Embodiment 109. The method of any one of embodiments 104 to 108, wherein the administration is intramuscular administration.

[0620] Embodiment 110. Use of a coding RNA according to any one of embodiments 1 to 74, a pharmaceutical composition according to any one of embodiments 75 to 96, a vaccine according to any one of embodiments 97 to 99 or a kit or kit of parts according to embodiment 100 for the manufacture of a medicament for increasing an immune response in a mammal, for example a medicament for treating and / or preventing a disease, for example an E. coli infection. EXAMPLES

[0621] Below are given specific examples illustrating various embodiments and aspects of the present disclosure. However, the present disclosure is not limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and practice the present disclosure. However, the present disclosure is not limited in scope by the illustrated embodiments, which are intended only as illustrations of single aspects of the present disclosure, and functionally equivalent methods are within the scope of the present disclosure. Indeed, in addition to those described herein, various modifications of the present disclosure will be readily apparent to those skilled in the art from the foregoing description, the accompanying figures, and the following examples. All such modifications are within the scope of the appended claims.

[0622] Example 1: Preparation of DNA and RNA constructs, compositions, and vaccines This example provides methods for obtaining the coding RNA of the present disclosure, as well as methods for producing a composition or vaccine of the present disclosure.

[0623] 1.1. Preparation of DNA and RNA Constructs DNA sequences encoding differently designed E. coli FimH proteins were prepared and used in the subsequent RNA in vitro transcription reaction. The aforementioned DNA sequences were prepared by modifying wild-type or reference coding DNA sequences by introducing G / C-optimized or modified coding sequences (e.g., "cds opt1") for stabilization and expression optimization. The sequences were introduced into pUC-derived DNA vectors and contain stabilizing 3'-UTR and 5'-UTR sequences, as well as a stretch of adenosines (e.g., A100), and optionally a histone stem loop (hSL) structure (see Table 3, see Table 1 for an overview of antigen design).

[0624] The resulting plasmid DNA constructs were transformed and propagated in bacteria using common protocols known in the art, and finally, the plasmid DNA constructs were extracted, purified, and used for subsequent RNA in vitro transcription (see section 1.2).

[0625] 1.2. In vitro transcription of RNA from plasmid DNA templates DNA plasmids prepared according to section 1.1 were enzymatically linearized using restriction enzymes and used for DNA-dependent RNA in vitro transcription with T7 RNA polymerase in the presence of a sequence-optimized nucleotide mixture (ATP / GTP / CTP / UTP) and cap analogs (for cap1: m7G(5')ppp(5')(2'OmeA)pG; TriLink) under suitable buffer conditions. The resulting RNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008077592) and used for in vitro and in vivo experiments. To obtain chemically modified mRNAs, in vitro transcription of RNA was performed in the presence of modified nucleoside mixtures containing N1-methylpseudouridine (m1ψ) or pseudouridine (ψ) instead of uridine. The resulting m1ψ or ψ chemically modified RNA was purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008077592) and used for further experiments.

[0626] RNA for clinical development is produced under current good manufacturing practices, e.g. according to WO2016180430, and various quality control steps are performed at the DNA and RNA level.

[0627] (Example RNA constructs) The generated RNA sequences / constructs are provided in Table 3, where the encoded antigenic proteins and respective UTR elements are shown. Unless otherwise indicated, the RNA sequences / constructs in Table 3 were produced using RNA in vitro transcription in the presence of m7G(5')ppp(5')(2'OMeA)pG cap analog; thus, the RNA sequences / constructs contain a 5'cap1 structure. Unless otherwise indicated, the RNA sequences / constructs in Table 3 were produced in the absence of chemically modified nucleotides (e.g., pseudouridine (ψ) or N1-methylpseudouridine (m1ψ)).

[0628] [Table 3-1] [Table 3-2] [Table 3-3]

[0629] 1.4. Preparation of LNP-Formulated mRNA Compositions LNPs were prepared using cationic lipids, structured lipids, PEG-lipids, and cholesterol. The lipid solution (in ethanol) was mixed with the RNA solution (in aqueous buffer) using a microfluidic mixer. The resulting LNPs were rebuffered with carbohydrate buffer by dialysis and concentrated to the target concentration using ultracentrifuge tubes. The mRNA prepared in the LNPs was stored at -80°C before use in in vitro or in vivo experiments.

[0630] Suitably, lipid nanoparticles have been prepared and tested according to the general procedures described in PCT publications WO2015199952, WO2017004143 and WO2017075531, the entire disclosures of which are incorporated herein by reference. Lipid nanoparticles (LNPs) forming mRNA were prepared using ionizable amino lipids (cationic lipids), phospholipids, cholesterol, and PEGylated lipids. LNPs were prepared as follows: Cationic lipid according to formula III-3 (ALC-0315), DSPC, cholesterol, and PEG-lipid according to formula IVa (ALC-0159) were solubilized in ethanol in a molar ratio of approximately 47.5:10:40.8:1.7 (see Table 4). Lipid nanoparticles (LNPs) containing compound III-3 were prepared with a ratio of mRNA (sequence see Table 3) to total lipid of 0.03-0.04 w / w. Briefly, mRNA was diluted to 0.05–0.2 mg / ml in 10–50 mM citrate buffer, pH 4. Using a pump, the ethanol lipid solution was mixed with the aqueous mRNA solution at a ratio of approximately 1:5–1:3 (vol / vol) at a total flow rate of 15 ml / min or more. Then, the ethanol was removed and the external buffer was replaced with PBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. The particle size of the lipid nanoparticles was 50–120 nm, determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

[0631] [Table 4]

[0632] 1.5 Preparation of Combination mRNA Vaccines (Bivalent or Multivalent Vaccine Compositions) Containing Combinations of Antigens Combination mRNA vaccines were formulated with LNPs either separately or by co-formulation. For separately mixed or formulated mRNA vaccines, each mRNA component was prepared and LNP-formulated separately as described in Example 1.4, and then the different LNP-formulated components were mixed. For co-formulated mRNA vaccines, the different mRNA components were first mixed together and then co-formulated into LNPs as described in Example 1.4.

[0633] Example 2: Analysis of E. coli FimH antigen design 2.1 In vitro analysis of expression and secretion of antigen designs using Western blot To measure the in vitro protein expression of several mRNA constructs, HeLa cells were transfected with 2 μg of unformulated mRNA encoding different antigen designs using Lipofectamine 2000 and 6-well plates. 24 hours after transfection, cell lysates and cell culture supernatants were subjected to SDS-PAGE and Western blot analysis using goat anti-rabbit IgG IRDye® 680RD antibody (1:10000; Li-Cor) plus mouse anti-FimC serum (1:1000), mouse anti-FimHLcys serum (1:1000) or rabbit anti-α-tubulin antibody (1:1000; Cell Signaling) as primary antibodies and goat anti-mouse IgG IRDye® 800CW antibody (1:10000; Li-Cor) as secondary antibody. Anti-FimC and anti-FimHLcys sera were obtained by subcutaneous immunization of CD1 mice on days 0, 21, and 35, and serum was collected on day 49. FimHLcys was obtained as described in Kisiela DI, et al. Proc Natl Acad Sci US A. 2013 Nov 19;110(47):19089-94. Detection and quantification were performed using the Li-Cor detection system (Odyssey CLx imaging system) in combination with Image Studio Lite software. Table 5 contains the mRNA constructs used in the experiments, and Figure 1 shows the experimental results.

[0634] [Table 5-1] [Table 5-2]

[0635] (result) For most of the RNA constructs, expression was demonstrated in the corresponding cell lysates (see Figure 1A). By analyzing the supernatants of transfected HeLa cells, secretion of the tested E. coli FimH antigen designs was detected for constructs 1, 2, 3, 6, 7, 8, 9, 10 and 11 (see Figure 1B).

[0636] 2.2 Analysis of the immunogenicity of E. coli FimH antigen designs in mice The mRNA constructs (see Table 5) encoding the E. coli FimH antigen design were prepared according to Example 1. The mRNA was formulated in a lipid-based carrier (see Example 1.4. Preparation of LNP-formulated mRNA composition). As shown in Table 6, the different mRNA vaccine candidates were applied to female BALB / c mice on days 0, 21, and 35, and 2 μg or 4 μg of RNA was administered intramuscularly (im). A negative control group (A) received only buffer (0.9% NaCl) and one group (B) received the PHAD-adjuvanted FimHC protein complex subunit vaccine obtained as described in US9017698. Serum and urine samples were taken on days 1 (18 hours), 21, 35, and 49 to determine humoral immune responses.

[0637] ELISA was performed using recombinant FimHL for coating. FimHL was obtained by cloning amino acids 22-181 of UPEC J96 FimH (GenBank: ELL41155.1) into the Pet22b plasmid. Recombinant FimHL was expressed in E. coli BL21-DE3 and purified from the periplasmic space. The coated plates were incubated with the respective serum or urine dilutions, and specific antibody binding to FimHL was detected using a peroxidase-conjugated goat anti-mouse IgG (H+L) antibody (1:5000, Jackson ImmunoResearch) followed by Amplex® UltraRed reagent (1:200, Invitrogen) as substrate. The endpoint titers of IgG antibodies against the recombinant protein FimHL were measured by ELISA on days 21, 35, and 49.

[0638] [Table 6]

[0639] (result) As shown in Figure 2, the tested antigen designs induced substantial humoral immune responses in mice. FimHL-specific IgG end-point titers (analyzed by ELISA) were detectable in serum and urine on days 21, 35, and 49 in most groups. Early immune responses are crucial for rapid and robust protection against UTI. Although adaptive immune responses were already quite high after one vaccination, serum and urinary antibody titers induced by RNA vaccination or PHAD-adjuvanted FimHC protein complex subunit vaccine could be further boosted by a second and third vaccination.

[0640] 2.3 Analysis of functional serum antibody responses to E. coli FimH antigen designs The antibody responses generated by immunization with the E. coli FimH constructs described in Example 2.2 were characterized by bacterial adherence inhibition assay (BAI). BAI titers were determined using serum pools (8 mice per group) at days 21, 35 and 49.

[0641] The UTI89 E. coli UPEC strain was engineered to express the mCherry fluorescent marker and was cultured in stationary liquid culture for three passages. Bacteria were harvested, washed with PBS, and grown at 0.012 OD in F12K medium (Thermo Scientific) supplemented with 10% FBS without antibiotics. 600 / ml.

[0642] Serum samples were prepared at twice the final working concentration (2x) in F12K medium or F12K medium supplemented with 10% FBS and further diluted in serial dilutions. 20% D-(+)-mannose and antibiotic-free F12K medium supplemented with 10% FBS were used as positive and negative controls, respectively.

[0643] SV-HUC cells (ATCC) were cultured in F12K medium (Thermo Scientific) supplemented with 10% FBS and antibiotics. SV-HUC cells were plated in a 96-well plate at 3.5 × 10 4 The cells were seeded at a density of 100 cells / well (final volume 200 μl / well) and incubated at 37°C, 5% CO 2 Plates were incubated at 37 °C for 30 min at 4 °C for 1 h. The medium was replaced with antibiotic-free F12K medium supplemented with 10% FBS. The medium was removed and 50 μl of sample or control was added to each well, followed by 50 μl of 2× bacterial inoculum or medium as a negative control. Plates were incubated for 30 min and serum dilutions were added from 15% to 0.06%. Plates were incubated at 37 °C, 5% CO 2After incubation for 30 min at 4°C, the medium was removed and the wells were washed three times with PBS. Bacteria were fixed with 4% formaldehyde solution for 20 min and then stained with DAPI (62248, ThermoScientific) as known in the art. Microscopic analysis was performed with OPERA Phenix. Data were analyzed with Harmony software. Total bacterial fluorescent area (single object ≦100 μm 2 ) was calculated as the adhesion value. The titer of each sample was calculated as the dilution corresponding to the inflection point of the dose-response curve.

[0644] (result) As shown in Table 7, BAI titers were detected in almost all samples at day 21. Higher inhibitory titers were detected for constructs 13, 14 and 15, which encode antigen clustering domains. No specific titers were observed for the PHAD adjuvant FimHC protein complex, as it was below th...

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 fimbria D-mannose specific adhesin (FimH).

2. 2. The coding RNA of claim 1, wherein the E. coli FimH comprises an amino acid sequence identical to or 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 SEQ ID NOs: 177-186, 247-256, or is an immunogenic fragment or variant thereof.

3. The coding RNA of claim 1 or 2, wherein the coding sequence further encodes one or more additional peptide or protein elements selected from a donor chain peptide, a signal peptide, an antigen clustering domain, or a transmembrane domain.

4. The coding RNA of claim 3, wherein the one or more additional peptide or protein elements are donor chain peptides, and optionally 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 chain peptide.

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

338.

6. The coding RNA of claim 4 or 5, wherein the coding sequence further codes for a peptide linker, and optionally the coding sequence codes for the following elements in the N-terminal to C-terminal direction: an antigenic polypeptide selected from or derived from E. coli FimH; the peptide linker element; and the donor chain peptide.

7. The coding RNA of 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 further codes for an antigen clustering domain, optionally wherein the antigen clustering domain is selected from or derived from ferritin or lumazine synthase.

9. The encoded RNA of claim 8, wherein the amino acid sequence of the antigen clustering domain 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 any one of the amino acid sequences SEQ ID NOs: 457-459, 443, 444, or a fragment or variant thereof.

10. The encoded RNA according to any one of claims 3 to 9, wherein the coding sequence further codes for a peptide linker, and optionally the signal peptide is selected from or derived from IgE or IgK, and the amino acid sequence of the signal peptide 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 any one of the amino acid sequences SEQ ID NOs: 394, 395, or fragments or variants thereof.

11. The coding RNA according to any one of claims 3 to 10, wherein the coding sequence optionally codes for the following elements in the N-terminal to C-terminal direction: a) signal peptide, antigenic polypeptide; b) signal peptides, antigenic polypeptides, peptide linkers, donor chain peptides; c) antigen clustering domain, peptide linker, antigenic polypeptide, peptide linker, donor chain peptide; d) signal peptides, antigen clustering domains, peptide linkers, antigenic polypeptides, peptide linkers, donor chain peptides; e) a signal peptide, an antigenic polypeptide, a peptide linker, a donor chain peptide, a peptide linker, an antigen clustering domain; or f) signal peptide, antigenic polypeptide, peptide linker, donor chain peptide, peptide linker, transmembrane domain.

12. The encoded RNA according to any one of claims 1 to 11, wherein the coding sequence encodes 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 any one of SEQ ID NOs: 177-186, 247-256, 498-520, 1277, or an immunogenic fragment or immunogenic variant thereof.

13. The encoded RNA according to any one of claims 1 to 12, wherein said coding sequence comprises a nucleic 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 a sequence according to any one of SEQ ID NOs: 187-246, 257-316, 523-545, 548-570, 573-595, 598-620, 623-645, 648-670, or a fragment or variant thereof.

14. The coding RNA according to any one of claims 1 to 13, wherein the coding sequence comprises at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ), and optionally, essentially all uracil nucleotides are replaced with pseudouridine (ψ) and / or N1-methylpseudouridine (m1ψ) nucleotides.

15. The coding RNA according to any one of claims 1 to 14, wherein said coding sequences are codon-modified coding sequences, and the amino acid sequence encoded by said at least one codon-modified coding sequence is not optionally modified compared to the amino acid sequence encoded by a corresponding wild-type coding sequence, and optionally said 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 adapted coding sequence, a G / C content modified coding sequence, and a G / C optimized coding sequence, or any combination thereof.

16. The coding RNA is an mRNA, and optionally is selected from the group consisting of SEQ ID NOs: 673-695, 698-720, 723-745, 748-770, 773-795, 798-820, 823-845, 848-870, 873-895, 898-920, 923-945, 948-970, 973-995, 998-1020, 1023-1045, 1048-1070, 1073-1095, 1098-1120, 1123-1145, 1148-1170, 1173-1195 16. The encoded RNA according to any one of claims 1 to 15, comprising or consisting of a nucleic acid sequence which is identical to or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence according to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 ...20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:29, SEQ ID NO:29, SEQ ID NO:29, SEQ ID NO:

17. A pharmaceutical composition comprising a coding RNA according to any one of claims 1 to 16.

18. 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 a coding RNA according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17 or 18.

20. A kit or kit-of-parts comprising a coding RNA according to any one of claims 1 to 16, a pharmaceutical composition according to any one of claims 17 or 18 and / or a vaccine according to claim 19, optionally comprising a liquid vehicle for solubilization and optionally technical instructions providing information regarding the administration and dosing of the components.

21. A coding 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 19 or a kit or kit of parts according to claim 19 for use as a medicament.

22. A coding RNA according to any one of claims 1 to 17, a pharmaceutical composition according to claim 18 or 19, a vaccine according to claim 20 or a kit or kit of parts according to claim 21 for use in the treatment or prevention of one or more symptoms associated with a urinary tract infection (UTI) in a subject in need thereof.

23. A coding RNA according to any one of claims 1 to 16, a pharmaceutical composition according to claims 17 or 18, a vaccine according to claim 19 or a kit or kit of parts according to claim 20 for use in the treatment or prevention of a disease caused by E. coli.

24. A method for treating or preventing a disorder, comprising administering to a subject in need thereof an effective amount of a coding RNA according to any one of claims 1 to 16, a pharmaceutical composition according to claim 17 or 18, a vaccine according to claim 21, or a kit or kit of parts according to claim 20.

Images