Methods for stabilizing RNA

Abstract

This invention relates to RNA molecules encoding the fimbriae H antigen (FimH) of Escherichia coli (E. coli). This disclosure further relates to compositions comprising RNA molecules formulated in lipid nanoparticles (RNA-LNPs). This disclosure further relates to the use of RNA molecules, RNA-LNPs, and compositions for the prevention of Escherichia coli (E. coli) infections, including urinary tract infections.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 508,060 filed on 14 June 2023, U.S. Provisional Application No. 63 / 610,042 filed on 14 December 2023, and U.S. Provisional Application No. 63 / 649,495 filed on 20 May 2024. The entire contents of each of the above applications are thus incorporated herein by reference.

[0002] The present invention relates to a composition of a ribonucleic acid vaccine comprising a polynucleotide molecule encoding one or more Escherichia coli (E. coli) fimbriae antigens, such as fimbriae H antigen (FimH), and to a method for preparing, producing, and therapeutically using the ribonucleic acid vaccine.

[0003] Sequence listing reference This application was filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing named "PC72992A Sequence Listing.xml", created on May 29, 2024, and having a size of 378KB. The sequence listing contained in this .xml file is part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Urinary tract infections (UTIs) affect one in five women at least once in their lifetime, are a significant cause of morbidity and death, and result in a substantial burden on healthcare systems. While several different bacteria can cause UTIs, the most common cause (90–95% of cases) is the bacterium Escherichia coli (E. coli). Escherichia coli (E. coli) is a Gram-negative bacterium that colonizes the human gut flora or causes severe invasive disease (Bonten, M. et al., Clin infect dis, 2021.72(7):1211–1219). E. coli (E. coli) is one of the most common causes of bacteremia and UTIs. Uropathogenic Escherichia coli (UPEC) is the most prevalent virulence factor, causing 80–90% of uncomplicated UTI cases (Bonten, M. et al., Clin infect dis, 2021.72(7):1211–1219; and Flores-Mireles, AL et al., Nat rev microbiol, 2015.13(5):269–284). When the infection is limited to the bladder, it is called cystitis. These bacterial infections can ascend from the bladder to the kidneys, potentially leading to pyelonephritis. It is estimated that 50% of women will experience at least one symptomatic UTI in their lifetime (Terlizzi, ME, G. Gribaudo, and MEMaffei. Front microbiol, 2017.8:1566). Children and the elderly are also at significant risk of developing these infections. UTIs have a high incidence and a recurrence rate of 27%–44%. As antibiotic resistance rates and pathogenic isolates increase, multidrug-resistant strains (e.g., Escherichia coli ST131) emerge.

[0005] UPECs typically originate in the gut, then adhere to host urothelial cells, and migrate to the urogenital tract by rapidly replicating upon reaching the bladder (Flores-mireles, AL et al., Nat rev microbiol, 2015.13(5):269~284; and Klein, RD and SJHultgren. Nat rev microbiol, 2020.18(4):211~226). Adhesion is facilitated by ciliary adhesins, including type 1 fimbriae, which bind to mannosylated glycoproteins expressed on the surface of host urothelial cells. Type 1 fimbriae are highly conserved among clinical UPEC isolates and are encoded by a cluster of genes called fim, which encode accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG), and an adhesin called FimH. In mouse and porcine models of UTI, FimH is essential for establishing bladder infection (Staerk, K. et al., Microbiology (Reading), 2021.167(10); Schwartz, DJ et al., Infect Immun, 2011.79(10):4250~4259; and Hannan, TJ et al., Plos Pathog, 2010.6(8):e1001042). Small molecule inhibitors that target FimH by mimicking mannosylated receptors have further investigated the role of FimH in UTI and shown promise as therapeutic agents in animal models (Cusumano, CK et al., Sci transl med., 2011.3(109):109ra115). Additionally, FimH is positively selected in human cystitis isolates of Escherichia coli (Chen, SL et al., Proc natl acad sci USA, 2009.106(52):22439~44), and the positively selected residues may influence pathogenicity in mouse models of cystitis (Schwartz, DJ et al., Proc natl acad sci USA, 2013.110:15530~15537).

[0006] FimH is a lectin-binding domain that is responsible for binding to mannosylated glycoproteins (FimH LD It consists of two domains: the FimH domain and the pyrin domain. The pyrin domain helps to link FimH to other structural subunits of the fimbria, such as FimG, through a mechanism called donor strand exchange (Le Trong, I. et al., J. Struct Biol., 2010;172(3):380~388). The FimH pyrin domain results in a groove that provides a binding site for the N-terminal β-strand of FimG, which forms an incomplete immunoglobulin fold and creates a strong intermolecular linkage between FimH and FimG. LD FimH can be expressed in a soluble, stable form, but full-length FimH is unstable on its own, except when it is in a complex with the chaperone FimC, or when it complements the donor chain peptide of FimG in peptide form or as a fusion protein (Barnhart MM et al., Proc Natl Acad Sci US A.2000;97(14):7709~14; Sauer MM et al., Nat Commun.2016;7:10738; Barnhart MM et al., J Bacteriol.2003;185(9):2723~30) (Vetsch, M. et al., J.Mol.Biol.322:827~840(2002); Barnhart MM et al., Proc Natl Acad Sci US A.2000;97(14):7709~7714). The design and expression of a full-length FimH molecule by linking a FimG donor peptide to full-length FimH via a glycine-serine linker has been previously described (published May 6, 2021, PCT international publication number WO2021 / 084429) and is referred to as FimH-DSG.

[0007] FimH LD It is considered a poor immunogen in terms of its ability to stimulate functional immunogenicity. Some studies have shown that the binding antibody titer is different with and without FimH LDWhile it can be induced, the functional neutralizing titer was only observed in the presence of an adjuvant, suggesting this (PCT International Publication No. 2021 / 084429, published May 6, 2021). The study suggests that locking of FimH in an open conformation with reduced affinity for mannoside ligands improves functional immunogenicity (Kisiela, DI et al., Proc Natl Acad Sci USA 110, 19089-19094 (2013)).

[0008] While some progress has been made in the development of UTI vaccines, no licensed vaccines are currently available. Therefore, there is a need for immunogenic compositions containing FimH antigens with improved biochemical properties that result in reduced affinity for mannoside ligands and improved functional immunogenicity compared to wild-type FimH. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] RNA technology, particularly mRNA technology, is especially advantageous as a vaccine or therapeutic platform. For effective RNA vaccines or therapeutics, it is crucial to maximize protein expression so that the desired amount of protein or antigen is produced from a minimum amount of RNA. However, mRNA-based therapies may suffer from challenges including low manufacturing efficiency, short half-life of administered mRNA in circulation, and low translation efficiency. Therefore, there is a need for RNA compositions with improved stability and translation efficiency, including methods for improving protein expression and enabling high levels of expression by optimizing the sequence and structure of the 5' untranslated region of mRNA. [Means for solving the problem]

[0010] The present invention, as provided herein, satisfies an unmet need for an improved immunogenic composition against Escherichia coli (E. coli) infections.

[0011] In one embodiment, the Disclosure provides immunogenic compositions and methods for preventing, treating or alleviating an infection, disease, or condition in a subject, comprising the administration of an amino acid sequence, such as an RNA molecule encoding an immunogenic antigen, such as an immunogenic RNA polynucleotide, comprising the E. coli (E. coli) FimH protein ("FimH"), its immunogenic variant, or an immunogenic fragment of the FimH protein or its immunogenic variant, such as an antigen peptide or protein. Thus, the immunogenic antigen comprises an epitope of the FimH protein for inducing an immune response to FimH in the subject. The RNA polynucleotide encoding the immunogenic antigen is administered to provide the antigen (after expression of the polynucleotide by appropriate target cells) for induction of an immune response, such as the induction of antibodies and / or immune effector cells, such as stimulation, priming, and / or proliferation. In one embodiment, the immune response induced according to the Disclosure is both a B cell-mediated immune response, such as an antibody-mediated immune response, and a T cell-mediated immune response. In one embodiment, the immune response is an anti-FimH immune response.

[0012] The immunogenic compositions described herein include an RNA molecule (as an active ingredient) containing RNA that can be translated into one or more proteins in recipient cells. In addition to wild-type, codon-optimized, or mutant sequences encoding an antigen sequence, the RNA molecule may contain one or more structural elements (5' cap, 5' UTR, subgenome promoter, 3' UTR, poly-A tail) optimized for the maximum efficacy of the RNA in terms of stability and translation efficiency. In one embodiment, the RNA molecule contains all of these elements. The RNA molecules described herein may be complexed with lipids and / or proteins to generate RNA particles (e.g., lipid nanoparticles (LNPs)) for administration. In one embodiment, the RNA molecule described herein is complexed with lipids to generate RNA-lipid nanoparticles (e.g., RNA-LNPs) for administration. In one embodiment, the RNA molecule described herein is complexed with proteins for administration. In one embodiment, the RNA molecule described herein is complexed with lipids and proteins for administration. When different RNA molecule combinations are used, the RNA molecules may be complexed with lipids and / or proteins to produce RNA particles for administration, or they may be complexed separately from lipids and / or proteins.

[0013] This disclosure provides RNA molecules and RNA-LNPs comprising at least one open reading frame (ORF) encoding a FimH antigen and a 5' untranslated region (5'UTR), wherein the 5'UTR comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence described in any one of SEQ ID NOs. 95-101. In some embodiments, the FimH antigen is a FimH polypeptide. In some embodiments, the FimH polypeptide is its full length, cleaved form, fragment, or variant. In some embodiments, the FimH polypeptide comprises at least one mutation.

[0014] This disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF encoding a FimH polypeptide, wherein the FimH polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-64, 77, 79, 81, or 83. In some embodiments, the FimH polypeptide has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to any of the amino acid sequences described in SEQ ID NOs: 1-64, 77, 79, 81, or 83, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity, or has up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity. In some embodiments, the FimH polypeptide consists of any of the amino acid sequences described in SEQ ID NOs: 1-64, 77, 79, 81, or 83.

[0015] In another embodiment, the present disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF encoding a FimH polypeptide, wherein the FimH polypeptide comprises FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), its immunogenic fragment, or any two or more combinations thereof. In some embodiments, the FimH polypeptide has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to any of the amino acid sequences of SEQ ID NOs. 1-64, 77, 79, 81, or 83, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.

[0016] This disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF transcribed from at least one DNA nucleic acid. In some embodiments, the RNA molecule is transcribed from a nucleic acid sequence selected from SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138. In some embodiments, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher % identity to any of the nucleic acid sequences of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher % identity, or having up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher % identity. In some embodiments, the RNA molecule includes an ORF transcribed from a nucleic acid sequence consisting of any of the nucleic acid sequences of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138.

[0017] This disclosure further provides RNA molecules and RNA-LNPs comprising at least one ORF containing an RNA nucleic acid sequence. In some embodiments, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs. 90. In some embodiments, the RNA molecule comprises a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the nucleic acid sequences of SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs. 90, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or having up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the RNA molecule comprises a nucleic acid sequence consisting of any of the nucleic acid sequences of SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs. In some embodiments, the RNA molecule having any of SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs. 90 comprises at least one modified nucleotide. In another embodiment, each uridine in any of SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs. 90 is replaced by a modified nucleotide (e.g., modified RNA; modRNA). In one embodiment, the modified nucleotide is 1-methyl-3'-pseudolyl (also known as pseudouridine) (Ψ). In another embodiment, the modified nucleotide is N 1 -It is methylpseudridine (m1Ψ).

[0018] This disclosure further provides RNA molecules and RNA-LNPs comprising a 5' untranslated region (5'-UTR) and / or a 3' untranslated region (3'-UTR). In some embodiments, the RNA molecule comprises a 5' untranslated region (5'-UTR). In some embodiments, the 5'UTR comprises a sequence selected from any of SEQ ID NOs. 95 to 102. In some embodiments, the 5'UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any of SEQ ID NOs. 95 to 102. In some embodiments, the 5'UTR comprises a sequence consisting of any of SEQ ID NOs. 95 to 102. In another embodiment, the 5'UTR comprises a nucleic acid sequence that is at least 92% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 95, 98, 99, and 101. In some embodiments, the 5'UTR includes a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 95, 99, and 101. In some embodiments, the 5'UTR includes a nucleic acid sequence that is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 99, 101. In some embodiments, the 5'UTR includes a nucleic acid sequence that is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 99, 101. In some embodiments, the 5'UTR includes, Sequence ID 99 (5'UTR_BMD562) and Sequence ID 101 (5'UTR_BMD576) It includes a nucleic acid sequence selected from the group consisting of the following.

[0019] In some embodiments, RNA molecules and RNA-LNPs include a 3' untranslated region (3'-UTR). In some embodiments, the 3'UTR includes a sequence selected from any of SEQ ID NOs. 103 to 106. In some embodiments, the 3'UTR includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to any of SEQ ID NOs. 103 to 106. In some embodiments, the 3'UTR includes a sequence selected from any of SEQ ID NOs. 103 to 106. In some embodiments, the 3'UTR includes a sequence consisting of any of SEQ ID NOs. 103 to 106. In preferred embodiments, the 3'UTR is described in SEQ ID NO. 103.

[0020] This disclosure further provides RNA molecules and RNA-LNPs including a 5' cap portion. This disclosure further provides RNA molecules and RNA-LNPs including a 3' poly-A tail. In some embodiments, the poly-A tail includes a sequence having SEQ ID NO: 93 or SEQ ID NO: 140.

[0021] In some embodiments, the RNA molecule includes a 5'UTR and a 3'UTR. In some embodiments, the RNA molecule includes a 5' cap, a 5'UTR, and a 3'UTR. In some embodiments, the RNA molecule includes a 5' cap, a 5'UTR, a 3'UTR, and a poly-A tail. In some embodiments, the RNA molecule includes a 5' cap, a 3'UTR, and a poly-A tail. In some embodiments, one, two, three, or more of the above elements may be excluded from the RNA molecule. In some embodiments, each uridine in either the 5'UTR, 3'UTR, or poly-A tail is replaced by a modified base. In some embodiments, the modified base is pseudouridine (Ψ). In another embodiment, the modified base is N1-methylpseudridine (m 1 It is Ψ).

[0022] In some embodiments, the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG or (m27,3’-O )Gppp(m 2’-O It's ApG.

[0023] In some embodiments, the length of the poly-A tail may contain +1 / -1 A.

[0024] This disclosure provides RNA molecules as listed in Table 20. In some embodiments, the RNA molecule comprises the 5'UTR of SEQ ID NO: 95, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 96, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In yet another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 97, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 104 and / or the polyA tail of SEQ ID NO: 140. In yet another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 98, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 99, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 100, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 105 and / or the polyA tail of SEQ ID NO: 140. In some embodiments, the RNA molecule comprises the 5'UTR of SEQ ID NO: 95, the FimH ORF of SEQ ID NO: 117, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 102, the FimH ORF of SEQ ID NO: 117, the 3'UTR of SEQ ID NO: 106 and / or the polyA tail of SEQ ID NO: 140. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 95, the FimH ORF of SEQ ID NO: 118, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 102, the FimH ORF of SEQ ID NO: 119, the 3'UTR of SEQ ID NO: 106 and / or the polyA tail of SEQ ID NO: 93. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO: 95, the FimH ORF of SEQ ID NO: 139, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140.In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 99, the FimH ORF of SEQ ID NO: 118, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In some aspects, the RNA molecule comprises the 5'UTR of SEQ ID NO: 99, the FimH ORF of SEQ ID NO: 139, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 101, the FimH ORF of SEQ ID NO: 118, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO: 140. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 101, the FimH ORF of SEQ ID NO: 139, the 3'UTR of SEQ ID NO: 103 and / or the polyA tail of SEQ ID NO:140. In some aspects, the FimH ORF further comprises a stop codon as described herein. In some aspects, the length of the polyA tail can contain +1 / -1 A or +2 / -2 A. In some aspects, each uridine of the RNA molecule is replaced by pseudouridine (Ψ). In some aspects, each uridine of the RNA molecule is replaced by N1-methylpseudouridine (m. 1 Ψ).

[0025] This disclosure further provides RNA molecules comprising at least one open reading frame generated from codon-optimized DNA. In some embodiments, the open reading frame comprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%, up to a maximum of 50%, 51%, 52%, 5 3%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75%, exactly 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6 Includes G / C content of 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%, or between any two of the following (including or excluding both ends): for example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 50% to 75% or approximately 50% to 75%, or 55% to 70% or approximately 55% to 70%. In some embodiments, the G / C content is 58% or approximately 58%, 66% or approximately 66%, or 62% or approximately 62%.

[0026] This disclosure further provides RNA molecules comprising at least one codon-optimized open reading frame. This disclosure further provides RNA molecules comprising stabilized RNA. This disclosure further provides RNA molecules comprising RNA having at least one modified nucleotide. In some embodiments, the modified nucleotide is pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the modified nucleotide is pseudouridine(Ψ). In some embodiments, one, two, three, four, five, or more of the above-mentioned modified nucleotides may be excluded from the RNA molecule.

[0027] This disclosure further provides RNA molecules that are messenger RNA (mRNA) and can be nucleoside-modified RNA (modRNA). In some embodiments, the RNA is mRNA. In other embodiments, the RNA is modRNA.

[0028] This disclosure further provides immunogenic compositions comprising RNA molecules as described herein. The RNA molecules may be formulated in lipid nanoparticles (LNPs) (e.g., FimH RNA-LNPs) in such immunogenic compositions, encapsulated within them, complexed with them, bound to them, or adsorbed thereto. In some embodiments, the lipid nanoparticles comprise at least one of cationic lipids, polymer-conjugated lipids (e.g., PEG-lipids), and at least one structural lipid (e.g., neutral lipids and steroids or steroid analogs). In some embodiments, one, two, three, or more of these lipids may be excluded from the lipid nanoparticles.

[0029] In some embodiments, the lipid nanoparticles include cationic lipids. In some embodiments, the cationic lipid is (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0030] In some embodiments, lipid nanoparticles include lipids conjugated to a polymer. In some embodiments, lipid nanoparticles include PEG lipids, also referred to as PEG-modified lipids. In some embodiments, PEG lipids include glycol lipids containing PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEG-modified diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy-polyethylene glycol)-2,3-di Myristoyl glycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), for example, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, for example, co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the PEG lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0031] In some embodiments, the lipid nanoparticles include at least one structural lipid, such as a neutral lipid. In some embodiments, the neutral lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4- The following are selected from (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and / or 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0032] In some embodiments, the lipid nanoparticles include a second structural lipid, such as a steroid or steroid analog. In some embodiments, the steroid or steroid analog is cholesterol.

[0033] In some embodiments, lipid nanoparticles range from approximately 1 to approximately 500 nm, for example, at least 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, or 500nm, up to 1nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 28 0nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, or 500nm, exactly 1nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm m, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, or 500nm, or 1nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm,It has an average diameter between any two of the following (including or excluding both ends): 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, or 500nm.

[0034] In some embodiments, the RNA-LNP immunogenic composition is encapsulated in an LNP having a lipid composition of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, and up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, and exactly 0. A liquid RNA-LNP composition comprising an RNA polynucleotide encoding a FimH polypeptide disclosed herein in a concentration of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, and further comprising a buffering composition comprising a first buffer at a concentration of 0.15 to 0.3 mg / mL, a second buffer at a concentration of 1.25 to 1.4 mg / mL, and a stabilizer at a concentration of 95 to 110 mg / mL. In some embodiments, the RNA-LNP immunogenic composition contains 0.8 to 0.95 mg / mL or approximately 0.8 to 0.95 mg / mL (for example, at least 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg / mL, and at most 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg / mL). Cationic lipids at concentrations of exactly 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg / mL, or between any two of 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg / mL (with or without the two ends), 0.05-0.15 mg / mL, or approximately 0.0.05-0.15 mg / mL (for example, at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, and at most 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or exactly 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or 0.05, 0.06, 0.07, 0.08, 0.09, 0 PEGylated lipids at concentrations between any two of 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg / mL (with or without including both ends), 0.1–0.25 mg / mL or approximately 0.1–0.25 mg / mL (e.g., at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg / mL, with a maximum of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.1 6, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, exactly 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, or any two of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL A concentration of the first structural lipid (including or excluding both ends), and 0.3–0.45 mg / mL or approximately 0.3–0.45 mg / mL (e.g., at least 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, with a maximum of 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.0.7 mg / mL encapsulated in an LNP having a lipid composition containing a second structural lipid at a concentration of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.7 mg / mL, precisely between any two of the following (including or excluding both ends): 45 mg / mL, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg / mL. A liquid RNA-LNP composition comprising an RNA molecule / polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of 5 or 0.90 mg / mL, up to a maximum of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL (with or without both ends), preferably at a concentration of 0.01 to 0.09 mg / mL or about 0.01 to 0.09 mg / mL. In some embodiments, the liquid composition contains 0.1 to 0.3 mg / mL or about 0.1 to 0.3 mg / mL (for example, at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30 mg / mL, and at most 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.1 7, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30 mg / mL, precisely 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30 mg / mL, or 0.10, 0.11, 0.12, 0.A first buffer at a concentration between any two of the following (including or excluding both ends): 13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 mg / mL; 1.25–1.4 mg / mL or approximately 1.25–1.4 mg / mL (for example, at least 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg / mL L, up to 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg / mL, exactly 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1. 32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg / mL, or 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.A second buffer at a concentration between any two of the 40 mg / mL ranges (with or without the ends), and a concentration of 95–110 mg / mL or approximately 95–110 mg / mL (for example, at least 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg / mL, with a maximum of 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 The buffering composition further comprises a stabilizer at a concentration of 109 or 110 mg / mL, precisely 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg / mL, or between any two of the following concentrations (with or without the ends): 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg / mL. In some embodiments, one, two, three, four, five or more of the above elements may be excluded from the liquid RNA-LNP composition. In some embodiments, one, two, three, four, five or more of the above element concentrations may be excluded from the liquid RNA-LNP composition.

[0035] In a specific embodiment, the liquid RNA-LNP immunogenic composition contains 0.8 to 0.95 mg / mL or about 0.8 to 0.95 mg / mL (for example, at least 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, and at most 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL). (4-hydroxybutyl)azanediyl) bis(b) at concentrations exactly 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, or between any two of 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL (with or without the ends). (Hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 0.05-0.15 mg / mL or approximately 0.05-0.15 mg / mL (for example, at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, up to 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, exactly 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.1 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at concentrations of 1, 0.12, 0.13, 0.14 or 0.15 mg / mL, or between any two of 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL (including or excluding both ends), 0.1 to 0.25 mg / mL or approximately 0.1 to 0.25 mg / mL (e.g., at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, up to 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, exactly 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, or 0.1 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in concentrations between any two of the following (including or excluding the two extremes): 0, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg / mL, and 0.3-0.45 mg / mL or approximately 0.3-0.45 mg / mL (for example, at least 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, up to a maximum of 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, exactly 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, or 0.30, 0.31, 0.32, 0.33, 0.34, 0. Encapsulated in LNPs having a lipid composition containing cholesterol at concentrations between any two of the following (including or excluding the ends): 35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg / mL, with a maximum of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, and exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.60, 0.90 mg / mL.The solution comprises an RNA molecule / polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of 75 or 0.90 mg / mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL (with or without the ends), preferably 0.01 to 0.09 mg / mL or about 0.01 to 0.09 mg / mL. In some embodiments, the liquid composition contains 0.1 to 0.3 mg / mL or about 0.1 to 0.3 mg / mL (for example, at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30 mg / mL, and at most 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0. 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30 mg / mL, precisely 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29 or 0.30 mg / mL, or 0.10 Tromethamine in concentrations between any two of the following (including or excluding the two extremes): 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 mg / mL, and 1.25–1.4 mg / mL or approximately 1.25–1.4 mg / mL (for example, at least 1.25, 1.26 , 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg / mL, up to a maximum of 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg / mL, exactly 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.Tris hydrochloride (HCl) at concentrations of 31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg / mL, or between any two of 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg / mL (with or without the ends), and Tris hydrochloride (HCl) at concentrations of 95-110 mg / mL or approximately 95-110 mg / mL (for example, at least 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 The Tris buffering composition further comprises sucrose in a concentration of 109 or 110 mg / mL, up to a maximum of 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg / mL, exactly 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg / mL, or between any two of 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg / mL (with or without the ends). In some embodiments, one, two, three, four, five, or more of the above elements may be excluded from the liquid RNA-LNP composition. In some embodiments, one, two, three, four, five, or more of the above element concentrations may be excluded from the liquid RNA-LNP composition.

[0036] In some embodiments, the liquid RNA-LNP immunogenic composition is encapsulated within an LNP having a lipid composition of ALC-0315 at a concentration of 0.8-0.95 mg / mL, ALC-0159 at a concentration of 0.05-0.15 mg / mL, DSPC at a concentration of 0.1-0.25 mg / mL, and cholesterol at a concentration of 0.3-0.45 mg / mL, with a maximum of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL. The Tris buffering composition further comprises an RNA polynucleotide encoding a FimH polypeptide disclosed herein in a concentration of 90 mg / mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, preferably 0.01 to 0.09 mg / mL or about 0.01 to 0.09 mg / mL, and tromethamine in a concentration of 0.1 to 0.3 mg / mL, Tris HCl in a concentration of 1.25 to 1.4 mg / mL, and sucrose in a concentration of 95 to 110 mg / mL.In some embodiments, the LNP is 5–15 mM or approximately 5–15 mM of Tris buffer (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mM, up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mM, exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mM, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mM (with or without the ends)) and 200–400 mM or approximately 200-400 mM sucrose (for example, at least 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 mM, up to 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 mM, exactly 200, 210, 220, 230, 2 40, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 mM, or between any two of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 mM (with or without the ends)), between 7.0 and 8.0 or approximately 7.0 and 8.0 (for example, at least 7.0, 7 pH further includes 0.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, up to a maximum of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, exactly 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, or between any two of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0 (with or without the ends). In some embodiments, one, two, three or more of the above elements may be excluded from the liquid RNA-LNP composition.In some embodiments, one, two, three, four, five or more of the above element concentrations may be excluded from the liquid RNA-LNP composition.

[0037] In a specific embodiment, the RNA-LNP immunogenic composition contains 0.8 to 0.95 mg / mL (for example, at least 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, and at most 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, exactly 0.80, 0.81, 0.82, 0. Cationic lipids at concentrations of 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, or between any two of 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL (with or without the ends), and 0.05 to 0.15 mg / mL (for example, at least 0.05, 0.06, 0.07, 0.08 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, up to a maximum of 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, exactly 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or any two of 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL PEGylated lipids at concentrations between two ends (including or excluding both ends), 0.1–0.25 mg / mL (e.g., at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, and at most 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, exactly 0.10, 0.A first structural lipid at a concentration of 11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL, or between any two of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg / mL (with or without including both ends), and 0.3~0.45 mg / mL of (for example, at least 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, and at most 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, exactly 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36 , 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, or 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL encapsulated in an LNP having a lipid composition of a second structural lipid at a concentration between any two of these (including or excluding both ends), at least 0.01, 0.15, 0.30, 0 0.45, 0.60, 0.75 or 0.90 mg / mL, up to a maximum of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL (with or without both ends), preferably between 0.01 and 0.09 mg / mL or about 0.01 and 0.The lyophilized (restored) RNA-LNP composition comprises an RNA polynucleotide encoding the FimH polypeptide disclosed herein at a concentration of 0.9 mg / mL. In some embodiments, the lyophilized composition contains 0.01 to 0.15 mg / mL of (e.g., at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, and up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.1) for restoration. 3. 0.14 or 0.15 mg / mL, exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or any two of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL ( A first buffering agent of a concentration of 0.5 to 0.65 mg / mL (including or excluding both ends), for example, at least 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64 or 0.65 mg / mL, with a maximum of 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.6 1, 0.62, 0.63, 0.64, or 0.65 mg / mL, exactly 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg / mL, or 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.A second buffer at a concentration between any two of the 65 mg / mL ranges (including or excluding both ends), 35-50 mg / mL (for example, at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL, at most 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL, exactly 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL, or 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, The mixture further comprises a stabilizer in a concentration between any two of 46, 47, 48, 49, or 50 mg / mL (with or without the ends), and a salt in a concentration between 5 and 15 mg / mL (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL, with a maximum of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL, exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL (with or without the ends)). In a specific embodiment, the freeze-dried composition contains 0.6 to 0.75 mL of carrier or diluent (for example, at least 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.75 mL, and a maximum of 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.75 mL). 1, 0.72, 0.73, 0.74 or 0.75 mL, or exactly 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.75 mL, or 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.The reconstitution occurs within any two of the 75 mL values ​​(including or excluding both ends). The concentration in the lyophilized RNA-LNP composition is determined after reconstitution. In some embodiments, one, two, three, four, five, or more of the above elements may be excluded from the lyophilized RNA-LNP composition. In some embodiments, one, two, three, four, five, or more of the above element concentrations may be excluded from the lyophilized RNA-LNP composition.

[0038] In a specific embodiment, the freeze-dried (restored) RNA-LNP composition contains 0.8 to 0.95 mg / mL or approximately 0.8 to 0.95 mg / mL (for example, at least 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, and at most 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0. ALC-0315 at concentrations of 0.95 mg / mL, precisely 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL, or between any two of 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg / mL (with or without the two ends), and 0.05~0.15 mg / mL Or approximately 0.05-0.15 mg / mL (for example, at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, and at most 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or exactly 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or 0.05, 0.06, 0.07, 0.08, 0. ALC-0159 in concentrations between any two of 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg / mL (with or without including both ends), 0.1 to 0.25 mg / mL, or approximately 0.1 to 0.25 mg / mL (for example, at least 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg / mL, with a maximum of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg / mL, exactly 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg / mL, or any two of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg / mL DSPC at concentrations of 0.3–0.45 mg / mL or approximately 0.3–0.45 mg / mL (e.g., at least 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, and at most 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg Encapsulated in LNPs having a cholesterol lipid composition at concentrations of exactly 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL, or between any two of 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg / mL (with or without the two ends), At least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, and at most 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg / mL (with or without both ends), preferably 0.01 to 0.09 mg / mL or about 0.01 to 0.The present invention provides an RNA polynucleotide encoding the FimH polypeptide disclosed herein at a concentration of 0.9 mg / mL, and for restoration, 0.01–0.15 mg / mL or about 0.01–0.15 mg / mL (e.g., at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, with a maximum of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0. 0.7, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, precisely 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL, or 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg / mL Tromethamine in concentrations between any two of L (including or excluding both ends), and 0.5–0.65 mg / mL or approximately 0.5–0.65 mg / mL (e.g., at least 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64 or 0.65 mg / mL, and at most 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0. 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg / mL, exactly 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg / mL, or 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.Tris concentrations between any two of the 65 mg / mL ranges (including or excluding both ends) HCl, 35-50 mg / mL or approximately 35-50 mg / mL (for example, at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL, at most 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL, exactly 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL, or between any two of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL (both ends) The Tris buffering composition further comprises sucrose in a concentration of (including or excluding both ends) and a sodium chloride (NaCl) diluent in a concentration of 5 to 15 mg / mL or about 5 to 15 mg / mL (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL, up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL, exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL (including or excluding both ends)). In a specific embodiment, the freeze-dried composition contains 0.6 to 0.75 mL or about 0.6 to 0.75 mL of sodium chloride (for example, at least 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.75 mL, and at most 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66 , 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.75 mL, exactly 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74 or 0.75 mL, or 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.The concentration is restored between any two of the following volumes: 69, 0.70, 0.71, 0.72, 0.73, 0.74, or 0.75 mL (with or without including both ends). The concentration in the lyophilized RNA-LNP composition is determined after restoration. In some embodiments, one, two, three, four, five, or more of the above elements may be excluded from the lyophilized RNA-LNP composition. In some embodiments, one, two, three, four, five, or more of the above element concentrations may be excluded from the lyophilized RNA-LNP composition.

[0039] This disclosure provides RNA molecules, RNA-LNPs, and immunogenic compositions in which FimH RNA encapsulated in LNPs can be administered to a subject in doses of at least 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more, and up to 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more, or exactly between any two of the following (including or excluding both ends): 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more. In some embodiments, one, two, three, four, five or more of the above concentrations of FimH RNA encapsulated in the LNP may be excluded.

[0040] This disclosure provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered in a single dose. This disclosure further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered in two doses (for example, on day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 1 month later, day 0 and 2 months later, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later). This disclosure further provides RNA molecules, RNA-LNPs, and immunogenic compositions that may be administered twice, on day 0 and 2 months later. This disclosure further provides RNA molecules, RNA-LNPs, and immunogenic compositions that may be administered twice, on day 0 and 6 months later. This disclosure further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more doses. In some embodiments, periodic boosters at intervals of 1 to 5 years may be desired to maintain protective levels of the antibody. This disclosure further provides administration of at least one booster dose. In some embodiments, one, two, three, four, five, or more of the above dosing regimens may be excluded.

[0041] This disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP, and / or immunogenic composition described herein. This disclosure further provides the use of the RNA molecule, RNA-LNP, and / or immunogenic composition described herein in the production of a pharmaceutical for use in inducing an immune response in a subject.

[0042] This disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of an RNA molecule and / or RNA-LNP or composition containing at least one open reading frame encoding the FimH polypeptide described herein. This disclosure further provides the use of an RNA molecule and / or RNA-LNP or composition containing at least one open reading frame encoding the FimH polypeptide described herein in the production of a pharmaceutical for use in inducing an immune response in a subject.

[0043] This disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of an RNA molecule and / or RNA-LNP or composition comprising at least one open reading frame encoding a polypeptide of a gene of interest described herein. This disclosure further provides the use of an RNA molecule and / or RNA-LNP or composition comprising at least one open reading frame encoding a polypeptide of a gene of interest described herein in the production of a pharmaceutical for use in inducing an immune response in a subject.

[0044] This disclosure provides a method for preventing, treating or alleviating an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP, and / or immunogenic composition described herein. This disclosure further provides the use of the RNA molecule, RNA-LNP, and / or immunogenic composition described herein in the production of a pharmaceutical for use in the prevention, treatment, or alleviation of an infection, disease, or condition in a subject. In some embodiments, the infection or condition is associated with Escherichia coli (E. coli) FimH. In some embodiments, the infection, disease, or condition is a urinary tract infection (UTI), urinary tract sepsis, cystitis, or pyelonephritis.

[0045] This disclosure provides a method for preventing, treating or alleviating an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule and / or RNA-LNP or immunogenic composition containing at least one open reading frame encoding the FimH polypeptide described herein. This disclosure further provides the use of an RNA molecule and / or RNA-LNP or immunogenic composition containing at least one open reading frame encoding the FimH polypeptide described herein in the production of a medicament for use in the prevention, treatment, or alleviation of an infection, disease, or condition in a subject. In some embodiments, the infection, disease, or condition is associated with Escherichia coli (E. coli) FimH. In some embodiments, the infection, disease, or condition is a urinary tract infection (UTI), urinary tract sepsis, cystitis, or pyelonephritis.

[0046] This disclosure further provides a method for preventing, treating or alleviating an infection, disease, or condition in a subject, comprising administering to the subject an RNA molecule and / or RNA-LNP or immunogenic composition containing at least one open reading frame encoding a polypeptide of a gene of interest described herein in an effective amount. This disclosure further provides the use of an RNA molecule and / or RNA-LNP or immunogenic composition containing at least one open reading frame encoding a polypeptide of a gene of interest described herein in the production of a medicament for use in the prevention, treatment, or alleviation of an infection, disease, or condition in a subject. In some embodiments, the infection, disease, or condition is associated with the gene of interest.

[0047] In some embodiments, subjects are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of age or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years or older, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of age or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years or older, exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of age or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years or older, or between any two of the following ages (including or excluding both ends): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of age or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years or older. In some embodiments, subjects are under approximately 1 year of age, approximately 1 year or older, approximately 5 years or older, approximately 10 years or older, approximately 20 years or older, approximately 30 years or older, approximately 40 years or older, approximately 50 years or older, approximately 60 years or older, approximately 70 years or older, or older, at least these ages, or at most these ages. In some embodiments, subjects are approximately 50 years or older. In further embodiments, subjects are 6 months to 1 year, 1 year to 2 years, 1 year to 3 years, 1 year to 4 years, 1 year to 5 years, 6 months to 5 years, or 60 years or older. The whole birth cohort is included as a relevant population for immunization. This can be done, for example, by initiating an immunization regimen in pregnant women (or women of childbearing age) to protect infants by passive transfer of antibodies, at any point between birth and 6 months of age, between 6 months and 5 years of age, and in subjects over 50 years of age. In some embodiments, one, two, three, four, five or more of the above age groups are not administered RNA molecules and / or RNA-LNPs.

[0048] In some embodiments, the subject is human. In some specific embodiments, the human is a child, for example, an infant. In some other specific embodiments, the human is a woman, in particular a pregnant woman. In some embodiments, the subject is immunocompetent. In some embodiments, the subject is immunodeficient.

[0049] This disclosure provides methods or uses described herein in which RNA molecules, RNA-LNPs and / or immunogenic compositions are administered as vaccines.

[0050] This disclosure provides methods or uses described herein for administering RNA molecules, RNA-LNPs, and / or immunogenic compositions by intradermal or intramuscular injection.

[0051] One embodiment of the present invention provides an Escherichia coli (E. coli) vaccine comprising at least one ribonucleic acid polynucleotide having an open reading frame encoding at least one FimH antigen polypeptide (RNA) or an immunogenic fragment thereof, formulated in lipid nanoparticles.

[0052] In one embodiment of the Escherichia coli (E. coli) vaccine, the RNA further comprises a 5' cap analog. In a preferred embodiment, the 5' cap analog is m7G(5')ppp(5')(2'OMeA)pG or N 1 Contains -methylpseudridine-5'-triphosphate

[0053] In another embodiment of the E. coli vaccine, the RNA further comprises modified nucleotides.

[0054] In another embodiment of the Escherichia coli (E. coli) vaccine, at least one antigen polypeptide is FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), its immunogenic fragment, or any two or more of the above in combination.

[0055] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine comprises: a) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding FimH-DSG (SEQ ID NO: 59); b) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62); or c) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54).

[0056] In another embodiment of the E. coli vaccine, the RNA molecule encoding the FimH polypeptide is fused to the C-terminal membrane targeting domain.

[0057] In another embodiment of the E. coli vaccine, the RNA encoding the FimH polypeptide is fused to a C-terminal membrane targeting domain, and the two are separated by a linker. In a preferred embodiment of the E. coli vaccine, the encoded linker has the amino acid sequence GSSGSGSS (SEQ ID NO: 94).

[0058] In another embodiment of the E. coli vaccine, the C-terminal membrane targeting domain is a glycoprotein. In another embodiment of the E. coli vaccine, the membrane targeting sequence is derived from a human DAF protein GPI sequence or a synthetic GPI sequence.

[0059] In another embodiment of the Escherichia coli (E. coli) vaccine, FimH is secreted and lacks a C-terminal membrane targeting domain.

[0060] In another aspect of the E. coli vaccine, the RNA-encoded open reading frame is codon-optimized.

[0061] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine further comprises cationic lipids.

[0062] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine comprises lipid nanoparticles containing RNA molecules.

[0063] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine comprises: a) lipid nanoparticles containing at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding FimH-DSG; b) lipid nanoparticles containing at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a FimH-DSG triple mutant (G15A, G16A, V27A); or c) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54).

[0064] In another embodiment of the E. coli vaccine, the lipid nanoparticle size is at least 40 nm. In yet another embodiment of the E. coli vaccine, the lipid nanoparticle size is up to 180 nm.

[0065] In another embodiment of the E. coli vaccine, at least 80% of the total RNA in the composition is encapsulated.

[0066] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine contains ALC-0315(4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0067] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine contains ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

[0068] In another embodiment of the Escherichia coli (E. coli) vaccine, the vaccine contains 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0069] In another embodiment of the Escherichia coli (E. coli) vaccine, the RNA polynucleotide comprises a 5' cap, a 5' UTR, a 3' UTR, and a poly-A tail.

[0070] In another embodiment of the E. coli vaccine, each uridine is replaced with a modified base, which is pseudouridine (Ψ) or N. 1 -methyl-pseudridine(m 1 It is Ψ).

[0071] In another embodiment of the E. coli vaccine, the polyA tail is 80 nucleotides long.

[0072] In another embodiment of the Escherichia coli (E. coli) vaccine, the FimH polypeptide includes serine substitutions at positions N228 and N235.

[0073] All methods described herein may be performed in any preferred order, unless otherwise indicated herein or otherwise clearly contradict the context. Any and all examples or exemplary expressions provided herein (e.g., “For Example”) are intended solely to illustrate further examples of the disclosure and do not impose any limitation on the scope of the claims. Nothing expressed herein should be construed as referring to any unclaimed element essential to the practice of the disclosure.

[0074] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in the use-claim language, such as "use" of any of the compounds, compositions, or agents described herein, in order to achieve or perform the therapeutic, diagnostic, or physiological purpose or effect described herein. The use of one or more compositions may be performed based on any of the methods described herein.

[0075] Several documents are referenced throughout the text of this disclosure. Each of the documents referenced herein (including all patents, patent applications, scientific publications, producer specifications, instructions, etc.), whether above or below, is incorporated into this specification by reference in its entirety. Nothing in this specification should be construed as an acknowledgment that this disclosure did not have any rights prior to such disclosure in terms of date.

[0076] Any aspect discussed herein may be carried out with respect to any method or composition of the Disclosure, and vice versa. Furthermore, any composition of the Disclosure may be used to achieve any method of the Disclosure.

[0077] Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while pointing to specific aspects of this disclosure, are given only as examples, and various modifications and alterations within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0078] [Figure 1] Figures 1A and 1B describe the FimH gene variants of the present invention. Figure 1A shows the tertiary structure of FimH highlighting the positions of glycine loop mutations G15A and G16A and the native variant V27A. A 7-residue Gly-Ser linker separates the pyrin domain C-terminus from the donor chain G(DsG) peptide. Figure 1B shows a linear representation of the FimHLD and FimHDSG genes having an N-terminal IgK signal peptide ([SP]), lectin and pyrin domain mutations, a G peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor. Legend: SP, mouse IgG copper signal peptide; FimHLD, FimH lectin domain; gpi, glycosylphosphatidylinositol anchor. This refers to FimH amino acid substitutions introduced to prevent N-glycosylation (N7S, N70S, N228S, and N235S) or to stabilize conformation (G15A, G16A, V27A, also known as the triple mutant "TM"); G, a stabilizing donor chain peptide added to the C-terminus of the full-length FimH protein. [Figure 2] Figure 2 shows the positive surface expression of FimH using the modRNA LNP of the present invention on the surface of Expi293 suspension cells, as determined by flow cytometry. [Figure 3]Figures 3A and 3B describe the neutralizing antibody titers induced by FimH modRNA LNP in PD2 (6-week) mouse serum. The results of the BMD2 and WHO vector comparison are shown in Figure 3A and summarized in Figure 3B, demonstrating that FimHDSG modRNA is more immunogenic than modRNA expressing FimHLD or secreted FimHDSG. p-values ​​were derived from pairwise comparisons of log-transformed data using an unpaired t-test with Welch correction (GraphPad Prism): ***p<0.0001, *p<0.05, ns-not significant. [Figure 4] Figures 4A and 4B demonstrate that the FimHDSG-GPI constructs described herein are equivalent to the benchmarks. Figure 4A shows the functional titers induced by various full-length FimHDSG-GPI modRNA LNPs (including BMD2 and the benchmark group shown in Figures 3A and 3B), which are summarized in Figure 4B. [Figure 5] Figures 5A to 5D show that the membrane-targeted FimH construct of the present invention induces a robust Th1 response. [Figure 6] Figure 6 shows that the membrane-targeted FimH construct of the present invention induces a strong CD8 T cell response. [Figure 7] Figures 7A and 7B illustrate the substitution of a GPI-anchored peptide by the Gly-Ser linker (SEQ ID NO: 94). Figure 7A shows the covalent attachment of the glycolipid anchor to the ω-site serine, where the N-terminal signal peptide (black box) and C-terminal GPI domain are processed in the endoplasmic reticulum (ER), followed by cleavage of the C-terminal polypeptide by GPI transamidase (white box). The illustration is from Kinoshita T. 2020. Open Biol 10:190290. Figure 7B shows the substitution of eight DAF amino acid residues proximal to the ω-site serine by the serine / glycine linker. mlgK, mouse IgK signal peptide. Not drawn to exact scale. [Figure 8]Figures 8A and 8B demonstrate that substitution of the DAF GPI peptide with the GlySer linker does not affect FimH expression on the surface of transfected Expi293 cells. Expi293 cells were transfected with modRNA, and FimH surface expression was detected by flow cytometry using FimH mAb 926. Figure 8A shows the mean fluorescence intensity (MFI) of surface staining. Figure 8B shows the percentage of stain-positive cells based on interpolated EC50 titers. Titers were determined using 4-parameter sigmoid curve fitting (GraphPad Prism). [Figure 9] Figures 9A and 9B show the neutralizing titers at PD2 (week 6). Neutralizing titers against *E. coli* were assessed two weeks after mice received a second 1 μg dose of modRNA LNP (results are shown in Figure 9A and summarized in Figure 9B). The indicated p-values ​​were derived from pairwise comparisons of log-transformed data using an unpaired t-test with Welch correction (GraphPad Prism). [Figure 10] Figures 10A–10D describe antigen-specific T cell responses in spleen cells derived from vaccinated mice. The results indicate that membrane-targeted FimHDSG candidates induce a robust Th1 response. p-values ​​were derived from pairwise comparisons of log-transformed data using an unpaired t-test with Welch correction (GraphPad Prism): ***p<0.0001, **p<0.005, *p<0.05. [Figure 11] Figure 11 depicts the antigen-specific T cell response in vaccinated mouse spleen cells, demonstrating that membrane-targeted FimHDSG candidates induce a strong CD8+ T cell response. p-values ​​were derived from pairwise comparisons of log-transformed data using an unpaired t-test with Welch correction (GraphPad Prism): ***p<0.0001, **p<0.005, *p<0.05, ns-not significant. [Figure 12]Figures 12A–12D demonstrate that FimH-DSG gpi-anchored modRNA and FimH-DSG secreted modRNA are immunogenic in non-human primates. Figure 12A depicts a schematic schedule of immunization, bleeding, and loading (single experiment with n=9 NHP / group). Figures 12B–12D show various titers from NHP immunized with placebo (circles), FimH-DSG V27A G15A G16A (subunit proteins) and LiNA-2 adjuvant (squares), FimH-DSG gpi-anchored modRNA (triangles), and FimH-DSG secreted modRNA (inverted triangles) at weeks 0, 4, and 14. Figure 12B depicts neutralization titers (Log IC50) in the yeast mannan neutralization assay at pre-vaccination and post-dose 2 and post-dose 3. Figure 12C depicts serum anti-FimH IgG titers measured 2 and 3 post-dose. Each symbol represents an individual animal. Error bars indicate the geometric mean by 95% CI. The dotted line indicates the limit of quantification (LLOQ) of the assay. Figure 12D depicts urinary anti-FimH IgG titers measured 2 and 3 post-dose. [Figure 13] Figures 13A and 13B show reduced bacteriuria in NHP immunized with FimH modRNA and FimH subunit protein. Bacteriuria was quantified by qPCR over a 28-day period (Figure 13A). NHP was immunized at weeks 0, 4, and 14 with placebo (circles), FimH subunit protein (squares), FimH gpi-anchored modRNA (triangles), and FimH secreted modRNA (inverted triangles). At week 19, UPEC isolate PFEEC0578 with 10⁸ colony-forming units (CFUs) was injected via catherization. Urine samples were collected via catherization at the indicated post-infection days. Viable bacterial colonies were also evaluated using urine samples (Figure 13B). Each symbol represents an individual animal. Error bars indicate the geometric mean by 95% CI. The ratio indicates the number of NHPs with urine samples containing 100 bacteria across the total number of animals tested. The dotted line indicates the limit of quantification (LLOQ) of the assay. [Figure 14] Figure 14 shows reduced inflammatory biomarkers in NHP vaccinated with FimH-DSG G15A, G16A, and V27A, and FimH modRNA. The concentration of interleukin-8 (IL-8) in urine over a 28-day period post-infection is shown. Each symbol represents an individual animal. Error bars indicate the geometric mean by 95% confidence interval. The dotted line represents the lower limit of quanitization (LLOQ) of the assay. [Figure 15] Figures 15A-15C show the FimH-induced CD4+ T cell response. The vaccination schedule is shown in Figure 12A. PBMCs obtained at week 0 (pre-prime) and week 16 (7 days after the third boost for FimHgpi modRNA, FimH secreted modRNA, and FimH subunit protein) were stimulated with FimH peptide, and cytokine production by CD4+ and CD8+ T cells was evaluated by flow cytometry. The percentages of CD4+ T cells producing TNFα (Figure 15A), IFN-γ (Figure 15B), and IL-2 (Figure 15C) are shown. After immunization one week after the third boost following peptide stimulation, no CD8+ T cell response was induced (data not shown). [Modes for carrying out the invention]

[0079] Array identification symbol Sequence ID 1 is wild-type Escherichia coli (E. coli) FimH LD The amino acid sequence of (FimHLD_WT) is listed below.

[0080] Sequence ID 2 describes the amino acid sequence of the mutant E. coli (FimHLD_G65A_V27A).

[0081] Sequence ID 3 describes the amino acid sequence of the mutant E. coli (FimHLD_F1I).

[0082] Sequence ID 4 describes the amino acid sequence of the mutant E. coli (FimHLD_F1L).

[0083] Sequence ID 5 describes the amino acid sequence of the mutant E. coli (FimHLD_F1V).

[0084] Sequence ID 6 describes the amino acid sequence of the mutant E. coli (FimHLD_F1M).

[0085] Sequence ID 7 describes the amino acid sequence of the mutant E. coli (FimHLD_F1Y).

[0086] Sequence ID 8 describes the amino acid sequence of the mutant E. coli (FimHLD_F1W).

[0087] Sequence ID 9 describes the amino acid sequence of the mutant E. coli (FimHLD_Q133K).

[0088] Sequence ID 10 describes the amino acid sequence of the mutant E. coli (FimHLD_G15A).

[0089] Sequence ID 11 describes the amino acid sequence of the mutant E. coli (FimHLD_G15P).

[0090] Sequence ID 12 describes the amino acid sequence of the mutant E. coli (FimHLD_G16A).

[0091] Sequence ID 13 describes the amino acid sequence of the mutant E. coli (FimHLD_G16P).

[0092] Sequence ID 14 describes the amino acid sequence of the mutant E. coli (FimHLD_G15A_G16A).

[0093] Sequence ID 15 describes the amino acid sequence of the mutant E. coli (FimHLD_R60P).

[0094] Sequence ID 16 describes the amino acid sequence of the mutant E. coli (FimHLD_G65A).

[0095] Sequence ID 17 describes the amino acid sequence of the mutant E. coli (FimHLD_P12C_A18C).

[0096] Sequence ID 18 describes the amino acid sequence of the mutant E. coli (FimHLD_G14C_F144C).

[0097] Sequence ID 19 describes the amino acid sequence of the mutant E. coli (FimHLD_P26C_V35C).

[0098] Sequence ID 20 describes the amino acid sequence of the mutant E. coli (FimHLD_P26C_V154C).

[0099] Sequence ID 21 describes the amino acid sequence of the mutant E. coli (FimHLD_P26C_V156C).

[0100] Sequence ID 22 describes the amino acid sequence of the mutant E. coli (FimHLD_V27C_L34C).

[0101] Sequence ID 23 describes the amino acid sequence of the mutant E. coli (FimHLD_V28C_N33C).

[0102] Sequence ID 24 describes the amino acid sequence of the mutant E. coli (FimHLD_V28C_P157C).

[0103] Sequence ID 25 describes the amino acid sequence of the mutant E. coli (FimHLD_Q32C_Y108C).

[0104] Sequence ID 26 describes the amino acid sequence of the mutant E. coli (FimHLD_N33C_L109C).

[0105] SEQ ID NO: 27 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_N33C_P157C.

[0106] SEQ ID NO: 28 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_V35C_L107C.

[0107] SEQ ID NO: 29 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_V35C_L109C.

[0108] SEQ ID NO: 30 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_S62C_T86C.

[0109] SEQ ID NO: 31 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_S62C_L129C.

[0110] SEQ ID NO: 32 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_Y64C_L68C.

[0111] SEQ ID NO: 33 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_Y64C_A127C.

[0112] SEQ ID NO: 34 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_L68C_F71C.

[0113] SEQ ID NO: 35 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_V112C_T158C.

[0114] SEQ ID NO: 36 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_S113C_G116C.

[0115] SEQ ID NO: 37 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_S113C_T158C.

[0116] Sequence ID 38 describes the amino acid sequence of the mutant E. coli (FimHLD_V118C_V156C).

[0117] Sequence ID 39 describes the amino acid sequence of the mutant E. coli (FimHLD_A119C_V155C).

[0118] Sequence ID 40 describes the amino acid sequence of the mutant E. coli (FimHLD_L34N_V27A).

[0119] Sequence ID 41 describes the amino acid sequence of the mutant E. coli (FimHLD_L34S_V27A).

[0120] Sequence ID 42 describes the amino acid sequence of the mutant E. coli (FimHLD_L34T_V27A).

[0121] Sequence ID 43 describes the amino acid sequence of the mutant E. coli (FimHLD_A119N_V27A).

[0122] Sequence ID 44 describes the amino acid sequence of the mutant E. coli (FimHLD_A119S_V27A).

[0123] Sequence ID 45 describes the amino acid sequence of the mutant E. coli (FimHLD_A119T_V27A).

[0124] Sequence ID 46 describes the amino acid sequence of the mutant E. coli (FimH-DSG_A115V).

[0125] Sequence ID 47 describes the amino acid sequence of the mutant E. coli (FimH-DSG_V163I).

[0126] Sequence number 48 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimH-DSG_V185I.

[0127] Sequence number 49 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimH-DSG_DSG_V3I.

[0128] Sequence number 50 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G15A_V27A.

[0129] Sequence number 51 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G16A_V27A.

[0130] Sequence number 52 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G15P_V27A.

[0131] Sequence number 53 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G16P_V27A.

[0132] Sequence number 54 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G15A_G16A_V27A.

[0133] Sequence number 55 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_V27A_R60P.

[0134] Sequence number 56 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G65A_V27A.

[0135] Sequence number 57 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_V27A_Q133K.

[0136] Sequence number 58 describes the amino acid sequence of mutant Escherichia coli (E. coli) FimHLD_G15A_G16A_V27A_Q133K.

[0137] Sequence ID 59 describes the amino acid sequence of full-length wild-type Escherichia coli (E. coli) FimH (FimH-DSG_WT) containing a donor chain FimG peptide linked via a linker.

[0138] Sequence ID 60 describes the amino acid sequence of the mutant E. coli (FimH-DSG_V27A).

[0139] Sequence ID 61 describes the amino acid sequence of the mutant E. coli (FimH-DSG_G15A_V27A).

[0140] Sequence ID 62 is a mutant strain of Escherichia coli (E. coli) FimH. DSG The amino acid sequences for _G15A_G16A_V27A are listed below.

[0141] Sequence ID 63 is a mutant strain of Escherichia coli (E. coli) FimH. DSG The amino acid sequence for _V27A_Q133K is described below.

[0142] Sequence ID 64 is a mutant strain of Escherichia coli (E. coli) FimH. DSG The amino acid sequence for _G15A_G16A_V27A_Q133K is described below.

[0143] Sequence ID 65 describes the amino acid sequence of the mouse Ig kappa signal peptide.

[0144] Sequence ID 66 is BMD2 / FimH DSG The nucleic acid sequence for GPI / hHBB_80pA is described below.

[0145] Sequence ID 67 is BMD70 / FimH DSG- The nucleic acid sequence for GPI / hHBB_80pA is described below.

[0146] Sequence ID 68 is BMD91 / FimH DSG-The nucleic acid sequence for GPI / CYP2E1_80pA is described below.

[0147] Sequence ID 69 is BMD105 / FimH DSG- The nucleic acid sequence for GPI / hHBB_80pA is described below.

[0148] Sequence ID 70 is BMD562 / FimH DSG- The nucleic acid sequence for GPI / hHBB_80pA is described below.

[0149] Sequence ID 71 is BMD3 / FimH DSG- The nucleic acid sequence for GPI / hHBB-AES_80pA is described below.

[0150] Sequence ID 72 is BMD2 / FimH LD- The nucleic acid sequence for GPI / hHBB_80pA is described below.

[0151] Sequence ID 73 is from WHO / FimH LD- The nucleic acid sequence for GPI / WHO_80pA is described below.

[0152] Sequence ID 74 is BMD2 / FimH DSG- The nucleic acid sequence of Sec / hHBB_80pA is described.

[0153] Sequence ID 75 is from WHO / FimH DSG- The nucleic acid sequence of GPI / WHO_30L70 is described.

[0154] Sequence ID 76 is FimH LD - Describe the nucleic acid sequence of CtDAFGPI.

[0155] Sequence ID 77 is the same as FimH described in Sequence ID 76. LD - Describe the amino acid sequence of CtDAFGPI.

[0156] Sequence ID 78 is FimH DSG - Describe the secreted nucleic acid sequence.

[0157] Sequence ID 79 is the same as FimH described in Sequence ID 78. DSG - Secretory type, BMD562 / FimH described in Sequence ID No. 84 DSG- BMD576 / FimH as described in Sec / hHBB_80pA and Sequence ID No. 88 DSG- The amino acid sequence for Sec / hHBB_80pA is described below.

[0158] Sequence ID 80 is FimH DSG - Describe the nucleic acid sequence of CtDAFGPI.

[0159] Sequence ID 81 is the same as FimH described in Sequence ID 80. DSG - Describe the amino acid sequence of CtDAFGPI.

[0160] Sequence ID 82 is BMD2 / FimH DSG- The nucleic acid sequence for SerGlyGPI / hHBB_80pA is described below.

[0161] Sequence ID 83 is BMD2 / FimH as described in Sequence ID 82. DSG- SerGlyGPI / hHBB_80pA, BMD562 / FimH as described in Sequence ID No. 86. DSG- BMD576 / FimH described in SerGlyGPI / hHBB_80pA and Sequence ID No. 90 DSG- The amino acid sequence for SerGlyGPI / hHBB_80pA is described below.

[0162] Sequence ID 84 is BMD562 / FimH DSG- The nucleic acid sequence for Sec / hHBB_80pA is described below.

[0163] Sequence ID 86 is BMD562 / FimH DSG- The nucleic acid sequence for SerGlyGPI / hHBB_80pA is described below.

[0164] Sequence ID 88 is BMD576 / FimH DSG- The nucleic acid sequence for Sec / hHBB_80pA is described below.

[0165] Sequence ID 90 is BMD576 / FimH DSG- The nucleic acid sequence for SerGlyGPI / hHBB_80pA is described below.

[0166] Sequence ID 92 describes the nucleic acid sequence for the 80A polyA tail.

[0167] Sequence ID 93 describes the nucleic acid sequence of a split polyA tail, referred to as the "30L70" polyA tail.

[0168] Sequence ID 94 describes the amino acid sequence of an 8-amino acid glycine-serine linker substitution in a DAF GPI anchor.

[0169] Sequence ID 95 describes the nucleic acid sequence for 5'UTR_BMD2.

[0170] Sequence ID 96 describes the nucleic acid sequence for 5'UTR_BMD70.

[0171] Sequence ID 97 describes the nucleic acid sequence for 5'UTR_BMD91.

[0172] Sequence ID 98 describes the nucleic acid sequence for 5'UTR_BMD105.

[0173] Sequence ID 99 describes the nucleic acid sequence for 5'UTR_BMD562.

[0174] Sequence ID 100 describes the nucleic acid sequence for 5'UTR_BMD3.

[0175] Sequence ID 101 describes the nucleic acid sequence for 5'UTR_BMD576.

[0176] Sequence ID 102 describes the nucleic acid sequence for 5'UTR_WHO.

[0177] Sequence ID 103 describes the nucleic acid sequence for 3'UTR_hHBB.

[0178] Sequence ID 104 describes the nucleic acid sequence for 3'UTR_CYP2E1.

[0179] Sequence ID 105 describes the nucleic acid sequence for 3'UTR_hHBB-AES.

[0180] Sequence ID 106 describes the nucleic acid sequence for 3'UTR_WHO.

[0181] Detailed description of the invention The present invention can be more readily understood by referring to the following detailed description of embodiments of the invention and the examples included herein. It should be understood that the present invention is not limited to specific manufacturing methods, which may, of course, be modified. It should also be understood that the technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting.

[0182] Exemplary embodiments (E) of the invention provided herein include:

[0183] E1. An RNA molecule comprising at least one open reading frame (ORF) encoding a fimbriae H antigen (FimH) polypeptide and a 5' untranslated region (5'UTR), wherein the 5'UTR comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence described in any one of sequence numbers 95-101.

[0184] E2. The RNA molecule according to Embodiment E1, comprising a nucleic acid sequence in which the 5'UTR is at least 92% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 95, 98, 99, and 101.

[0185] E3. An RNA molecule according to any one of Embodiments E1 to E2, comprising a nucleic acid sequence in which the 5'UTR is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 95, 99, and 101.

[0186] E4. An RNA molecule according to any one of Embodiments E1 to E3, comprising a nucleic acid sequence in which the 5'UTR is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 99 and 101.

[0187] E5. An RNA molecule according to any one of Embodiments E1 to E4, comprising a nucleic acid sequence in which the 5'UTR is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 99 and 101.

[0188] E6. 5'UTR is, Sequence ID 99 (5'UTR_BMD562) and Sequence ID 101 (5'UTR_BMD576) An RNA molecule according to any one of embodiments E1 to E5, comprising a nucleic acid sequence selected from the group consisting of the following.

[0189] In one embodiment of Embodiment E6, the 5'UTR includes the nucleic acid sequence described in Sequence ID No. 99. In another embodiment of Embodiment E6, the 5'UTR includes the nucleic acid sequence described in Sequence ID No. 101.

[0190] E7. An RNA molecule according to any one of embodiments E1 to E6, further comprising a 3' untranslated region (3'UTR).

[0191] E8. The RNA molecule according to Embodiment E7, wherein the 3'UTR contains a nucleotide having the sequence described in SEQ ID NO: 103 (3'UTR_hHBB).

[0192] E9. The RNA molecule according to any one of embodiments E1 to E8, wherein the FimH polypeptide encoded by the RNA molecule is its full length, cleaved form, fragment, or variant.

[0193] E10. An RNA molecule according to any one of embodiments E1 to E9, wherein the FimH polypeptide encoded by the RNA molecule contains at least one mutation.

[0194] E11. The RNA molecule according to any one of Embodiments E1 to E10, wherein the FimH polypeptide encoded by the RNA molecule has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NOs: 1 to 64.

[0195] E12. The RNA molecule according to any one of embodiments E1 to E11, wherein the FimH polypeptide encoded by the RNA molecule has an amino acid sequence selected from SEQ ID NOs: 1 to 64.

[0196] E13. The RNA molecule according to any one of Embodiments E1 to E12, wherein the FimH polypeptide encoded by the RNA molecule is selected from the group consisting of FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), and FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), or immunogenic fragments thereof.

[0197] E14. An RNA molecule according to any one of embodiments E1 to E13, wherein a FimH polypeptide encoded by the RNA molecule is fused to a C-terminal membrane targeting domain.

[0198] E15. An RNA molecule according to any of embodiments E1 to E14, wherein the C-terminal membrane targeting domain is DAFgpi or a variant thereof.

[0199] E16. The RNA molecule according to Embodiment E15, wherein DAFgpi is a variant comprising a serine / glycine linker substitution of eight DAF amino acid residues proximal to the ω-site serine by a serine / glycine linker having the amino acid sequence GSSGSGSS (SEQ ID NO: 94).

[0200] E17. The RNA molecule according to any one of embodiments E14 to E16, wherein the FimH polypeptide encoded by the RNA molecule has an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NOs. 77, 79, 81, or 83.

[0201] E18. The RNA molecule according to any one of embodiments E14 to E17, wherein the FimH polypeptide encoded by the RNA molecule is selected from the group consisting of SEQ ID NOs: 77, 79, 81, and 83.

[0202] In one embodiment of Embodiment E18, the FimH polypeptide encoded by an RNA molecule is described in SEQ ID NO: 77. In another embodiment of Embodiment E18, the FimH polypeptide encoded by an RNA molecule is described in SEQ ID NO: 79. In yet another embodiment of Embodiment E18, the FimH polypeptide encoded by an RNA molecule is described in SEQ ID NO: 81. In a further embodiment of Embodiment E18, the FimH polypeptide encoded by an RNA molecule is described in SEQ ID NO: 83.

[0203] E19. The RNA molecule according to any one of Embodiments E1 to E18, wherein the open reading frame is transcribed from a nucleic acid containing a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the sequences of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138.

[0204] E20. The RNA molecule according to Embodiment E19, wherein the open reading frame is transcribed from a nucleic acid containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO: 138.

[0205] In one embodiment of Embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence described in Sequence ID No. 76. In another embodiment of Embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence described in Sequence ID No. 78. In yet another embodiment of Embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence described in Sequence ID No. 80. In a further embodiment of Embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence described in Sequence ID No. 138.

[0206] E21. An RNA molecule according to any one of Embodiments E1 to E19, wherein the open reading frame contains a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the sequences described in SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 139.

[0207] E22. The RNA molecule according to Embodiment E21, wherein the open reading frame contains a nucleic acid sequence selected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 139.

[0208] In one embodiment of Embodiment E22, the open reading frame includes the nucleic acid sequence described in Sequence ID No. 117. In another embodiment of Embodiment E22, the open reading frame includes the nucleic acid sequence described in Sequence ID No. 118. In yet another embodiment of Embodiment E22, the open reading frame includes the nucleic acid sequence described in Sequence ID No. 119. In a further embodiment of Embodiment E22, the open reading frame includes the nucleic acid sequence described in Sequence ID No. 139.

[0209] E23. An RNA molecule according to any one of embodiments E1 to E22, further comprising a 5' cap portion or a 3' poly-A tail.

[0210] E24. The 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG or (m2 7,3’-O )Gppp(m 2’-O An RNA molecule that is ApG, as described in any of embodiments E1 to E23.

[0211] In one embodiment of Embodiment E24, the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG. In another embodiment of Embodiment E24, the 5' cap is (m2 7,3’-O )Gppp(m 2’-O It's ApG.

[0212] E25. The RNA molecule according to Embodiment E24, wherein the poly-A tail contains a sequence having SEQ ID NO: 93 or SEQ ID NO: 140.

[0213] E26. An RNA molecule according to any one of Embodiments E1 to E25, comprising a nucleotide having a sequence described in SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs. 90.

[0214] In one embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 66. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 67. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 68. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 69. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 70. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 71. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 72. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 73. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 74. In another embodiment of Embodiment E26, the RNA molecule includes a nucleotide having the sequence described in Sequence ID No. 75. In another embodiment of Embodiment E26, the RNA molecule comprises a nucleotide having the sequence described in Sequence ID No. 82. In another embodiment of Embodiment E26, the RNA molecule comprises a nucleotide having the sequence described in Sequence ID No. 84. In another embodiment of Embodiment E26, the RNA molecule comprises a nucleotide having the sequence described in Sequence ID No. 86. In another embodiment of Embodiment E26, the RNA molecule comprises a nucleotide having the sequence described in Sequence ID No. 88. In a further embodiment of Embodiment E26, the RNA molecule comprises a nucleotide having the sequence described in Sequence ID No. 90.

[0215] E27. The RNA molecule according to Embodiment E26, transcribed from a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the sequences selected from SEQ ID NOs. 107-116 or SEQ ID NOs. 120-124.

[0216] In one embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in any one of SEQ ID NOs: 107-116 or SEQ ID NOs: 120-124. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 107. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 108. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 109. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 110. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 111. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 112. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in SEQ ID NOs: 113. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 114. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 115. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 116. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 120. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 121. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 122. In another embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 123. In a further embodiment of Embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence described in Sequence ID No. 124.

[0217] E28. An RNA molecule according to any one of Embodiments E1 to E27, wherein the open reading frame contains at least 55%, 60%, 65%, 70%, or 75%, or 50% to 75%, or 55% to 70%, or about 50% to 75%, or 55% to 70% G / C content.

[0218] E29. An RNA molecule according to any one of Embodiments E1 to E28, wherein the encoded FimH polypeptide is localized in the cell membrane, localized in the Golgi, and / or secreted.

[0219] E30. An RNA molecule according to any one of Embodiments E1 to E29, wherein the RNA comprises at least one modified nucleotide.

[0220] E31. The RNA molecule according to Embodiment E30, wherein the modified nucleotide is pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, or 2'-O-methyluridine.

[0221] E32. Modified nucleotides are pseudouridine (Ψ) or N 1 - The RNA molecule described in Embodiment E31, which is methylpseudridine (m1Ψ).

[0222] E33. Each uridine in an RNA molecule is pseudouridine (Ψ) or N 1 - The RNA molecule described in Embodiment E32, which is replaced with methylpseudridine (m1Ψ).

[0223] E34. An RNA molecule according to any one of embodiments E1 to E33, wherein RNA is mRNA.

[0224] E35. The RNA molecule described in Embodiment E34, wherein the RNA is modRNA.

[0225] E36. A composition comprising an RNA molecule as described in any one of Embodiments E1 to E35, wherein the RNA molecule is formulated in lipid nanoparticles (RNA-LNP).

[0226] E37. The composition according to Embodiment E36, wherein the lipid nanoparticles comprise at least one of cationic lipids, PEGylated lipids, neutral lipids, and steroids or steroid analogs.

[0227] E38. The composition according to Embodiment E37, wherein the cationic lipid is (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0228] E39. PEGylated lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), and glycol lipids including PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG), e.g., 1-(monometh The composition according to Embodiment E37 or E38, wherein the composition is xy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), for example, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, for example, co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate.

[0229] E40. The composition according to Embodiment E39, wherein the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0230] E41. Neutral lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)- A composition according to any one of Embodiments E37 to E40, wherein the composition is chlorohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), or 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

[0231] E42. The composition according to Embodiment E41, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0232] E43. The composition according to any one of embodiments E37 to E42, wherein the steroid or steroid analog is cholesterol.

[0233] E44. A vaccine, the composition described in any one of embodiments E36 to E43.

[0234] E45. A mutant FimH polypeptide containing at least 80% identity to any one of the amino acid sequences described in SEQ ID NO: 77, SEQ ID NO: 81, or SEQ ID NO: 83.

[0235] E46. The mutant FimH polypeptide according to embodiment (embodimen) E45, comprising an amino acid having the sequence described in SEQ ID NO: 81 or SEQ ID NO: 83.

[0236] E47. A polynucleotide encoding a mutant FimH polypeptide that has at least 80% identity to any one of the amino acid sequences described in SEQ ID NO: 77, SEQ ID NO: 81, or SEQ ID NO: 83.

[0237] E48. A polynucleotide encoding a mutant FimH polypeptide containing a nucleic acid having the sequence described in SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO: 139.

[0238] E49. The polynucleotide according to Embodiment E47, wherein the polynucleotide encoding the mutant FimH polypeptide is transcribed from a nucleic acid containing the nucleotide sequence described in SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 138.

[0239] E50. A method for (i) inducing an immune response to extraenteric pathogenic Escherichia coli (E. coli) in a subject, or (ii) inducing the production of opsonized phagocytic antibodies and / or neutralizing antibodies specific to extraenteric pathogenic Escherichia coli (E. coli), comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP and / or vaccine described in any one of embodiments E1 to E44.

[0240] E51. The method according to embodiment E50, wherein the subject is at risk of developing a urinary tract infection.

[0241] E52. The method according to embodiment E50, wherein the subject is at risk of developing bacteremia.

[0242] E53. The method according to embodiment E50, wherein the subject is at risk of developing urinary tract sepsis.

[0243] E54. The method according to Embodiment E50, wherein the subject is at risk of developing cystitis.

[0244] E55. Use of RNA molecules, RNA-LNPs and / or compositions according to any one of Embodiments E1 to E44 in the production of pharmaceuticals for use in (i) in inducing an immune response against extraenteropathogenic Escherichia coli (E. coli) in a subject, or (ii) in inducing the production of opsonized phagocytic antibodies and / or neutralizing antibodies specific to extraenteropathogenic Escherichia coli (E. coli) in a subject.

[0245] E56. Use according to Embodiment E55, where the infection, disease, or condition is a urinary tract infection.

[0246] E57. Use as described in Embodiment E55, in which the subject is at risk of developing bacteremia.

[0247] E58. Use as described in Embodiment E55, in which the subject is at risk of developing sepsis.

[0248] E59. Use as described in Embodiment E55, in which the subject is at risk of developing cystitis.

[0249] E60. The method or use of any one of Embodiments E50 to E59, wherein the subject is under approximately 1 year of age, approximately 1 year or older, approximately 5 years or older, approximately 10 years or older, approximately 20 years or older, approximately 30 years or older, approximately 40 years or older, approximately 50 years or older, approximately 60 years or older, approximately 70 years or older, or older.

[0250] E61. The method or use of any one of Embodiments E50 to E59, wherein the subject is approximately 50 years of age or older.

[0251] E62. The method or use of any one of embodiments E50 to E59, wherein the subject is a pregnant woman.

[0252] E63. The method or use according to any one of Embodiments E50 to E62, wherein an RNA molecule or composition is administered as a vaccine.

[0253] E64. The method or use according to any one of Embodiments E50 to E63, wherein the RNA molecule or composition is administered by intradermal or intramuscular injection.

[0254] E65. The method or use according to any one of Embodiments E50 to E64, wherein the subject is administered a single dose, two doses, three doses or more, and optionally a booster dose, of an RNA molecule, composition, or vaccine.

[0255] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0256] All references made herein, including patent applications, patent publications, and UniProtKB accession numbers, are incorporated herein by reference in the same way as each individual reference is specifically and individually indicated to be incorporated in whole by reference.

[0257] I. Examples of Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have meanings that are generally understood by those skilled in the art.

[0258] Throughout this application, the terms “about,” “approximately,” and “substantially” are used in accordance with their plain and common meanings in the field of cell and molecular biology, indicating a deviation of ±10% from the value in which they are set. Accordingly, in any disclosed embodiment, the terms may be replaced by “within [a certain percentage] of” the specified one. In one non-limiting embodiment, this percentage includes 0.1, 0.5, 1, 5, and 10 percent.

[0259] The descriptions of value ranges in this specification are merely intended to serve as a simplified way of referring individually to each separate value that falls within that range. Unless otherwise indicated herein, each individual value is incorporated herein in the same way as if it were individually listed herein.

[0260] The use of the words "a" or "an," when used in combination with the term "contains," can mean "one," but also coincides with the meanings of "one or more," "at least one," and "one or more."

[0261] The phrase "and / or" means "and" or "or." For example, A, B, and / or C includes A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" functions as an inclusive "or."

[0262] The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group possess a certain characteristic, then at least 95% of the members of the group possess that characteristic. In some embodiments, “essentially all” means that the members of the group possess the characteristic if they are equal to or at least one of 95, 96, 97, 98, 99, or 100%, or if they are between two of 95, 96, 97, 98, 99, or 100%.

[0263] Compositions and methods of use thereof may “contain,” “essentially consist of,” or “consist of” any of the components or steps disclosed throughout this Spec. Throughout this Spec. unless the context otherwise requires, the words “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are understood to be inclusive or open-ended, implying that they include the steps or elements or groups of steps or elements described, but not exclude any other steps or elements or groups of steps or elements. The embodiments described herein in the context of the term “contains” are also expected to be implemented in the context of the terms “consisting of” or “essentially consisting of.” Compositions and methods “essentially consisting of” any of the disclosed components or steps limit the scope of the claims to identified materials or steps that do not substantially affect the basic and novel features of the claimed disclosure. The word “consisting of” (and any form of “consisting of,” e.g., “consist of” and “consists of”) means including and being limited to everything that precedes the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that other elements cannot be present.

[0264] Any reference throughout this Specification to “one aspect,” “aspect,” “a particular aspect,” “related aspect,” “a certain aspect,” “additional aspect,” or “further aspects,” or any combination thereof, means that the particular features, structures, or characteristics described in connection with such aspects are included in at least one aspect of this disclosure. Therefore, the occurrence of these terms in various places throughout this Specification does not necessarily mean that all of them refer to the same aspect. Furthermore, particular features, structures, or characteristics may be combined in any preferred manner in one or more aspects.

[0265] The terms “inhibit,” “reduce,” or “mitigate,” or any variation thereof, include any measurable reduction (e.g., a reduction of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) or complete inhibition to achieve the desired outcome. The terms “enhance,” “promote,” or “increase,” or any variation thereof, include any measurable increase (e.g., an increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) to achieve the desired result or production of a protein or molecule.

[0266] Where used herein, the terms “reference,” “standard,” or “control” refer to a value against which a comparison is made. For example, an agent, subject, population, sample, or value of interest is compared to a reference, standard, or control of the agent, subject, population, sample, or value of interest. The reference, standard, or control may be tested and / or determined substantially simultaneously with and / or together with the test or determination of the agent, subject, population, sample, or value of interest, and / or determined or characterized under conditions or circumstances equivalent to those of the agent, subject, population, sample, or value of interest being evaluated.

[0267] The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free from the source cellular material, bacterial material, viral material, or culture medium (if produced by recombinant DNA technology), or chemical precursors or other chemicals (if chemically synthesized). Furthermore, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, a compound may not be simply considered “isolated” if it is attached to a column or embedded in an agarose gel. Furthermore, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that does not exist naturally as a fragment and / or is not typically in a functional state and / or has been modified or extracted from its natural state through human intervention. For example, DNA that exists naturally in a living animal is not “isolated,” but synthetic DNA, or DNA that is partially or completely separated from the material that coexists with it in its natural state, is “isolated.” Isolated nucleic acids may exist in a substantially purified form or in a non-native environment, such as in the cell to which the nucleic acid is delivered.

[0268] When used herein, “nucleic acid” refers to a molecule containing nucleic acid components, and means a DNA or RNA molecule. It may be used interchangeably with the term “polynucleotide.” A nucleic acid molecule is a polymer containing or consisting of nucleotide monomers covalently linked to one another by phosphodiester bonds of a sugar / phosphate backbone. Nucleic acids may also include modified nucleic acid molecules, such as DNA or RNA molecules that have been modified by base modification, sugar modification, or backbone modification. Nucleic acids may exist in various forms, e.g., polypeptides, isolated segments and incorporated sequences encoding one or both chains of an antigen or antibody, or fragments thereof, derivatives, mutaines, or variants, or recombinant vectors of recombinant polynucleotides, hybridization probes for identification, analysis, mutation or amplification of polynucleotides encoding polypeptides, sufficient polynucleotides for use as PCR primers or sequencing primers, more polynucleotides than described herein, mRNA, modRNA, and antisense nucleic acids for inhibiting the expression of complementary sequences. Nucleic acids may encode epitopes to which antibodies can bind.

[0269] The term “epitope” refers to a portion of an antigen that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on the surface when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0270] Nucleic acids may be single-stranded or double-stranded and may include RNA and / or DNA nucleotides as well as their artificial variants (e.g., peptide nucleic acids). In some cases, the nucleic acid sequence may encode the polypeptide sequence together with additional heterologous coding sequences to enable, for example, the purification, transport, secretion, post-translational modification of the polypeptide, or therapeutic benefits such as targeting or efficacy. Tags or other heterologous polypeptides may be appended to the modified polypeptide coding sequence, and “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0271] The term "polynucleotide" refers to nucleic acid molecules that may be recombinant or isolated from whole-genome nucleic acids. Recombinant vectors, including oligonucleotides (nucleic acids with a length of 100 residues or fewer), such as plasmids, cosmids, phages, and viruses, are included in the term "polynucleotide." Polynucleotides, in certain embodiments, contain regulatory sequences substantially isolated from their naturally occurring gene or protein-coding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), their analogues, or a combination thereof. Additional coding or non-coding sequences may, but are not required, be present within the polynucleotide.

[0272] In a particular embodiment, a polynucleotide variant having substantial identity with respect to the sequence disclosed herein; equal to any one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher in sequence identity when compared to the polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters), or at least 70%, 75%, 80%, 85%, 90%. There are polynucleotide variants that have sequence identity of any one of %, 95%, 96%, 97%, 98%, or 99% or higher, or up to any one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, or sequence identity among any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher. In a particular embodiment, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90% identity with the amino acid sequences described herein across the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 95% identity with the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0273] Nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, and other coding segments, and as a result, their overall length may vary considerably. Nucleic acids may be of any length. They may be equal to one of the lengths of, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides, or at least 5, 10, 15, 2 The nucleotide length can be any one of the following: 0, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more, or up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 7 Any of the following nucleotide lengths: 5, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more, or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250 , 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotide lengths may be between any two of these lengths and / or may include one or more additional sequences, e.g., regulatory sequences, and / or may be larger nucleic acids, e.g., portions of a vector.Therefore, nucleic acid fragments of almost any length may be used, and the full length is expected to be limited by ease of preparation and use in the intended recombinant nucleic acid protocol.

[0274] In this regard, the term “gene” is used to refer to nucleic acids (including any sequences required for proper transcription, post-translational modification, or localization) that encode proteins, polypeptides, or peptides. As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller, manipulated nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or part of such polypeptide. A particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences, but nevertheless, it is also conceivable that they encode the same or substantially similar polypeptides.

[0275] As used herein, the term “expression” of a nucleic acid sequence refers to the production of any gene product from a nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence involves one or more of the following: (1) the production of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) the translation of the RNA into a polypeptide or protein; and / or (4) post-translational modifications of the polypeptide or protein.

[0276] Generally, the term "manipulated" refers to a state in which something has been manipulated by human hands. For example, a polynucleotide can be considered "manipulated" if it is manipulated by human hands so that two or more sequences that are not linked in their natural order are directly linked to each other in the manipulated polynucleotide, and / or if a particular residue in the polynucleotide is linked to an entity or part that does not exist in nature and / or is not linked in nature, through the action of human hands.

[0277] As used herein, the term "DNA" refers to nucleic acid molecules comprising nucleotides, such as deoxy-adenosine monophosphate, deoxy-thymidine monophosphate, deoxy-guanosine monophosphate, and deoxy-cytidine monophosphate monomers, which are composed of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and which polymerize by a characteristic skeletal structure. The skeletal structure is typically formed by a phosphodiester bond between the sugar moiety of a nucleotide of a first monomer, e.g., deoxyribose, and the phosphate moiety of a second adjacent monomer. The characteristic order of monomers, e.g., the order of bases linked to the sugar / phosphate backbone, is called the DNA sequence. DNA may be single-stranded or double-stranded. In the double-stranded form, nucleotides of the first strand typically hybridize with nucleotides of the second strand, e.g., by A / T base pairing and G / C base pairing. DNA may contain all or most deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. Without limitation, DNA may include double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinant DNA, and modified DNA.

[0278] The term "RNA," as used herein, means a nucleic acid molecule comprising nucleotide monomers, such as adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate, and cytidine-monophosphate, linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar of a first monomer, such as ribose, and the phosphate moiety of a second adjacent monomer. RNA may be obtained, for example, by the transcription of a DNA sequence inside a cell. In eukaryotic cells, transcription typically occurs inside the nucleus or mitochondria. In vivo, the transcription of DNA may result in immature RNA that is processed into messenger RNA (mRNA). Processing of immature RNA, for example in eukaryotes, includes various post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. Mature messenger RNA provides a nucleotide sequence that can be processed and translated into the amino acid sequence of a peptide or protein. Mature mRNA may include a 5' cap, 5' UTR, open reading frame, 3' UTR, and poly-A tail sequence. RNA may contain all or most ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. In one embodiment, RNA may be messenger RNA (mRNA) relating to an RNA transcript encoding a peptide or protein. As is known to those skilled in the art, mRNA generally contains a 5' untranslated region (5' UTR), a polypeptide coding region, and a 3' untranslated region (3' UTR). Without limitation, RNA may include double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinant RNA, and modified RNA (modRNA).

[0279] "Isolated RNA" is defined as an RNA molecule that may be recombinant or isolated from whole-genome nucleic acids. Isolated RNA molecules or proteins may exist in a substantially purified form or in a non-native environment, such as in a host cell.

[0280] "Modified RNA" or "modRNA" refers to an RNA molecule having at least one addition, deletion, substitution, and / or modification of one or more nucleotides compared to naturally occurring RNA. Such modification may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the 5' and / or 3' ends of the RNA. In one embodiment, such modRNA contains at least one modified nucleotide, e.g., a modification to the base of a nucleotide. For example, the modified nucleotide may replace one or more uridine and / or cytidine nucleotides. For example, these substitutions may occur for any case of uridine and / or cytidine in the RNA sequence, or only for selected uridine and / or cytidine nucleotides. Such modifications to standard nucleotides in RNA may include non-standard nucleotides, e.g., chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide may be replaced with N1-methylpseudridine in the RNA sequence. Other such modified nucleotides are known to those skilled in the art. Such modified RNA molecules are considered analogs of naturally occurring RNA. In some embodiments, RNA is produced by in vitro transcription using a DNA template, where DNA refers to nucleic acids containing deoxyribonucleotides. In some embodiments, RNA may be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA).

[0281] Without limitation, as assumed herein, RNA may be used as a therapeutic modality to treat and / or prevent a number of conditions in mammals, including humans. The methods described herein include the administration of the RNA described herein to a mammal, e.g., a human. For example, in one embodiment, the method of use of such RNA includes an antigen-coding RNA vaccine to achieve protective immunization by inducing robust neutralizing antibodies and a concomitant / contingent T cell response. In some embodiments, a minimum vaccine dose is administered to achieve protective immunization by inducing robust neutralizing antibodies and a concomitant / contingent T cell response. In one embodiment, the RNA administered is RNA transcribed in vitro. For example, such RNA may be used to encode at least one antigen intended to produce an immune response in the mammal. The pathogenic antigen is a peptide or protein antigen derived from a pathogen associated with an infectious disease. In a specific embodiment, the pathogenic antigen is a peptide or protein antigen derived from Escherichia coli (E. coli) FimH. Conditions and / or diseases that can be treated with RNA disclosed herein include, but are not limited to, those caused and / or affected by bacterial infections. Such bacteria include, but are not limited to, Escherichia coli (E. coli).

[0282] When used herein in connection with the onset of a disease, disorder, and / or condition, “prevent” or “prevention” means reducing the risk of developing a disease, disorder, and / or condition, and / or delaying the onset of one or more characteristics or symptoms of a disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed over a predetermined period of time.

[0283] As understood from the context, the “risk” of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100%, or at least these percentages, or at most these percentages. In some embodiments, the risk is expressed as a risk compared to a risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or group of reference samples is from individuals comparable to a particular individual. In some embodiments, the risk may reflect one or more genetic attributes, e.g., one or more genetic attributes that may give an individual a predisposition to developing (or not developing) a particular disease, disorder, and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes. Susceptibility: Individuals "susceptible to" a disease, disorder, and / or condition are individuals who have a higher risk of developing a disease, disorder, and / or condition than members of the general population. In some embodiments, individuals susceptible to a disease, disorder, and / or condition do not have to be diagnosed with the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition do not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition develop the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition do not develop the disease, disorder, and / or condition.

[0284] The terms “protein,” “polypeptide,” or “peptide” are used herein as synonyms and refer to polymers of amino acid monomers, for example, molecules containing at least two amino acid residues. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogs thereof. Polypeptides may be single molecules or multimolecular complexes, such as dimers, trimers, or tetramers. Proteins may comprise one or more peptides or polypeptides and may be folded into a three-dimensional form that may be required for the protein to perform its biological function.

[0285] As used herein, the terms “wild-type,” “WT,” or “native” refer to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, the wild-type version of a protein or polypeptide is used, while in other embodiments of this disclosure, a modified protein or polypeptide is used to generate an immune response. The terms described above may be used interchangeably.

[0286] "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, in particular its amino acid sequence, is altered relative to a wild-type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is particularly assumed that a modified / variant protein or polypeptide may be modified with respect to one activity or function, but may retain wild-type activity or function in other respects, such as immunogenicity. Where a protein is specifically referred to herein, it is generally a reference to a native (wild-type) or recombinant (modified) protein. A protein may be isolated directly from an organism native to it, or may be produced by recombinant DNA / exogenous expression methods, solid-phase peptide synthesis (SPPS), or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antigens or fragments thereof). The term “recombinant” may be used in combination with the name of a polypeptide or a specific polypeptide, and generally refers to a polypeptide produced from a nucleic acid molecule that is manipulated in vitro, or a polypeptide produced from a replica product of such a molecule.

[0287] The term "fragment" refers to a portion of an amino acid sequence (peptide or protein), for example, a sequence representing a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminus shortened fragment (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminus shortened fragment (C-terminal fragment) can also be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, insofar as the truncated open reading frame contains a start codon that helps initiate translation. A fragment of an amino acid sequence can contain, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the amino acid residues from the amino acid sequence. In this disclosure, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence is defined as a fragment of which at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, This refers to sequences that have sequence identity between any two of the following percentages: 96%, 97%, 98%, or 99%, exactly 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0288] In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 70% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 80% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 85% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 90% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 95% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 97% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 99% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived.

[0289] When used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that exhibits significant structural identity with a reference molecule but is structurally different from the reference molecule, for example, in the presence or absence of one or more chemical parts compared to the reference entity, or at a certain level. In some embodiments, a variant may also be functionally different from its reference molecule. Generally, whether a particular molecule is considered appropriate to be a “variant” of a reference molecule depends on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant is, by definition, a distinct molecule that shares one or more such characteristic structural elements but is different from the reference molecule in at least one embodiment. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequences and / or one or more differences in chemical parts (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, and at most 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, exactly 85%. The variant polypeptide or nucleic acid exhibits overall sequence identity with the reference polypeptide or nucleic acid, which is between any two of the following percentages: 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities.In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduction in the level of one or more biological activities compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference amino acid or nucleotide sequence, except for a small number of sequence changes at specific positions. Preferably, a variant polypeptide or nucleic acid sequence has at least one modification, e.g., 1 to about 20 modifications, compared to a reference polypeptide or nucleic acid sequence. In one embodiment, a variant polypeptide or nucleic acid sequence has 1 to about 10 modifications compared to a reference polypeptide or nucleic acid sequence. In one embodiment, a variant polypeptide or nucleic acid sequence has 1 to about 5 modifications compared to a reference polypeptide or nucleic acid sequence. In one embodiment, a variant polypeptide or nucleic acid sequence has 1 to about 4 modifications compared to a reference polypeptide or nucleic acid sequence. Typically, approximately 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or less than 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In many cases, the variant polypeptide or nucleic acid contains a very small number of substitutions, insertions, or deletions of functional residues (e.g., residues that participate in specific biological activities) compared to the reference (e.g., approximately 5, 4, 3, 2, or less than 1). In some embodiments, the variant polypeptide or nucleic acid contains approximately 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue substitutions compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains fewer than approximately 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, or 6 additions or deletions compared to the reference, and generally fewer than approximately 5, 4, 3, or 2 additions or deletions.In some embodiments, the variant polypeptide or nucleic acid contains about 5, about 4, about 3, about 2, or less than 1 addition or deletion compared to the reference, and in some embodiments, it contains neither addition nor deletion.

[0290] In some embodiments, the reference polypeptide or nucleic acid is a naturally occurring “wild-type” or “WT” or “native” sequence, including allele variations. A wild-type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For the purposes of this disclosure, “variant” of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. “Variant” of a nucleotide sequence includes nucleotide insertion variants, nucleotide addition variants, nucleotide deletion variants, and / or nucleotide substitution variants. The term “variant” includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allele variants, species variants, and species homologs, in particular those occurring in nature. The term “variant” includes, in particular, fragments of amino acid or nucleic acid sequences.

[0291] The changes may be introduced into nucleic acids by mutation, which may lead to changes in the amino acid sequence of the polypeptide encoded by the nucleic acid (e.g., an antigen or antibody or antibody derivative). Mutations may be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, for example, using a random mutagenesis protocol. In some embodiments, the mutant polypeptide, however it is made, may be screened for expressed and desired properties.

[0292] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues may be introduced. Alternatively, one or more mutations that selectively alter the biological activity of the polypeptide encoded by the nucleic acid may be introduced into the nucleic acid. For example, the mutation may alter the biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing, or eliminating activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0293] "Sequence similarity" refers to the percentage of amino acids that are identical or represent a conserved amino acid substitution. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0294] The terms “% identical,” “% identity,” or similar terms are intended to specifically refer to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may, but do not need to be, randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences after optimal alignment with respect to a segment or “comparison window” to identify local regions of the corresponding sequences. Optimal alignment for comparison may be performed manually, or with the assistance of a local homology algorithm by Smith and Waterman, 1981, Ads App.Math.2, 482, by Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443, by Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 88, 2444, or with the assistance of a computer program using such algorithms (Wisconsin Genetics Software Package, GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Genetics Computer Group). In some embodiments, the percentage of identity between two sequences is determined using the BLASTN or BLASTP algorithm available on the United States National Center for Biotechnology Information (NCBI) website.

[0295] The identity percentage is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0296] In some embodiments, the degree of similarity or identity is given for regions that are at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the total length of the reference sequence, at most about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, exactly about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. For example, if a reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, at most about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, exactly about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, or a number of nucleotides between any two of about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, in some embodiments, for consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0297] Homologous amino acid sequences may exhibit identity in at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of amino acid residues, up to a maximum of 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, exactly 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, or any two of the following percentages: 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, homologous amino acid sequences exhibit identity in at least 95% of amino acid residues. In one embodiment, homologous amino acid sequences exhibit identity in at least 98% of amino acid residues. In one embodiment, homologous amino acid sequences exhibit identity in at least 99% of amino acid residues.

[0298] A fragment or variant of an amino acid sequence (peptide or protein) may be called a “functional fragment” or “functional variant.” The terms “functional fragment” or “functional variant” in relation to an amino acid sequence relate to any fragment or variant that exhibits one or more functional properties identical or similar to one or more functional properties of an amino acid sequence from which it is derived, for example, functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities demonstrated by the amino acid sequence from which the fragment or variant is derived. When used herein, the terms “functional fragment” or “functional variant” specifically refer to a variant molecule or sequence that includes an amino acid sequence modified by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, and which still possesses one or more of the functions of the parent molecule or sequence, for example, the ability to induce an immune response. In one embodiment, the modification of the amino acid sequence of the parent molecule or sequence does not substantially affect or substantially alter the characteristics of the molecule or sequence. The terms "mutant" of wild-type E. coli (E. coli) FimH protein, "mutant" of E. coli (E. coli) FimH protein, "E. coli (E. coli) FimH protein mutant," or "modified E. coli (E. coli) FimH protein" refer to polypeptides that exhibit the introduction of mutations compared to wild-type FimH protein and are immunogenic to wild-type FimH protein.

[0299] The amino acid sequence (peptide, protein, or polypeptide) “derived” from a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence is identical, essentially identical, or homologous to that particular sequence or fragment. An amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or fragment. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified so that their sequences vary from the naturally occurring or native sequences from which they are derived, while retaining the desired activity of the native sequences.

[0300] In this disclosure, "vector" refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. A vector may be used to incorporate a nucleic acid sequence, such as a nucleic acid sequence including an open reading frame. A vector includes, but is not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors. A vector may be an RNA vector or a DNA vector. In some embodiments, the vector is a DNA molecule. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector. Typically, an expression vector contains a desired coding sequence and other appropriate sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Cloning vectors are commonly used to manipulate and amplify a particular desired fragment (typically a DNA fragment) and may lack the functional sequences required for the expression of the desired fragment.

[0301] As used herein, the term “pharmaceutical composition” refers to an activator formulated with one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be an immunogenic composition. In some embodiments, the activator is present in an appropriate unit dose for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for parenteral administration, for example, as a sterile solution or suspension, or as a sustained-release formulation, for example, by subcutaneous, intramuscular, intravenous, or epidural injection.

[0302] As used herein, the term “vaccination” refers to the administration of an immunogenic composition intended to produce an immune response to, for example, a disease-related (e.g., disease-inducing) factor (e.g., bacteria). In some embodiments, vaccination may be administered before, during, and / or after exposure to a disease-related factor, and in certain embodiments, before, during, and / or immediately after exposure to said factor. In some embodiments, vaccination comprises multiple doses of the vaccine composition, appropriately time-intervened. In some embodiments, vaccination produces an immune response to an infectious agent. In some embodiments, vaccination produces an immune response to a tumor; and in some such embodiments, vaccination is “personalized” in that it is directed, in part or whole, to an epitope (e.g., one or more neoepitopes, or a combination thereof) that has been determined to be present in the tumor of a particular individual.

[0303] The immune response refers to a humoral response, a cellular response, or both humoral and cellular responses in an organism. The immune response may be measured by assays that measure the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays that measure T cell activation or proliferation, and / or assays that measure modulation in terms of the activity or expression of one or more cytokines.

[0304] As used herein, the term “combination therapy” refers to a situation in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of the first regimen are administered prior to any dose of the second regimen); in some embodiments, such agents are administered in overlapping drug regimens. In some embodiments, the “administration” of combination therapy may involve the administration of one or more agents or modalities to a subject receiving other agents or modalities in combination. For clarity, combination therapy does not require that the individual agents be administered together (or necessarily simultaneously) in a single composition; however, in some embodiments, two or more agents, or their active portions, may be administered together in a combination composition, or even in a combination compound (e.g., as a single chemical complex or as part of a covalent entity).

[0305] Those skilled in the art will understand that the term “medication regimen” may be used to refer to a set of unit doses (typically more than one) administered individually to a subject, typically separated by a period of time. In some embodiments, a given therapeutic agent has a recommended medication regimen, which may consist of one or more doses. In some embodiments, a medication regimen comprises multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of the same length; in some embodiments, a medication regimen comprises multiple doses and at least two different periods of time separating the individual doses. In some embodiments, all doses within a medication regimen are in the same unit dose amount. In some embodiments, the different doses within a medication regimen are different amounts. In some embodiments, a medication regimen comprises a first dose in the amount of a first dose, followed by one or more additional doses in the amount of a second dose different from the amount of the first dose. In some embodiments, the drug regimen comprises a first dose in the amount of the first dose, followed by one or more additional doses in the amount of the second dose, which is the same as the amount of the first dose. In some embodiments, the drug regimen correlates with a desired or beneficial outcome when administered across a relevant population (e.g., a therapeutic drug regimen).

[0306] II. Escherichia coli (E. coli) fimbriatal antigen H (FimH) As used herein, the term “FimH antigen polypeptide” includes, but is not limited to, any FimH polypeptide or its immunogenic mutants, as described in SEQ ID NOs: 1-64, 77, 79, 81, or 83.

[0307] As used herein, the term “E. coli polypeptide” includes any E. coli polypeptide. In a preferred embodiment, the E. coli polypeptide is a fimbriatal antigen. In a preferred embodiment, the E. coli fimbriatal antigen is FimH.

[0308] The FimH antigen polypeptide is described in PCT International Publication No. 2022 / 137078, which is incorporated in its entirety herein by reference.

[0309] Embodiments of this disclosure provide RNA (e.g., mRNA) vaccines comprising polynucleotides encoding the Escherichia coli (E. coli) FimH antigen. Such Escherichia coli (E. coli) FimH RNA vaccines may be used to induce a balanced immune response, including both cellular and humoral immunity.

[0310] Some embodiments provide an Escherichia coli (E. coli) vaccine comprising one or more RNA polynucleotides having an open reading frame encoding the FimH protein, formulated within cationic lipid nanoparticles, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the FimH protein is FimH-DSG, FimH-DSG triple mutant (G15A, G16A, V27A), or FimH LD Selection is made from triple mutants (G15A, G16A, V27A).

[0311] As used herein, the term "TM" means, when used in relation to an antigen, a triple mutant, in particular FimH having mutations at amino acid positions G15A, G16A, and V27A. LD Alternatively, it refers to the triple mutant of the FimH-DSG polypeptide. Therefore, the terms "FimH-DSG triple mutant (G15A, G16A, V27A)" and "FimH-DSG™" are interchangeable. Additionally, the term "FimH LD Triple mutant (G15A, G16A, V27A) and "FimH LD "TM" is replaceable.

[0312] As used herein, the abbreviation "Ct" means the C-terminal domain of a polypeptide or polynucleotide.

[0313] Some embodiments provide a method for preventing or treating Escherichia coli (E. coli) infection, comprising administering one of the vaccines described herein to a subject. In some embodiments, the antigen-specific immune response includes a T-cell response. In some embodiments, the antigen-specific immune response includes a B-cell response. In some embodiments, the antigen-specific immune response includes both a T-cell response and a B-cell response. In some embodiments, the method for generating an antigen-specific immune response involves a single dose of the vaccine. In some embodiments, the vaccine is administered to a subject by intradermal, intramuscular, subcutaneous, intranasal injection, or oral administration.

[0314] In some embodiments, the RNA (e.g., mRNA) polynucleotide or portion thereof may encode one or more polypeptides or fragments of Escherichia coli (E. coli) FimH as antigens.

[0315] III.RNA molecule In some embodiments, the RNA molecules described herein are coding RNA molecules. Coding RNA includes functional RNA molecules that can be translated into peptides or polypeptides. In some embodiments, the coding RNA molecule includes at least one open reading frame (ORF) encoding at least one peptide or polypeptide. The open reading frame includes a sequence of codons that can be translated into peptides or proteins. The coding RNA molecule may include 1 (monocistronic), 2 (dipistronic), or more (multicistronic) ORFs, which may be sequences of codons that can be translated into polypeptides or proteins of interest.

[0316] Numerous mRNA vaccine platforms are available in prior art. The basic structure of in vitro transcribed (IVT) mRNA closely resembles that of “mature” eukaryotic mRNA, consisting of (ii) 5' and 3' uncoding regions (UTRs) adjacent to each other, (i) a protein-coding open reading frame (ORF), as well as (iii) a 5' cap structure and (iv) a 3' poly(A) tail at the terminal end. The non-coding structural features play a significant role in mRNA pharmacology and can be individually optimized to modulate mRNA stability, translation efficiency, and immunogenicity.

[0317] By incorporating modified nucleosides, mRNA transcripts referred to as "nucleoside-modified mRNA" or "modRNA" can be manufactured with reduced immunostimulatory activity, thus potentially yielding an improved safety profile. Additionally, modified nucleosides can bypass direct antimicrobial pathways induced by type IFNs and programmed to degrade and inhibit invasive mRNA, thus enabling the design of mRNA vaccines with strongly enhanced stability and translational capacity. For example, substitution of uridine with pseudouridine in in vitro transcribed (IVT) mRNA reduces the activity of 2'-5'-oligoadenylate synthase, which modulates mRNA cleavage by RNase L. Furthermore, lower activity is measured for protein kinase R, an enzyme associated with inhibition of the mRNA translation process.

[0318] In addition to the incorporation of modified nucleotides, other approaches have been investigated to increase the translational capacity and stability of mRNA. One example is the development of “sequence-engineered mRNA,” where mRNA expression can be strongly increased by sequence optimization in the mRNA’s ORF and UTR, for example by enriching the GC content, or by selecting the UTR of a naturally long-lived mRNA molecule.

[0319] Furthermore, several modifications are performed on the terminal structure of mRNA. Anti-reverse cap (ARCA) modifications can ensure precise cap orientation at the 5' end, resulting in a nearly complete fraction of mRNA that can efficiently bind to ribosomes. Other cap modifications, such as phosphorothioate cap analogs, can further improve affinity for eukaryotic translation initiation factor 4E and increase resistance to RNA decapping complexes.

[0320] Conversely, modifying its structure can further improve the potency of mRNA that triggers innate immune responses, but at the expense of translational capacity. Stabilizing mRNA using a phosphorothioate backbone or precipitation with the cationic protein protamine can reduce antigen expression but potentially yield stronger immunostimulatory capacity.

[0321] In one embodiment, the present invention relates to an immunogenic composition comprising an mRNA molecule encoding one or more polypeptides or fragments of Escherichia coli (E. coli) FimH as antigens. In some embodiments, the mRNA molecule comprises nucleoside-modified mRNA. The RNA molecule may encode one or more polypeptides of interest, e.g., one or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides. Alternatively or additionally, one RNA molecule may also encode one or more polypeptides of interest, e.g., antigens, e.g., a 2-cistronic or 3-cistronic RNA molecule encoding different or identical antigens.

[0322] The sequence of an RNA molecule may be codon-optimized or deoptimized for expression in a desired host, e.g., human cells. In some embodiments, the gene of interest (e.g., an antigen) described herein is codon-optimized and / or encoded by a coding sequence in which its guanosine / cytidine (G / C) content is increased compared to the wild-type coding sequence. In some embodiments, one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of the wild-type coding sequence. In some embodiments, codon optimization and / or increased G / C content do not alter the sequence of the encoded amino acid sequence.

[0323] Those skilled in the art will understand that the term “codon optimized” refers to modifying codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, in some embodiments, the coding region is codon optimized for optimal expression in the subject treated with the RNA polynucleotides described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies of tRNA molecules present in a cell. Thus, the RNA sequence may be modified so that codons where frequently present tRNA molecules are available are inserted in place of “rare codons.”

[0324] In some embodiments, the G / C content of the coding region of an RNA (e.g., the sequence of a gene of interest) is increased compared to the G / C content of the corresponding coding sequence of the wild-type RNA encoding the gene of interest, and in some embodiments, the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region being translated is important for the efficient translation of its mRNA. Sequences with increased G (guanosine) / C (cytidine) content are more stable than sequences with increased A (adenosine) / U (uridine) content. With respect to the fact that several codons encode one identical amino acid (so-called degeneracy of the genetic code), the most favorable codon for stability may be determined (so-called selective codon usage). Depending on the amino acids encoded by the RNA, there are various possibilities for modification of the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleosides may be modified by substituting these codons with other codons that encode the same amino acids but do not contain A and / or U, or that contain lower amounts of A and / or U nucleosides. Thus, in some embodiments, the G / C content of the coding region of the RNA described herein is increased by at least 10%, 20%, 30%, 40%, 50%, 55%, up to 10%, 20%, 30%, 40%, 50%, 55%, exactly 10%, 20%, 30%, 40%, 50%, 55%, or any two of 10%, 20%, 30%, 40%, 50%, 55%, or even higher percentages compared to the G / C content of the coding region of wild-type RNA.

[0325] In some aspects, RNA molecules have approximately 20 to approximately 100,000 nucleotides (e.g., 30-50, 30-100, 30-250, 30-500, 30-1,000, 30-1,500, 30-3,000, 30-5,000, 30-7,000, 30-10,000, 30-25,000, 30-50,000, 30-70,000, 100-250, 100-500, 100-1,000, 100- 1,500, 100-3,000, 100-5,000, 100-7,000, 100-10,000, 100-25,000, 100-50,000, 100-70,000, 100-100,000, 500-1,000, 500-1,500, 500-2,000, 500-3,000, 500-5,000, 500-7,000, 500-10,000, 500-25,000, 500-50,000 , 500~70,000, 500~100,000, 1,000~1,500, 1,000~2,000, 1,000~3,000, 1,000~5,000, 1,000~7,000, 1,000~10,000, 1,000~25,000, 1,000~50,000, 1,000~70,000, 1,000~100,000, 1,500~3,000, 1,500~5,000, 1,500~7,000 (Containing 0, 1,500-10,000, 1,500-25,000, 1,500-50,000, 1,500-70,000, 1,500-100,000, 2,000-3,000, 2,000-5,000, 2,000-7,000, 2,000-10,000, 2,000-25,000, 2,000-50,000, 2,000-70,000, and 2,000-100,000 nucleotides).

[0326] In some embodiments, RNA molecules are at least 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000 ,7200,7400,7600,7800,8000,8200,8400,8600,8800,9000,9200,9400,9600,9800,10000,10000,12000,14000,16000,18000,20000,22000,24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 44000, 46000, 48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 6600 0, 68000, 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000, with a maximum of 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 7 20, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400,3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400 , 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 36000 ,38000,40000,42000,44000,46000,48000,50000,52000,54000,56000,58000,60000,62000,64000,66000,68000,70000,72000,74000,76000,7800 0, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000, exactly 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 9 00, 920, 940, 960, 980, 1000, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5 000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 1 0000, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 44000, 46000, 48000,50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000, 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000 pieces, or approximately 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320 ,340,360,380,400,420,440,460,480,500,520,540,560,580,600,620,640,660,680,700,720,740,760,780,800,820,840,860,880,900,920,940,960,980,1000,1000,1200,1400,1600,1800,2000,2200,2400,2600,2800,3000,3200,3400,3600,3800,4000,4200,440 0, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 3600 It has a number of nucleotides between any two of the following: 0, 38000, 40000, 42000, 44000, 46000, 48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000, 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000.

[0327] In some embodiments, an RNA molecule contains at least 100 nucleotides. For example, in some embodiments, RNA has lengths of 100 to 15,000 nucleotides; 7,000 to 16,000 nucleotides; 8,000 to 15,000 nucleotides; 9,000 to 12,500 nucleotides; 11,000 to 15,000 nucleotides; 13,000 to 16,000 nucleotides; and 7,000 to 25,000 nucleotides. In some embodiments, RNA molecules are present in at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, and 200 0, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 59 50, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900,7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 103 50, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11 400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 1 2450, 12500, 12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, ​​13000, 13050, 13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500 , 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000 pieces, with a maximum of 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050,2100、2150、2200、2250、2300、2350、2400、2450、2500、2550、2600、2650、2700、2750、2800、2850、2900、2950、3000、3050、3100、3150、3200、3250、3300、3350、3400、3450、3500、3550、3600、3650、3700、3750、3800、3850、3900、3950、4000、4050、4100、4150、4200、4250、4300、4350、4400、4450、4500、4550、4600、4650、4700、4750、4800、4850、4900、4950、5000、5050、5100、5150、5200、5250、5300、5350、5400、5450、5500、5550、5600、5650、5700、5750、5800、5850、5900、5950、6000、6050、6100、6150、6200、6250、6300、6350、6400、6450、6500、6550、6600、6650、6700、6750、6800、6850、6900、6950、7000、7050、7100、7150、7200、7250、7300、7350、7400、7450、7500、7550、7600、7650、7700、7750、7800、7850、7900、7950、8000、8050、8100、8150、8200、8250、8300、8350、8400、8450、8500、8550、8600、8650、8700、8750、8800、8850、8900、8950、9000、9050、9100、9150、9200、9250、9300、9350、9400、9450、9500、9550、9600、9650、9700、9750、9800、9850、9900、9950、10000、10050、10100、10150、10200、10250、10300、10350、10400、10450、10500、10550、10600、10650、10700、10750、10800、10850、10900、10950、11000、11050、11100、11150、11200、11250、11300、11350、11400、11450、11500、11550、11600、11650、11700、11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12700, 12750 , 12800, 12850, 12900, 12950, ​​13000, 13050, 13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 1380 0, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 148 50, 14900, 14950, or 15000 pieces, exactly 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 , 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450 , 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700 ,3750,3800,3850,3900,3950,4000,4050,4100,4150,4200,4250,4300,4350,4400,4450,4500,4550,4600,4650,4700,4750,4800,4850,4900,4950 , 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200,6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450 , 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700 , 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950 , 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 1100 0, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12 050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, ​​13000, 13050, 1 3100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000 pieces, or approximately 100, 150,200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、1150、1200、1250、1300、1350、1400、1450、1500、155、 0、1600、1650、1700、1750、1800、1850、1900、1950、2000、2050、2100、2150、2200、2250、2300、2350、2400、2450、2500、2550、2600、2650、2700、2750、2800、2850、2900、2950、3000、3050、3100、3150、3200、3250、3300、3350、3400、3450、3500、3550、3600、3650、3700、3750、3800、3850、3900、3950、4000、4050、4100、4150、4200、4250、4300、4350、4400、4450、4500、4550、4600、4650、4700、4750、4800、4850、4900、4950、5000、5050、5100、5150、5200、5250、5300、5350、5400、5450、5500、5550、5600、5650、5700、5750、5800、5850、5900、5950、6000、6050、6100、6150、6200、6250、6300、6350、6400、6450、6500、6550、6600、6650、6700、6750、6800、6850、6900、6950、7000、7050、7100、7150、7200、7250、7300、7350、7400、7450、7500、7550、7600、7650、7700、7750、7800、7850、7900、7950、8000、8050、8100、8150、8200、8250、8300、8350、8400、8450、8500、8550、8600、8650、8700、8750、8800、8850、8900、8950、9000、9050、9100、9150、9200、9250、9300、9350、9400、9450、9500、9550、9600、9650、9700、9750、9800、9850、9900、9950、10000、10050、10100、10150、10200、10250、10300、10350、10400、10450、10500、10550、10600、10650、10700、10750、10800、10850、10900、10950、11000、11050、11100、11150、11200、11250、11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, ​​13000, 13050, 13100, 13150, 13200, 13250, It has a number of nucleotides between any two of the following: 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000.

[0328] An mRNA useful in this disclosure typically comprises a first region of a linked nucleoside encoding the polypeptide of interest (e.g., a coding region), a first facile region located at the 5' end of the first region (e.g., a 5'-UTR), a second facile region located at the 3' end of the first region (e.g., a 3'-UTR), at least one 5' cap region, and a 3' stabilization region. In some embodiments, the mRNA of the present invention further comprises a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the mRNA of the present invention may contain one or more intronic nucleotide sequences having the ability to be excised from polynucleotides. In some embodiments, the mRNA of the present invention may include a 5' cap structure, a chain-terminated nucleotide, a stem-loop, a poly-A sequence, and / or a polyadenylation signal. Any one of the regions of the nucleic acid may contain one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilizing region may contain alternative nucleosides, such as L-nucleosides, inverted thymidines, or 2'-O-methylnucleosides, and / or the coding region, 5'-UTR, 3'-UTR, or capping region may contain alternative nucleosides, such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methylpseudridine), and / or 5-substituted cytidines (e.g., 5-methylcytidine).

[0329] In some embodiments, the RNA disclosed herein comprises the following components in a 5'-to-3' orientation: a 5' cap containing the 5' cap disclosed herein; a 5' untranslated region (5'UTR) containing the cap proximal sequence, a sequence encoding the payload (e.g., Escherichia coli (E. coli) FimH protein); a 3' untranslated region (3'UTR); and a polyA sequence.

[0330] In some embodiments, the LNP comprises one or more RNAs, and the one or more RNAs, lipids, and their amounts may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in RNA. Generally, a lower N:P ratio is preferred. The one or more RNAs, lipids, and their amounts may be selected to provide an N:P ratio of about 2:1 to about 30:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio may be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio may be approximately 5.0:1, 5.5:1, 6.0:1, 6.5:1, or 7.0:1.

[0331] A. Modified nucleic acid bases In this disclosure, RNA molecules may include modified nucleosides and modified nucleic acid bases that can be incorporated into nucleotides. In some embodiments, RNA molecules may include one or more modified nucleotides. Naturally occurring nucleotide modifications are known in the art.

[0332] The mRNA of the present invention may contain one or more naturally occurring components, each comprising one of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, all or substantially all of the nucleotides constituting (a) 5'-UTR, (b) open reading frame (ORF), (c) 3'-UTR, (d) poly-A tail, and any combination of (a, b, c, or d) above, comprise the naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0333] The mRNA of the present invention may include one or more alternative components described herein that confer useful properties, including increased stability and / or substantial absence of induction of the innate immune response in cells into which polynucleotides have been introduced. For example, modRNA may exhibit reduced degradation in cells into which it has been introduced compared with the corresponding unmodified mRNA. These alternative species may have reduced immunogenicity in addition to enhancing the efficiency of protein production, intracellular retention of polynucleotides, and / or the viability of contacted cells.

[0334] The mRNA of the present invention may comprise one or more modified (e.g., altered or alternative) nucleic acid bases, nucleosides, nucleotides, or combinations thereof. Useful mRNAs in LNPs may include any useful modifications or alterations to, for example, nucleic acid bases, sugars, or nucleoside linkages (e.g., linking phosphate / phosphodiester linkages / phosphodiester skeletons). In certain embodiments, the alteration (e.g., one or more alterations) may be present in each of the nucleic acid bases, sugars, and nucleoside linkages. The alterations according to this disclosure may be alterations of ribonucleic acid (RNA), e.g., substitution of the 2'-OH of the ribofuranosyl ring to 2'-H, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0335] The mRNA of the present invention may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or one or more or all of A, G, U, and C) may or may not be uniformly modified in the mRNA or in a given predetermined sequence region thereof. In some examples, all nucleotide X in the mRNA (or in a given sequence region thereof) is modified, and X may be one of the nucleotides A, G, U, or C, or one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0336] Different sugar alterations and / or internucleoside linkages (e.g., skeletal structures) may be present at various positions within the polynucleotide. Those skilled in the art will understand that nucleotide analogs or other alterations may be located at any position within the polynucleotide such that the function of the polynucleotide is not substantially diminished. Alterations may also be 5' or 3' end alterations. In some embodiments, the polynucleotide includes alterations at the 3' end. Polynucleotides are present in amounts of approximately 1% to 100% or any intervening percentage (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-90%) relative to the overall nucleotide content, or relative to one or more types of nucleotides, e.g., A, G, U, or C. It may contain alternative nucleotides in the following percentages: 5%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%. Any remaining percentage is understood to be allocated to the presence of canonical nucleotides (e.g., A, G, U, or C).

[0337] A polynucleotide may contain at least 0 and at most 100% alternative nucleotides, or any intervening percentage, for example, at least 5% alternative nucleotides, at least 10% alternative nucleotides, at least 25% alternative nucleotides, at least 50% alternative nucleotides, at least 80% alternative nucleotides, or at least 90% alternative nucleotides. For example, a polynucleotide may contain alternative pyrimidines, such as alternative uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the polynucleotide is replaced with alternative uracil (e.g., 5-substituted uracil). The alternative uracil may be replaced by a compound having a single unique structure, or by multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures). In some cases, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in a polynucleotide are replaced with alternative cytosines (e.g., 5-substituted cytosines). These alternative cytosines may be replaced by a single compound having a unique structure, or by multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures).

[0338] In some cases, nucleic acids do not substantially induce an innate immune response in cells into which polynucleotides (e.g., mRNA) have been introduced. The innate immune response that is induced is characterized by 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.), and / or 3) termination or reduction of protein translation.

[0339] In some embodiments, mRNA comprises one or more alternative nucleosides or nucleotides. Alternative nucleosides and nucleotides may include alternative nucleic acid bases. The nucleic acid bases of the nucleic acid are organic bases, such as purines or pyrimidines or their derivatives. The nucleic acid bases may be canonical bases (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleic acid bases may be modified or replaced entirely to provide a polynucleotide molecule with enhanced properties, such as increased stability, e.g., resistance to nucleases. Non-canonical or modified bases may include, but are not limited to, one or more substitutions or modifications, including, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more condensations or open rings; oxidation; and / or reduction.

[0340] In some embodiments, the nucleic acid base is alternative uracil. Exemplary nucleic acid bases and nucleosides having alternative uracil include pseudouridine(ψ), pyridine-4-onribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, and 2-thiouracil(s) 2 U), 4-thiouracil(s 4 U), 4-thiopseudridine (s4ψ), 2-thiopseudridine (s2ψ), 5-hydroxyuracil (ho 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyluracil (m 3 U), 5-methoxyuracil (mo 5 U), uracil 5-oxyacetic acid (cmo 5 U), uracil 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uracil (cm 5 U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uracil (chm 5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uracil (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouracil (mcm 5 s 2 U), 5-aminomethyl-2-thiouracil (nmVu), 5-methylaminomethyl-uracil (mnm 5 U), 5-methylaminomethyl-2-thiouracil (mnmVu), 5-methylaminomethyl-2-selenouracil (mnm 5 se 2 U), 5-Carbamoylmethyl-uracil (ncm 5 U), 5-carboxymethylaminomethyl-uracil (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouracil (cmnmVu), 5-propynyl-uracil, 1-propynyl-pseudoluracil, 5-taurinomethyl-uracil (xm 5 U), 1-Taurinomethyl-Pseudouridine, 5-Taurinomethyl-2-Thio-Uracil (xm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyluracil (m 5 U (for example, those having the nucleic acid base deoxythymine), 1-methyl-pseudridine (mV), 5-methyl-2-thio-uracil (mV) 5 s 2U), 1-methyl-4-thio-pseuduridine (ms4ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m | / ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouracil (D), dihydropseuduridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseuduridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseuduridine, 4-methoxy-2-thio-pseuduridine, N1-methyl-pseuduridine, 3-(3-amino-3-carboxypropyl)uracil (acpU), l-methyl-3-(3-amino-3-carboxypropyl)pseuduridine (acp ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thiouracil (inm5s2U), 5,2'-0-dimethyluridine (m5Um), 2-thio-2'-0-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyluridine (mem This includes Um), 5-carbamoylmethyl-2'-0-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyluridine (cmnm5Um), 3,2'-0-dimethyluridine (mUm), and 5-(isopentenylaminomethyl)-2'-0-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-carbomethoxyvinyl)uracil, 5-(carbamoylhydroxymethyl)uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, and 5-[3-(lE-propenylamino)]uracil. Pseudouridine is an example of a modified nucleoside, an isomer of uridine, where uracil is attached to a pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0341] In some embodiments, the nucleic acid base is an alternative cytosine. Exemplary nucleic acid bases and nucleosides having an alternative cytosine include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-Hydroxymethylcytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-cytosine Pseudoisocytidine, Zebralin, 5-Aza-Zebralin, 5-Methyl-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxycytosine, 2-Methoxy-5-Methylcytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethylcytidine (m5Cm), N4 It contains -acetyl-2'-0-methylcytidine (ac4Cm), N4,2'-0-dimethylcytidine (m4Cm), 5-formyl-2'-0-methylcytidine (f5Cm), N4,N4,2'-0-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxycytosine, 5-(3-azidopropyl)cytosine, and 5-(2-azidoethyl)cytosine.

[0342] In some embodiments, the nucleic acid base is an alternative adenine. Exemplary nucleic acid bases and nucleosides having an alternative adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, and 1-methyl-adenine (ml A) 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine ( m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-0-dimethyl-adenosine (m6Am), N6,N6,2'-0-trimethyl-adenosine (m62Am), 1,2'-0-dimethyl-adenosine (ml It contains Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0343] In some embodiments, the nucleic acid base is an alternative guanine. Exemplary nucleic acid bases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mil), waiosine (imG), methylwaiosine (mimG), 4-demethylwaiosine (imG-14), isowyosine (imG2), waibutosine (yW), peroxywaibutosine (o2yW), hydroxywaibutosine (OHyW), low-modified hydroxywaibutosine (OHyW*), 7-deaza-guanine, and quaenosine (Q ), epoxy quenosine (oQ), galactosyl quenosine (galQ), mannosyl quenosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl), alkaeosin (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl 6-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (mlG), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-guanine It contains thio-6-guanine, N2-methyl-2'-0-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-0-methyl-guanosine (m22Gm), 1-methyl-2'-0-methyl-guanosine (mlGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'-0-methyl-inosine (Im), l,2'-0-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl-guanine.

[0344] Nucleic acid bases that can substitute for nucleotides can independently be purines, pyrimidines, or purine or pyrimidine analogs. For example, a nucleotide base can be a substitute for adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, nucleic acid bases also include, for example, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine This may include naturally occurring and synthetic derivatives of bases, including guanine, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinon, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazine-2-one, 1,2,4-triazine, pyridazine; or 1,3,5-triazine. When nucleotides are described using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives; for example, A includes adenine or adenine analogs, such as 7-deazaadenine.

[0345] In some embodiments, the RNA molecule contains a nucleic acid sequence in which at least one uridine is replaced by pseudouridine. In some embodiments, the RNA molecule contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56% , 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 1 7%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 7 6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, exactly 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 8 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 2 5%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% The RNA molecule contains nucleic acid sequences in which uridine is replaced by pseudouridine in a percentage between any two of the following percentages: 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA molecule contains nucleic acid sequences in which all uridine is replaced by pseudouridine.

[0346] B. 5' Cap mRNA may contain a 5' cap structure. The 5' cap structure of polynucleotides is involved in nuclear export and increased polynucleotide stability, binding to mRNA cap-binding proteins (CBPs), which in turn contribute to polynucleotide stability and translational competency in cells through association with poly(A)-binding proteins for the formation of mature circular mRNA species. The cap further assists in the removal of 5' proximal introns during mRNA splicing.

[0347] Endogenous polynucleotide molecules may be capped at their 5' end to create a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the transcribed sense nucleotide at the 5' end of the polynucleotide. This 5'-guanylate cap may then be methylated to produce an N7-methyl-guanylate residue. The terminal ribose sugar and / or the transcribed nucleotide at the anteterminal end of the polynucleotide may also be optionally 2'-O-methylated. 5' decapping through hydrolysis and cleavage of the guanylate cap structure may target polynucleotide molecules, such as mRNA molecules, for degradation.

[0348] Modifications to polynucleotides can prevent decapping by generating a hydrolyzable cap structure, thus increasing the polynucleotide half-life. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester linkage, alternative nucleotides may be used during the capping reaction. For example, the vaccinia capping enzyme from New England Biolabs (Ipswich, MA) may be used with α-thio-guanosine nucleotide according to the producer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.

[0349] Additional alternative guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, may be used. Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar at the 5' end and / or pre-5' end of a polynucleotide (as described above) on the 2'-hydroxyl group of the sugar. Multiple distinct 5' cap structures can be used to generate the 5' cap of an mRNA molecule.

[0350] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in chemical structure from natural (e.g., endogenous, wild-type, or physiological) 5' caps while retaining cap function. Cap analogs may be synthesized chemically (e.g., non-enzymatically) or enzymatically and may be linked to polynucleotides. For example, an anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, one of which contains a 3'-O-methyl group in addition to an N7-methyl group (e.g., N7'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7G-3'mppp-G, which can be equivalently referred to as 3'O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other unmodified guanosine is ligated to the 5' terminal nucleotide of the capped polynucleotide (e.g., mRNA). N7- and 3'-0-methylated guanosine provide the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on the guanosine (e.g., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0351] The cap may be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified with a boranophosphate group or a phosphoroselenoate group at different phosphate positions, such as the dinucleotide cap analog described in U.S. Patent No. 8,519,110, which is incorporated herein by reference, in which its cap structure is incorporated.

[0352] Alternatively, the cap analog may be an N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogs include the N7-(4-chlorophenoxyethyl)-G(5)ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5)ppp(5')G cap analogs (see, for example, Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570~4574, whose cap structure is incorporated herein by reference, various cap analogs and methods for synthesizing cap analogs). In other examples, a useful cap analog in the polynucleotides of this disclosure is a 4-chloro / bromophenoxyethyl analog.

[0353] Capping analogs enable contingent capping of polynucleotides in in vitro transcription reactions, although up to 20% of the transcript remains uncapped. Furthermore, structural differences between capping analogs and the endogenous 5' cap structure of polynucleotides generated by endogenous cellular transcription mechanisms may lead to reduced translational competency and decreased cellular stability.

[0354] Alternative polynucleotides may also be post-transcriptionally capped using enzymes to generate a more orthodox 5' cap structure. As used herein, the term “more orthodox” means a feature that closely mirrors or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a “more orthodox” feature is a better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure compared to a synthetic feature or analog of the prior art, or surpasses the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more orthodox 5' cap structures useful in the polynucleotides of this disclosure are, among others, those having enhanced binding of cap-binding proteins, increased half-life, reduced sensitivity to 5'-endonucleases, and / or reduced 5' decapping compared to synthetic 5' cap structures (or wild-type, natural or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can create a canonical 5'-5'-triphosphate linkage between the 5' terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, where the cap guanosine contains N7-methylation and the 5' terminal nucleotide of the polynucleotide contains 2'-O-methylation. Such a structure is referred to as a Capl structure. This cap results in higher translational competency, cell stability, and reduced activation of pro-inflammatory cytokines compared to, for example, other 5' cap analog structures known in the art. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p(cap 0), 7mG(5')ppp(5')NlmpNp(cap 1), 7mG(5')-ppp(5')NlmpN2mp(cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up(cap 4).

[0355] Another cap structure has the structure described below N1 -methylpsuduridine-5'-triphosphate(N 1 -methylpsuduridine-5'-triphosphate, N 1 meΨTP, m 1 ΨTP, 1-methyl-pseudolidine phosphoramidite or N 1 -Methyl-pseudridine-5'-triphosphate (also known as TriLink Biotechnologies):

[0356] [ka]

[0357] Since alternative polynucleotides can be capped post-transcriptionally, and this process is more efficient, nearly 100% of mRNA can be capped. This is in contrast to approximately 80% when the capping analog is ligated to the polynucleotide during the in vitro transcription reaction.

[0358] The 5' terminal cap may contain an endogenous cap or a cap analog. The 5' terminal cap may contain a guanosine analog. Useful guanosine analogs include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the polynucleotide contains a modified 5' cap. Modifications on the 5' cap may increase the stability of the polynucleotide, increase its half-life, and increase the translation efficiency of the polynucleotide. The modified 5' cap may include, but is not limited to, one or more of the following modifications: modifications at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), substitution of a sugar ring oxygen (forming a carbocyclic ring) at the methylene moiety (CH2), modifications at the triphosphate bridge portion of the cap structure, or modifications at the nucleoside (G) portion.

[0359] C. Untranslated region (UTR) The 5'UTR is a regulatory region located at the 5' end of a protein open reading frame that is transcribed into mRNA but not translated into an amino acid sequence, or a corresponding region in an RNA polynucleotide, such as an mRNA molecule. Untranslated regions (UTRs) may be present at the 5' (upstream) (5'UTR) and / or the 3' (downstream) (3'UTR) of the open reading frame.

[0360] In some embodiments, UTRs are derived from naturally abundant mRNA in specific tissues (e.g., lymphoid tissues) where mRNA expression is targeted. In some embodiments, UTRs increase protein synthesis. Although not bound by mechanism or theory, UTRs may increase protein synthesis by increasing the time mRNA remains in the translation polysome (message stability) and / or the rate at which ribosomes initiate translation on the message (message translation efficiency). Thus, UTR sequences may prolong protein synthesis in a tissue-specific manner.

[0361] In some embodiments, the 5'UTR and 3'UTR sequences are derived by computer. In some embodiments, the 5'UTR and 3'UTR are derived from naturally abundant mRNA in the tissue. The tissue may be, for example, the liver, stem cells, or lymphoid tissue. The lymphoid tissue may include, for example, lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or natural killer cells), macrophages, monocytes, dendritic cells, neutrophils, eosinophils, and reticulocytes. In some embodiments, the 5'UTR and 3'UTR are derived from alphavirus. In some embodiments, the 5'UTR and 3'UTR are from wild-type alphavirus.

[0362] In some embodiments, the RNA disclosed herein includes a 5'UTR. The 5'UTR, if present, is located at the 5' end and begins with the transcription start site upstream of the start codon of the protein coding region. The 5'UTR is downstream of the 5' cap (if present) and, for example, directly adjacent to the 5' cap. The 5'UTR may contain various regulatory elements, such as the 5' cap structure, the stem-loop structure, and internal ribosome entry sites (IRESs) that may play a role in controlling translation initiation.

[0363] In some embodiments, the 5'UTR disclosed herein includes, for example, a cap proximal sequence, as disclosed herein. In some embodiments, the cap proximal sequence includes a sequence adjacent to the 5' cap. In some embodiments, the cap proximal sequence includes nucleotides at the +1, +2, +3, +4, and / or +5 positions of the RNA polynucleotide.

[0364] In some embodiments, the cap structure comprises one or more polynucleotides of the cap proximal sequence. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide.

[0365] Those skilled in the art, having read this disclosure, will recognize that in some embodiments, one or more residues of the cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4 and / or +5) may be included in the RNA because they are contained within a cap entity (e.g., a cap 1 structure); alternatively, in some embodiments, at least a portion of the residues in the cap proximal sequence may be enzymatically added (e.g., by a polymerase such as T7 polymerase). For example, (m2 7,3’-O )Gppp(m 2’-OIn a particular illustrated embodiment in which the ApG cap is utilized, the +1 and +2 residues are (m2) of the cap. 7,3’-O These are A and G residues, and the +3, +4, and +5 residues are added by a polymerase (e.g., T7 polymerase).

[0366] In some embodiments, the cap proximal sequence includes N1 and / or N2 of the cap structure, where N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, the cap proximal sequence includes N1 and N2 of the cap structure, as well as N3, N4, and N5, where N1-N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some embodiments, N1, N2, N3, N4, or N5 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1N2 includes one of the following: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, or UU. In some embodiments, N1N2 includes AG, and N3N4N5 includes any one of the following: AAA, ACA, AGA, AUA, AAG, AGG, ACG, AUG, AAC, ACC, AGC, AUC, AAU, ACU, AGU, AUU, CAA, CCA, CGA, CUA, CAG, CGG, CCG, CUG, CAC, CCC, CGC, CUC, CAU, CCU, CGU, CUU, GAA, GCA, GGA, GUA, GAG, GGG, GCG, GUG, GAC, GCC, GGC, GUC, GAU, GCU, GGU, GUU, UAA, UCA, UGA, UUA, UAG, UGG, UCG, UUG, UAC, UCC, UGC, UUC, UAU, UCU, UGU, or UUU.

[0367] In some embodiments, the cap proximal sequence includes N1 and N2 of the cap structure, and a sequence containing A3A4X5 (sequence number 167; in the sequence, X5 is A, G, C, or U), where N1 and N2 are independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is selected from A, C, G, or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.

[0368] In some embodiments, the cap proximal sequence includes N1 and N2 of the cap structure, and a sequence containing C3A4X5 (sequence number 168; in the sequence, X5 is A, G, C, or U), where N1 and N2 are independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is selected from A, C, G, or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.

[0369] In some embodiments, the cap proximal sequence includes N1 and N2 of the cap structure, as well as a sequence containing X3Y4X5 (sequence number 169; in the sequence, X3 or X5 is independently selected from A, G, C, or U; Y4 is not C). In some embodiments, N1 and N2 are independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X3 and X5 are independently selected from A, C, G, or U. In some embodiments, X3 and / or X5 is A. In some embodiments, X3 and / or X5 is C. In some embodiments, X3 and / or X5 is G. In some embodiments, X3 and / or X5 is U. In some embodiments, Y4 is C. In other embodiments, Y4 is not C. In some embodiments, Y4 is A. In some embodiments, Y4 is G. In other embodiments, Y4 is not G. In some embodiments, Y4 is U.

[0370] In some embodiments, the cap proximal sequence includes N1 and N2 of the cap structure, as well as a sequence containing A3C4A5 (sequence number 170). In some embodiments, N1 and N2 are independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0371] In some embodiments, the cap proximal sequence includes N1 and N2 of the cap structure, as well as a sequence containing A3U4G5 (SEQ ID NO: 171). In some embodiments, N1 and N2 are independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0372] The 5'-UTR may be provided as an adjacency region to the mRNA. The 5'-UTR may be homogeneous or heterogeneous with respect to the coding region found in the polynucleotide. Multiple 5'-UTRs may be included in the adjacency region, and may be the same or different sequences. Any portion of the adjacency region, including the absence of 5'-UTRs, may be codon-optimized, and any may independently contain one or more different structural or chemical modifications before and / or after codon optimization.

[0373] A heterologous 5'UTR may be manipulated to alter one or more properties of the mRNA. The mRNA may then be administered to cells, tissues, or organisms, and outcomes such as protein levels, localization, and / or half-life may be measured to evaluate the beneficial effects that the heterologous 5'UTR may have on the mRNA. Variants of the 5'UTR in which one or more nucleotides, including A, T, C, or G, are added or removed from the terminal may be utilized. The 5'UTR may also be codon-optimized or modified in any manner described herein.

[0374] In some embodiments, the RNA molecule includes a 5' untranslated region (5'-UTR). In some embodiments, the 5'UTR includes a sequence selected from any of SEQ ID NOs. 95 to 102. In some embodiments, the 5'UTR includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to any of SEQ ID NOs. 95 to 102. In some embodiments, the 5'UTR includes a sequence selected from any of SEQ ID NOs. 95 to 102. In some embodiments, the 5'UTR includes a sequence consisting of any of SEQ ID NOs. 95 to 102.

[0375] In some embodiments, the RNA disclosed herein includes a 3'UTR. If present, the 3'UTR is located downstream of the proteincoding sequence open reading frame, for example, downstream of the stop codon in the proteincoding region. Typically, the 3'UTR is a portion of mRNA located between the proteincoding sequence and the poly-A tail. Therefore, in some embodiments, the 3'UTR is upstream of the poly-A sequence (if present), for example, directly adjacent to the poly-A sequence. The 3'UTR may be involved in regulatory processes including transcript cleavage, stability, and polyadenylation, translation, and mRNA localization.

[0376] The 3'UTR may also include elements that are not encoded in the template from which the RNA is transcribed but are added during post-transcriptional maturation, such as a poly-A tail. The 3'UTR of mRNA is not translated into an amino acid sequence. In some embodiments, the RNA disclosed herein includes a 3'UTR containing an F element and / or an I element. In some embodiments, the 3'UTR or a proximal sequence thereto includes a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an Xhol site.

[0377] In some embodiments, RNA molecules and RNA-LNPs include a 3' untranslated region (3'-UTR). In some embodiments, the 3'UTR includes a sequence selected from any of SEQ ID NOs. 103 to 106. In some embodiments, the 3'UTR includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to any of SEQ ID NOs. 103 to 106. In some embodiments, the 3'UTR includes a sequence selected from any of SEQ ID NOs. 103 to 106. In some embodiments, the 3'UTR includes a sequence consisting of any of SEQ ID NOs. 103 to 106.

[0378] mRNA may include stem-loops, for example, histone stem-loops, but are not limited to the following. The stem-loop may be a nucleotide sequence about 25 or about 26 nucleotides long. The histone stem-loop may be located 3' relative to the coding region (e.g., at the 3' end of the coding region). In a non-limiting example, the stem-loop may be located at the 3' end of the polynucleotide described herein. In some cases, mRNA may contain more than one stem-loop (e.g., two stem-loops). The stem-loop may be located in the second terminal region of the polynucleotide. In a non-limiting example, the stem-loop may be located within the untranslated region (e.g., the 3'-UTR) of the second terminal region. In some cases, mRNA containing histone stem-loops may be stabilized by the addition of a 3' stabilization region (e.g., a 3' stabilization region containing at least one strand-terminated nucleoside). While we do not wish to be bound by theory, the addition of at least one strand-terminal nucleoside may slow the degradation of the polynucleotide, thereby increasing its half-life. In other cases, mRNA containing a histone stem loop may be stabilized by modifications to the 3' region of the polynucleotide, which can prevent and / or inhibit the addition of oligo(U). In yet other cases, mRNA containing a histone stem loop may be stabilized by the addition of oligonucleotides ending at 3'-deoxynucleoside, 2',3'-dideoxynucleoside, 3'-O-methylnucleoside, 3-O-ethylnucleoside, 3'-arabinoside, and other alternative nucleosides known in the art and / or described herein. In some examples, the mRNAs of this disclosure may include a histone stem loop, a polyA region, and / or a 5' cap structure. The histone stem loop may be before and / or after the polyA region. A polynucleotide comprising a histone stem-loop and a poly(A) region sequence may also comprise a chain-terminated nucleoside as described herein. In other examples, the polynucleotides of this disclosure may also comprise a histone stem-loop and a 5' cap structure.The 5' cap structure may include, but is not limited to, those described herein and / or known in the art. In some cases, the preserved stem-loop region may include the miR sequence described herein. In an unspecified example, the stem-loop region may include the seed sequence of the miR sequence described herein. In another unspecified example, the stem-loop region may include the miR-122 seed sequence.

[0379] The mRNA may contain at least one histone stem-loop and a polyA region or polyadenylation signal. In certain cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode a pathogen antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode a therapeutic protein. In some cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode a tumor antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and the polyA region or polyadenylation signal may encode an allergenic antigen or an autoimmune autoantigen.

[0380] 5' Cap In some embodiments, the RNA molecules described herein include a 5' cap. In some embodiments, the 5' cap portion is a natural 5' cap.

[0381] A “natural 5' cap” is defined as a cap containing 7-methylguanosine attached to the 5' end of an mRNA molecule via a 5'-to-5' tripphosphate linkage. In some embodiments, the 5' cap portion is a 5' cap analog. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide having the structure m7G(5')ppp(5')N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (e.g., co-transcription capping) or enzymatically manipulated after RNA transcription (e.g., post-transcription capping), where “N” is any ribonucleotide. In some embodiments, the 5' end of the RNA molecule is capped with a modified ribonucleotide by an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification of the RNA molecule, e.g., tangential flow filtration. Exemplary enzymatic reactions for capping may include the use of vaccinia virus capping enzymes (VCEs) comprising mRNA triphotase, guanylyl-transferase, and guanine-7-methyl(methy)transferase to catalyze the construction of an N7-monomethylated cap 0 structure. The cap 0 structure can help maintain the stability and translational efficacy of the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2'-O-methyltransferase, resulting in the generation of a cap 1 structure (m7Gppp[m2'-O]N), which can further increase translational efficacy. In some embodiments, the RNA molecule may be enzymatically capped at the 5' end using vaccinia guanylyltransferase, guanosine triphotase, and S-adenosyl-L-methionine to produce a cap 0 structure. An inverted 7-methylguanosine cap is added by a 5'-to-5' triphot crosslink. Alternatively, the use of 2'O-methyltransferase and vaccinia-guanylyltransferase yields a cap 1 structure, in which the 2'OH group is methylated at the second to last nucleotide in addition to the cap 0 structure. S-adenosyl-L-methionine (SAM) is a cofactor used as a methyl transfer agent.Non-limiting examples of 5' cap structures include, among others, those having enhanced binding of cap-binding polypeptides, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' decapping compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can create a canonical 5'-5'-triphosphate linkage between the 5' terminal nucleotide of mRNA and the guanine cap nucleotide, where the cap guanine contains N7 methylation and the 5' terminal nucleotide of mRNA contains 2'-O-methylation. Such structures are referred to as cap 1 structures. This cap results in higher translational competency and cellular stability, as well as reduced activation of pro-inflammatory cytokines, compared to, for example, other 5' cap analog structures known in the art. The cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0) and 7mG(5')ppp(5')N1mpNp (cap 1). Cap 0 is N7-methylguanosine linked to the 5' nucleotide via a 5'-to-5' tripphosphate linkage, typically referred to as m7G cap or m7Gppp. In cells, the cap 0 structure can help provide efficient translation of cap-bearing mRNA. Additional methylation at the 2'O position of the start nucleotide generates cap 1, or is referred to as m7GpppNm-, where Nm represents any nucleotide having the 2'O methylation. In some embodiments, the 5' terminal cap may contain a cap analog; for example, the 5' terminal cap may contain a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.In some embodiments, the capping region may include a single cap or a series of nucleotides forming a cap. In this embodiment, the capping region may be equal to the length of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or at least 2 or 10 or less, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or at least 2 or 10 or less, or at least one of any two of nucleotides between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or at least 2 or 10 or less. In some embodiments, the cap is absent.In some embodiments, the first and second operating areas are 3 to 40, for example, 5 to 30, 10 to 20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or at least 4 or 30 or fewer, 3 ~40, for example, 5~30, 10~20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or at least one of 4 or 30 or less, 3~40, for example, 5~30, 10~20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or at least one of 4 or 30 or less, or 3 to 40, for example, 5 to 30, 10 to 20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 The sequences may be equal in length to 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or at least 4 or 30 or less, and may include one or more signal and / or restriction sequences in addition to start and / or stop codons.

[0382] In some embodiments, the 5' cap is represented by formula I:

[0383] [ka] (In the formula, R 1 and R 2 Each of them is independently H or Me, and B 1and B 2 is independently guanine, adenine, or uracil). In some embodiments, B 1 and B 2 are naturally occurring bases. In some embodiments, R 1 is methyl and R 2 is hydrogen. In some embodiments, B 1 is guanine. In some embodiments, B 1 is adenine. In some embodiments, B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, B 2 is uracil and at least 5% of the total population of uracil nucleotides in the molecule downstream of B 2 is replaced with one or more modified or non-natural nucleotides.

[0384] In some embodiments, the nucleotide immediately downstream of the 5' cap (in the 5' to 3' direction) contains guanine. In some embodiments, B 1 is adenine and B 2 is uracil. In some embodiments, B 1 is adenine and B 2 is uracil, R 1 is methyl, and R 2 is hydrogen. In some examples, the RNA does not contain a 5' cap. In some examples, the 5' cap is not represented by Formula I. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B 1 is adenine, B 2 is uracil, R[[ID=四十三]] 1 is methyl, and R 2 is hydrogen; this embodiment corresponds to CleanCap AU, and the inclusion of B 2 = uracil may optionally be B 2Uracil nucleotides downstream of the sequence may be substituted, but in some embodiments, this has been shown to improve RNA functionality. In some embodiments, the RNA molecule further comprises (1) an alphavirus 5' replication recognition sequence and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the RNA molecule encodes at least one antigen. In some embodiments, the RNA molecule contains at least 7,000 nucleotides. In some embodiments, the RNA molecule contains at least 8,000 nucleotides. In some embodiments, at least 80% of the total RNA molecule is full length. In some embodiments, the alphavirus is Venezuelan encephalitis virus. In some embodiments, the alphavirus is Semliki forest virus.

[0385] In some embodiments, the nucleotide directly downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and at least 50% of the total population of uridine nucleotides in the molecule are replaced with N1-methylpseudridine, and essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, the nucleotide directly downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 is hydrogen, and at least 50% of the total population of uridine nucleotides in the molecule are replaced with 5-methoxyuridine, and essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, the nucleotide directly downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2is hydrogen, and at least 50% of the total population of uridine nucleotides in the molecule are replaced with 5-methyluridine, and essentially all cytosine nucleotides in the molecule are replaced with 5-methylcytosine. In some embodiments, the nucleotide directly downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 It is hydrogen, and essentially all uridine nucleotides in the molecule are replaced by approximately 50% 5-methoxyuridine and approximately 50% N1-methylpseudolidine. In some embodiments, the nucleotide directly downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 The hydrogen is present, and essentially all uridine nucleotides in the molecule are replaced by approximately 75% 5-methoxyuridine and approximately 25% N1-methylpseudolidine. In some embodiments, the nucleotide directly downstream of the 5' cap (from 5' to 3') contains guanine, and B 1 It is adenine, and B 2 is uracil, and R 1 is methyl, and R 2 The hydrogen atom is present, and essentially all uridine nucleotides in the molecule are replaced by approximately 25% 5-methoxyuridine and approximately 75% N1-methylpseudolidine.

[0386] In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap includes:

[0387] [ka]

[0388] In some embodiments, the 5' cap contains m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, which is the CLEANCAP® reagent AG(3'OMe) for mRNA co-transcription capping.

[0389] [ka]

[0390] In an alternative embodiment, the 5' cap contains CLEANCAP® AU for self-amplifying mRNA and m7G(5')ppp(5')(2'OMeA)pU, which is CLEANCAP® reagent AU for mRNA co-transcription capping.

[0391] [ka]

[0392] D. Open Reading Frame (ORF) The 5' and 3' UTRs may be operably ligated to an open reading frame (ORF), which may be a sequence of codons capable of being translated into a polypeptide of interest. The open reading frame may be a sequence of several DNA or RNA nucleotide triplets that can be translated into a peptide or protein. The ORF may begin with a start codon at its 5' end, for example, a combination of three subsequent nucleotides (ATG or AUG) that typically codes for the amino acid methionine, as well as a subsequent region that typically exhibits a length of several three nucleotides. The open reading frame may end with at least one stop codon, including but not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof. In some embodiments, the open reading frame may end with one, two, three, four or more stop codons known in the art. The open reading frame may be isolated or incorporated into a longer nucleic acid sequence, such as a vector or mRNA. Open reading frames may also be referred to as "(protein) coding regions" or "coding sequences."

[0393] As described herein, RNA molecules may contain one (monocistronic), two (dicistronic), or more (multicistronic) open reading frames.

[0394] This disclosure provides RNA molecules comprising at least one open reading frame encoding the Escherichia coli (E. coli)FimH polypeptide described herein, and, in some aspects, RNA molecules comprising at least one open reading frame encoding the Escherichia coli (E. coli)FimH protein described herein.

[0395] E. Genes of interest The RNA molecules described herein may include genes of interest. Genes of interest encode polypeptides of interest. Non-exclusive examples of polypeptides of interest include, for example, biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell-permeable peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeted moieties, polypeptides encoded by the human genome that have no identified therapeutic indicators but are nevertheless useful in the fields of research and drug discovery, or combinations thereof. Sequences of specific genes of interest are readily identified by those skilled in the art using public and private databases, e.g., GENBANK®.

[0396] In some embodiments, the RNA molecule includes a coding region for a gene of interest. In some embodiments, the gene of interest is or includes an antigen polypeptide, an immunogenic variant thereof, or an immunogenic fragment thereof. In some embodiments, the antigen polypeptide includes one epitope from an antigen. In some embodiments, the antigen polypeptide includes multiple distinct epitopes from an antigen. In some embodiments, an antigen polypeptide including multiple distinct epitopes from an antigen is a polyepitope. In some embodiments, the antigen polypeptide includes an antigen polypeptide from an allergen, a viral antigen polypeptide, a bacterial antigen polypeptide, a fungal antigen polypeptide, a parasitic antigen polypeptide, an antigen polypeptide from an infectious agent, an antigen polypeptide from a pathogen, a tumor antigen polypeptide, or an autoantigen polypeptide.

[0397] The term “antigen” may refer to a substance that has the ability to be recognized by the immune system, such as the adaptive immune system, and that has the ability to induce an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. An antigen may be, or may contain, a peptide or protein that can be presented to T cells by MHC. An antigen may also be the translation product of a nucleic acid molecule provided, for example, an RNA molecule containing at least one coding sequence described herein. Additionally, fragments, variants, and derivatives of an antigen, such as peptides or proteins, containing at least one epitope are understood as antigens.

[0398] In some embodiments, the RNA encoding the gene of the object of interest, e.g., the antigen, is expressed in the target cells being treated in order to provide the gene of the object of interest, e.g., the antigen. In some embodiments, the RNA is transiently expressed in the target cells. In some embodiments, the expression of the gene of the object of interest, e.g., the antigen, occurs on the cell surface. In some embodiments, the gene of the object of interest, e.g., the antigen, is expressed and presented in the context of the MHC. In some embodiments, the expression of the gene of the object of interest, e.g., the antigen, occurs in the extracellular space, e.g., the antigen is secreted.

[0399] In some embodiments, the RNA molecule includes a coding region for a gene of interest, e.g., an antigen. In some embodiments, the RNA molecule includes a coding region for a gene of interest, e.g., an antigen derived from a pathogen associated with an infectious disease. In some embodiments, the RNA molecule includes a coding region for a gene of interest, e.g., an antigen derived from *E. coli* ciliated antigen (FimH).

[0400] In some embodiments, the RNA polynucleotides described herein or compositions or pharmaceutical preparations containing them contain the nucleotide sequences disclosed herein. In some embodiments, the RNA polynucleotides contain sequences having at least 80% identity to the nucleotide sequences disclosed herein. In some embodiments, the RNA polynucleotides contain sequences encoding polypeptides having at least 80% identity to the polypeptide sequences disclosed herein. In some embodiments, the RNA polynucleotides described herein or compositions or pharmaceutical preparations containing them are transcribed using a DNA template. In some embodiments, the DNA template used to transcribe the RNA polynucleotides described herein contains sequences complementary to the RNA polynucleotides. In some embodiments, the gene of interest described herein is encoded by the RNA polynucleotides described herein, which contain the nucleotide sequences disclosed herein. In some embodiments, the RNA polynucleotides encode polypeptides having at least 80% identity to the polypeptide sequences disclosed herein. In some embodiments, the polypeptides described herein are encoded by RNA polynucleotides transcribed using a DNA template containing sequences complementary to the RNA polynucleotides.

[0401] In some embodiments, the RNA molecule encodes a FimH protein containing one of the sequences of SEQ ID NOs: 1-64, 77, 79, 81, or 83, or a fragment or variant thereof.

[0402] In some embodiments, the RNA molecule encodes the E. coli (E. coli) FimH protein synthesized from a nucleic acid sequence containing one of the following: SEQ ID NOs: SEQ ID NOs: 76, 78, 80, 66-75, 82, 84, 86, 88, or 90, or a fragment or variant thereof.

[0403] F. Poly-A Tail In some embodiments, the RNA molecules disclosed herein include, for example, a polyadenylate (poly-A) sequence as described herein. In some embodiments, the poly-A sequence is located downstream of the 3'UTR, for example, adjacent to the 3'UTR. "Poly-A tail" or "poly-A sequence" refers to a stretch of consecutive adenine residues that may be attached to the 3' end of the RNA molecule. Poly-A sequences are known to those skilled in the art and may follow the 3'UTR in the RNA molecules described herein. The poly-A tail may increase the half-life of the RNA molecule.

[0404] mRNA may contain a polyA sequence and / or a polyadenylation signal. The polyA sequence may consist entirely or mostly of adenine nucleotides or their analogs or derivatives. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the nucleic acid. During RNA processing, a long chain of adenosine nucleotides (the polyA region) is typically added to the messenger RNA (mRNA) molecule to increase molecular stability. Immediately after transcription, the 3' end of the transcript is cleaved to a free 3'-hydroxyl group. PolyA polymerase then adds the chain of adenosine nucleotides to the RNA. The process called polyadenylation adds a polyA region of 100–250 residues in length. The unique length of the polyA region may provide certain advantages to the alternative polynucleotides of this disclosure. Generally, the length of the polyA region of this disclosure is at least 30 nucleotides. In another embodiment, the polyA region is at least 35 nucleotides long. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 70 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides.In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1700 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 1900 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In some examples, the polyA region may be 80, 120, or 160 nucleotides long on the alternative polynucleotide molecules described herein. In other examples, the polyA region may be 20, 30, 40, 80, 100, 120, 140, or 160 nucleotides long on the alternative polynucleotide molecules described herein. In some cases, the polyA region is designed in comparison to the overall length of the alternative polynucleotide. This design may be based on the length of the coding region of the alternative polynucleotide, the length of a particular feature or region of the alternative polynucleotide (e.g., mRNA), or the length of the final product expressed from the alternative polynucleotide.Compared to any feature of an alternative polynucleotide (e.g., the mRNA portion containing the polyA region), the polyA region may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the additional feature. The polyA region may also be designed as a ratio of the alternative polynucleotide to which it belongs. In this context, the polyA region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or greater of the total length of the construct or the total length of the construct excluding the polyA region.

[0405] In certain cases, the conjugation of mRNA for an engineered binding site and / or poly(A)-binding protein may be used to enhance expression. The engineered binding site may be a sensor sequence that can function as a binding site for a ligand in the local microenvironment of the mRNA. As a non-limiting example, the mRNA may include at least one engineered binding site to alter the binding affinity of poly(A)-binding protein (PABP) and its analogues. The incorporation of at least one engineered binding site may increase the binding affinity of PABP and its analogues.

[0406] Additionally, multiple distinct mRNAs may be joined together by ligating them to PABP (poly-A binding protein) via the 3' end using alternative nucleotides at the 3' end of the poly-A region. Transfection experiments are feasible in relevant cell lines, and protein production can be assayed by ELISA at 12, 24, 48, 72, and 7 days post-transfection. As a non-limiting example, transfection experiments may be used to evaluate the effect on the binding affinity of PABP or its analogues as a result of the addition of at least one manipulated binding site. In certain cases, the poly-A region may be used to modulate translation initiation. While we do not wish to be bound by theory, the poly-A region may be essential for protein synthesis because it mobilizes PABP, which can then interact with the translation initiation complex. In some cases, the poly-A region may also be used in this disclosure to protect against 3'-5'-exonuclease digestion. In some examples, the mRNA may contain a poly-AG quartet. A G-quartet is a cyclic, hydrogen-bonded array of four guanosine nucleotides that can be formed by a G-rich sequence in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of a poly-A region. The resulting mRNA may be assayed for other parameters, including stability, protein production, and half-life, at various time points. The poly-AG quartet has been found to result in protein production equivalent to at least 75% of that seen when a 120-nucleotide poly-A region is used alone. In some cases, the mRNA may contain a poly-A region and may be stabilized by the addition of a 3' stabilization region. mRNA having a poly-A region may further contain a 5' cap structure. In other cases, the mRNA may contain a poly-AG quartet. mRNA having a poly-AG quartet may further contain a 5' cap structure. In some cases, the 3' stabilization region that may be used to stabilize the mRNA may contain a poly-A region or a poly-AG quartet.In other cases, the 3' stabilizing region used in this disclosure may include a chain-terminated nucleoside, e.g., 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleoside, e.g., 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleoside, or O-methylnucleoside. In other cases, mRNA comprising a polyA region or polyAG quartet may be stabilized by modifications to the 3' region of the polynucleotide, which can prevent and / or inhibit the addition of oligo(U). In further examples, mRNA containing a poly-A region or poly-AG quartet may be stabilized by the addition of oligonucleotides ending at 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0407] In one embodiment, the RNA disclosed herein includes a polyA tail containing a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 93, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity, or identity between any two of the following: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity. In one embodiment, the polyA tail contains the sequence of SEQ ID NO: 93.

[0408] IV. RNA Transcription In some embodiments, the RNAs disclosed herein are produced by in vitro transcription or chemical synthesis. In the context of this disclosure, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptides or proteins.

[0409] According to this disclosure, “transcription” includes “in vitro transcription” or “IVT,” where “in vitro transcription” or “IVT” refers to the process by which transcription occurs in vitro in a non-cellular system, producing synthetic RNA products for use in various applications, including, for example, the production of proteins or polypeptides. Cloning vectors may be applied for the production of transcripts. These cloning vectors are commonly referred to as transcription vectors and are encompassed by the term “vector” in accordance with this invention. According to a particular embodiment, the RNA used is RNA transcribed in vitro (IVT-RNA), which may be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription may be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to this invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription may be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0410] Synthetic IVT RNA products may be translated in vitro or directly introduced into cells and translated in the cells. With respect to RNA, the terms “expression” or “translation” refer to the process in the ribosomes of a cell in which a chain of mRNA directs the assembly of amino acid sequences to make a peptide or protein. Such synthetic RNA products include, but are not limited to, mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, and small nucleolar RNA molecules. IVT reactions typically utilize DNA templates (e.g., linear DNA templates), ribonucleotides (e.g., unmodified ribonucleotide triphodes or modified ribonucleotide triphodes), and appropriate RNA polymerases as described and / or utilized herein.

[0411] In some embodiments, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In some embodiments, the RNA disclosed herein is in vitro transcribed RNA (IVT-RNA), which may be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription may be any promoter for any RNA polymerase. The DNA template for in vitro transcription may be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0412] In some embodiments, the starting material for IVT may include a linearized DNA template, nucleotides, an RNase inhibitor, a pyrophosphatase, and / or a T7 RNA polymerase. In some embodiments, the IVT process is carried out in a bioreactor. The bioreactor may include a mixer. In some embodiments, nucleotides may be added to the bioreactor throughout the entire IVT process.

[0413] In some embodiments, one or more post-IVT agents are added to the IVT mixture containing RNA in a bioreactor after the IVT process. Exemplary post-IVT agents may include DNAse I, configured to digest a linearized DNA template, and proteinase K, configured to digest DNAse I and T7 RNA polymerase. In some embodiments, the post-IVT agents are incubated with the mixture in a post-IVT bioreactor.In some embodiments, the bioreactor has at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500, up to 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500, exactly 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500, or 60, 70, 80, 90, 100, 110, The IVT mixture may contain an amount between or greater than any two of the following amounts: 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500 liters.The IVT mixture contains at least 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL, Large doses of 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL, exactly 3.3, 3.4, 3. 5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL, or 3.3, 3.4, 3.5, 3.6, 3.7, The RNA concentration may be between or higher than any two of the following values: 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg / mL.

[0414] In some embodiments, the IVT mixture may contain residual spermidine, residual DNA, residual protein, peptide, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+, Na+, Ca2+), RNA fragments, residual nucleotides, free phosphate, or any combination thereof.

[0415] In some embodiments, at least a portion of the IVT mixture is filtered. The IVT mixture may be filtered via ultrafiltration and / or diafiltration to remove at least some impurities from the IVT mixture and / or alter the buffer solution for at least a portion of the IVT mixture to produce a concentrated RNA solution as a retainate.

[0416] In some embodiments, both "ultrafiltration" and "diafiltration" refer to membrane filtration processes. Ultrafiltration is performed for at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 μm, up to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 μm, exactly 0.001, 0.0 Typically, membranes are used that have pore sizes between any two of the following: 0.2, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 μm. In some embodiments, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, MWCO has at least 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310 kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa, and 10000kDa, up to 30kDa, 40kDa, 50kDa,60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 24 0kDa,250kDa,260kDa,270kDa,280kDa,290kDa,300kDa,310kDa,320kDa,330kDa,340kDa,350kDa,360kDa,370kDa,380kDa,390kDa,400kDa,500kDa,6 00kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa, and 10000kDa, exactly 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 23 0kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa, and 10000kDa, or 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 2 20kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa,The filtration membrane may be between any two of 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 2000kDa, 3000kDa, 4000kDa, 5000kDa, 6000kDa, 7000kDa, 8000kDa, 9000kDa, and 10000kDa. Those skilled in the art will understand that, depending on the application, the filtration membrane may be made of different preferred materials, such as polymers, cellulose, and ceramics. In some embodiments, membrane filtration may be more desirable for large-scale purification processes.

[0417] In some embodiments, ultrafiltration and diafiltration of IVT mixtures for RNA purification may include (1) direct flow filtration (DFF), also known as "dead-end" filtration, in which the feedstream is applied perpendicular to the membrane surface and an attempt is made to pass 100% of the fluid through the membrane, and / or (2) tangential flow filtration (TFF), also known as cross-flow filtration, in which the feedstream passes parallel to the membrane surface, with one portion passing through the membrane (permeate) and the remaining portion (retainate) being retained and / or recycled to a feed tank.

[0418] In some embodiments, filtration of the IVT mixture is performed via a TFF comprising an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some embodiments, the first diafiltration step is performed in the presence of ammonium sulfate. The first diafiltration step may be configured to remove most of the impurities from the IVT mixture. In some embodiments, the second diafiltration step is performed without ammonium sulfate. The second diafiltration step may be configured to transfer RNA into the DS buffer formulation.

[0419] A filtration membrane with an appropriate MWCO may be selected for ultrafiltration in the TFF process. The MWCO of the TFF membrane determines which solutes can pass through the membrane into the filtrate and which are retained in the retainate. The MWCO of the TFF membrane may be selected so that substantially all of the solute of interest (e.g., the desired synthesized RNA species) remains in the retainate, while undesirable components (e.g., excess ribonucleotides, small nucleic acid fragments, e.g., digested or hydrolyzed DNA templates, peptide fragments, e.g., digested proteins and / or other impurities) pass into the filtrate. In some embodiments, the retainate containing the desired synthesized RNA species may be recycled to a feed reservoir and re-filtered in an additional cycle. In some embodiments, the TFF film may have an MWCO equal to at least 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, and up to 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, exactly 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, or between any two of 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, or greater kDa. In some embodiments, the TFF film may have an MWCO equal to at least 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, and up to 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, exactly 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, or between any two of 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, or greater kDa. In some embodiments, the TFF film may have MWCO of about 250-350 kDa. In some embodiments, the TFF film (e.g., a cellulose-based film) may have MWCO of about 30-300 kDa; in some embodiments, it may have MWCO of about 50-300 kDa, about 100-300 kDa, or about 200-300 kDa.

[0420] Diafiltration may be performed discontinuously or, alternatively, continuously. For example, in continuous diafiltration, the diafiltration solution may be added to the sample feed reservoir at the same rate as the filtrate is produced. In this way, the volume in the sample reservoir is kept constant, but low molecules (e.g., salts, solvents, etc.) that can freely permeate the membrane are removed. Using solvent removal as an example, each additional diafiltration volume (DV) further reduces the solvent concentration. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the desired concentration of low molecules (e.g., salts, solvents, etc.) remaining in the reservoir is reached. Each additional diafiltration volume (DV) further reduces the concentration of low molecules (e.g., solvents). Continuous diafiltration typically requires a minimum volume for a given reduction of the molecules being filtered. Discontinuous diafiltration, on the other hand, allows for rapid changes in retaining conditions, such as pH and salt content. In some embodiments, the first diafiltration step is performed in diavolumes equal to or greater than at least 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, or between any two of 2, 3, 4, 5, 6, 7, 8, 9, 10. In some embodiments, the second diafiltration step is performed with a dia volume equal to or greater than any two of the following: at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or between any two of the following, or greater than or equal to a dia volume. In some embodiments, the first diafiltration step is performed with a dia volume of 5, and the second diafiltration step is performed with a dia volume of 10.

[0421] In some embodiments, for ultrafiltration and / or diafiltration, the IVT mixture is at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 70 0, 800, 900, or 1000 L / m2, with a maximum of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L / m2, accurate. 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L / m2, or 100, 110, 120, 130, 140, 150 The material is filtered at a rate equal to the filter area per hour of any two of the following L / m2: 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L / m2, or a rate equal to the filter area per hour of a larger L / m2.The concentrated RNA solution may contain single-stranded RNA in amounts of at least 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / mL, and up to 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / mL, exactly 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / mL, or between any two of the following single-stranded RNA concentrations: 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / mL.

[0422] The bioburden of the RNA solution concentrated via filtration to obtain the RNA product solution may also be reduced in some embodiments. Filtration for bioburden reduction may be performed using one or more filters. One or more filters may include filters having pore sizes of at least 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, up to 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, exactly 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or between any two of 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or any other pore size configured to remove bioburden.

[0423] As one example, bioburden reduction may include draining a retainate tank containing retainate obtained from ultrafiltration and / or diafiltration to obtain retainate. Bioburden reduction may also include flushing the filtration system for ultrafiltration and / or diafiltration with a wash buffer solution to obtain a wash pool solution containing residual RNA remaining in the filtration system. The retainate may be filtered to obtain filtered retainate. The wash pool solution may be filtered using a first 0.2 μm filter to obtain filtered wash pool solution. The retainate may be filtered using a first 0.2 μm filter or another 0.2 μm filter.

[0424] The filtered wash pool solution and the filtered retainate may be combined to form a combined pool solution. The combined pool solution may be filtered using a second 0.2 μm filter to obtain a filtered combined pool solution, and the filtered combined pool solution may be further filtered using a third 0.2 μm filter to produce an RNA product solution.

[0425] V. RNA encapsulation RNA in an RNA product solution may be encapsulated, and the RNA solution may further contain at least one encapsulating agent. In one embodiment, the encapsulating agent includes lipids, lipid nanoparticles (LNPs), lipoplexes, polymer particles, polyplexes, and monolithic delivery systems, as well as combinations thereof.

[0426] Lipid nanoparticles may comprise lipid components and one or more additional components, such as therapeutic and / or prophylactic agents. LNPs may be designed for one or more specific applications or targets. The elements of an LNP may be selected based on a specific application or target and / or on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, a particular formulation of an LNP may be selected for a particular application or target, for example, according to the efficacy and toxicity of a particular combination of elements. The efficacy and tolerability of an LNP formulation may be influenced by the stability of the formulation.

[0427] Lipid nanoparticles may be designed for one or more specific applications or targets. For example, LNPs may be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups within the mammalian body.

[0428] The biochemical properties of lipid nanoparticles may be modified to increase selectivity for specific bodily targets. For example, particle size may be adjusted based on fenestration sizes of different organs. Therapeutic and / or prophylactic agents contained in LNPs may also be selected based on one or more desired delivery targets. For example, therapeutic and / or prophylactic agents may be selected for specific symptoms, conditions, diseases, or disorders and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., localized or specific delivery). In certain embodiments, the LNPs may contain mRNA encoding a polypeptide of interest that has the ability to be translated intracellularly to produce the polypeptide of interest. Such compositions may be designed to be specifically delivered to specific organs. In some embodiments, the compositions may be designed to be specifically delivered to the mammalian liver. In some embodiments, the compositions may be designed to be specifically delivered to lymph nodes. In some embodiments, the compositions may be designed to be specifically delivered to the mammalian spleen.

[0429] In one embodiment, the encapsulating agent is a lipid, and the product manufactured is lipid nanoparticle (LNP)-encapsulated RNA. While not intended to be bound by any theory, it is believed that cationic or cationically ionizable lipids or lipid-like materials and / or cationic polymers, in combination with nucleic acids, form aggregates, and these aggregates result in colloidally stable particles. Lipids may be naturally occurring or synthetic. However, lipids are typically biological substances. Biological lipids are well known in the art and include, for example, triglycerides, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, sphingoglycolipids, glucolipids, sulfatides, ethers and ester-linked fatty acids, as well as lipids having polymerizable lipids, and combinations thereof. Lipids are substances insoluble in water and extractable with organic solvents. Compounds other than those specifically described herein are understood as lipids by those skilled in the art and are encompassed by the compositions and methods of this disclosure. Lipid components and non-lipids may be attached to each other either covalently or non-covalently.

[0430] In some embodiments, LNPs may be designed to protect RNA molecules (e.g., mRNA) from extracellular RNases and / or manipulated for systemic delivery of RNA to target cells. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., mRNA, modRNA) when administered intravenously to a target that requires it. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., mRNA) when administered intramuscularly to a target that requires it.

[0431] In one embodiment, the RNA concentration in the RNA solution is less than 1 mg / mL. In another embodiment, the RNA concentration is at least about 0.05 mg / mL. In another embodiment, the RNA concentration is at least about 0.5 mg / mL. In another embodiment, the RNA concentration is at least about 1 mg / mL. In another embodiment, the RNA concentration is about 0.05 mg / mL to about 0.5 mg / mL. In another embodiment, the RNA concentration is at least 10 mg / mL. In another embodiment, the RNA concentration is at least 50 mg / mL. In some schematics, RNA is present in concentrations of at least 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, and up to 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, and exactly 0. The concentration is 0.5 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or between any two of the following concentrations, or higher.

[0432] This disclosure provides RNA solutions and lipid preparation mixtures or compositions thereof, comprising at least one RNA encoding an antigen (e.g., Escherichia coli (E. coli) FimH protein), which is complexed with one or more lipids, encapsulated within one or more lipids, and / or formulated using one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes, and / or nanoliposomes. In some embodiments, the composition comprises lipid nanoparticles.

[0433] Lipid nanoparticles or LNPs refer to any form of particles produced when a cationic lipid and optionally one or more further lipids are combined, for example, in an aqueous environment and / or in the presence of RNA. In some embodiments, lipid nanoparticles are included in formulations that may be used to deliver activators or therapeutic agents, such as nucleic acids (e.g., mRNA, modRNA), to target sites of interest (e.g., cells, tissues, organs, and tumors). In some embodiments, the lipid nanoparticles of this disclosure include nucleic acids. Such lipid nanoparticles typically include a cationic lipid and one or more excipients, such as one or more neutral lipids, charged lipids, steroids, lipids conjugated to polymers, or a combination thereof. In some embodiments, the activator or therapeutic agent, such as nucleic acids (e.g., mRNA, modRNA), may be encapsulated within the lipid portion of the lipid nanoparticle or within an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses. Nucleic acids (e.g., mRNA, modRNA) or their portions may also associate and complex with lipid nanoparticles. The lipid nanoparticles may contain any lipids capable of forming particles to which nucleic acids are attached or to which one or more nucleic acids are encapsulated.

[0434] In some embodiments, the provided RNA molecules (e.g., mRNA, modRNA) may be formulated using LNPs. In some embodiments, the lipid nanoparticles may have an average diameter of about 1 to 500 nm. In some embodiments, the lipid nanoparticles may have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or at least 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, up to 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, precisely 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 1 Having an average diameter between any two of the following: 45nm, or 150nm, or 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm, and being substantially nontoxic.The term "average diameter" refers to the average hydrodynamic diameter of a particle as measured by dynamic laser light scattering (DLS), along with data analysis using the so-called cumulant algorithm, which results in a so-called Z-mean with the dimension of length and a dimensionless multi-dispersion index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter," "diameter," "size," or "average size" of a particle are used synonymously with this value of the Z-mean.

[0435] The LNPs described herein may exhibit polyvariance indices lower than approximately 0.5, lower than approximately 0.4, lower than approximately 0.3, or approximately 0.2 or lower. For example, LNPs are at least 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0 0.5, up to 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5, exactly 0.1 , 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5, or 0.1, 0.11, 0.1 It may exhibit a multivariance index between any two of the following: 2, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5.The polydispersion index is calculated in some embodiments based on dynamic light scattering measurements by so-called cumulant analysis, which is referred to in the definition of "average diameter." Under certain requirements, it may also serve as an indicator of the size distribution of an ensemble of nanoparticles.

[0436] Lipid nanoparticles may be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to investigate the morphology and size distribution of LNPs. Dynamic light scattering or potentiometric measurements (e.g., potentiometric titration) may be used to measure the zeta potential. Dynamic light scattering may also be used to determine particle size. Instruments, such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK), may also be used to measure several features of LNPs, such as particle size, polydispersity index, and zeta potential.

[0437] The average size of LNPs may be in the range of tens to hundreds of nanometers, for example, measured by dynamic light scattering (DLS). For example, the average size may be approximately 40 nm to approximately 150 nm, such as approximately 40 nm, 45 nm, 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, or 150 nm. In some embodiments, the average size of the LNP may be approximately 50 nm to 100 nm, approximately 50 nm to 90 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, approximately 50 nm to 60 nm, approximately 60 nm to 100 nm, approximately 60 nm to 90 nm, approximately 60 nm to 80 nm, approximately 60 nm to 70 nm, approximately 70 nm to 100 nm, approximately 70 nm to 90 nm, approximately 70 nm to 80 nm, approximately 80 nm to 100 nm, approximately 80 nm to 90 nm, or approximately 90 nm to 100 nm. In a particular embodiment, the average size of the LNP may be approximately 70 nm to 100 nm. In a particular embodiment, the average size may be approximately 80 nm. In other embodiments, the average size may be approximately 100 nm.

[0438] LNPs may be relatively homogeneous. The polydispersity index may be used to indicate the homogeneity of LNPs, for example, the particle size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. LNPs may have a polydispersity index of about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs may be about 0.10 to about 0.20.

[0439] The zeta potential of LNPs may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of the LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements throughout the body. In some embodiments, the zeta potential of the LNP may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0440] In certain embodiments, nucleic acids (e.g., RNA molecules) are resistant to degradation by nucleases in aqueous solutions when present in the provided LNPs. In some embodiments, the LNPs are liver-targeting lipid nanoparticles. In some embodiments, the LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some embodiments, the cationic LNPs may comprise at least one cationic lipid, at least one lipid conjugated to a polymer, and at least one helper lipid (e.g., at least one neutral lipid).

[0441] In a particular manner, RNA solutions and their lipid preparation mixtures or compositions are available in concentrations of 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, and approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68 %, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% of specific lipids, lipid types, or non-lipid components, such as lipid-like materials and / or cationic polymers or adjuvants, antigens, peptides, polypeptides, sugars, nucleic acids or other materials disclosed herein or known to those skilled in the art, or at least 1%, approximately 2%, approximately 3%, approximately 4%Approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 3 0%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, Approximately 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, with a maximum of 1%, 2%, 3%, and 4%. Approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 3 0%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, Approximately 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, exactly 1%, 2%, 3%, 4%Approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30 %, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99%, or approximately 1%, approximately 2%, approximately 3%, approximately 4%Approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, Approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately The material may contain specific lipids, lipid types, or non-lipid components between any two of the following percentages: 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99%.

[0442] The LNPs described herein may be prepared using a wide range of methods that may involve obtaining colloids from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer, and mixing the colloids with nucleic acids to obtain nucleic acid particles. The term “colloid,” as used herein, refers to a type of homogeneous mixture in which dispersed particles do not precipitate. The insoluble particles in the mixture are microscopic in size and have a particle size of 1 to 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. In some cases, the term “colloid” refers only to the particles in the mixture and not the entire suspension.

[0443] Methods conventionally used and appropriately adapted for preparing liposome vesicles are applicable herein for the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer. The most commonly used method for preparing liposome vesicles shares the following basic stages: (i) dissolution of the lipid in an organic solvent, (ii) drying of the resulting solution, and (iii) hydration of the dried lipid (using various aqueous media). In the film hydration method, the lipid is first dissolved in a suitable organic solvent and dried down to yield a thin film at the bottom of the flask. The resulting lipid film is hydrated using a suitable aqueous media to produce a liposome dispersion. Further, an additional downsizing step may be included.

[0444] Reverse-phase evaporation is an alternative to film hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. A short ultrasonic treatment of this mixture is required for system homogenization. Removal of the organic phase under reduced pressure results in an emulsion gel that subsequently becomes a liposome suspension.

[0445] The term "ethanol injection technique" refers to the process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicle formation, or liposome formation. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in some embodiments as follows: an ethanol solution containing lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under agitation. In some embodiments, the RNA lipoplex particles described herein can be obtained without an extrusion step.

[0446] The term "extrude" or "extrude" refers to the creation of particles with a fixed cross-sectional profile. In particular, it refers to the downsizing of particles that are forced to pass through a filter with a defined pore.

[0447] Other methods having the characteristic of being free of organic solvents may also be used in accordance with this disclosure to prepare colloids.

[0448] In some embodiments, RNA encapsulated in LNPs may be produced by rapidly mixing an RNA solution (e.g., RNA product solution) and a lipid preparation (e.g., containing at least one cationic lipid and optionally one or more other lipid components in an organic solvent) described herein under conditions such that a sudden change in the solubility of the lipid components is triggered, driving the lipids toward self-assembly in the form of LNPs. In some embodiments, preferred buffering agents include tris, histidine, citrate, acetate, phosphate, or succinate. The pH of the liquid formulation is related to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale lower than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale higher than the pKa of the encapsulating agent (e.g., cationic lipid). In some embodiments, the properties of the cationic lipid are selected such that the new formation of the particle occurs by association with the oppositely charged backbone of the nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which can result in a much higher encapsulation efficiency than can be achieved, for example, in some embodiments, in the absence of interaction between the nucleic acid and at least one of the lipid components.

[0449] The encapsulation efficiency of therapeutic and / or prophylactic agents is described as the amount of encapsulated or otherwise associated therapeutic and / or prophylactic agent after preparation compared to the initial amount provided. The encapsulation efficiency is preferably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing lipid nanoparticles before and after disrupting the lipid nanoparticles with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For lipid nanoparticles described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the packaging efficiency may be at least 80%. In certain embodiments, the packaging efficiency may be at least 90%.

[0450] LNP may optionally include one or more coatings. For example, LNP may be formulated in a capsule, film, or tablet having a coating. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0451] Formulations comprising amphiphilic polymers and lipid nanoparticles may be formulated as a whole or in part as a pharmaceutical composition. A pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid nanoparticles. For example, a pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid nanoparticles comprising one or more different therapeutic and / or prophylactic agents. A pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or accessory components, e.g., those described herein. General guidelines for the formulation and production of pharmaceutical compositions and formulations are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and accessory components may be used in any pharmaceutical composition unless any conventional excipient or accessory component may be incompatible with one or more components of the LNPs or one or more amphiphilic polymers in the formulations of this disclosure. Excipients or accessory components may be incompatible with the components or amphiphilic polymers if their combination with the components or amphiphilic polymers of the form...

Claims

1. An RNA molecule comprising at least one open reading frame (ORF) encoding a fimbriae H antigen (FimH) polypeptide and a 5' untranslated region (5'UTR), wherein the 5'UTR comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence described in any one of sequence numbers 95 to 101.

2. The RNA molecule according to claim 1, wherein the 5'UTR comprises a nucleic acid sequence in which the 5'UTR is at least 92% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 95, 98, 99, and 101.

3. An RNA molecule according to any one of claims 1 to 2, wherein the 5'UTR comprises a nucleic acid sequence that is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 95, 99, and 101.

4. An RNA molecule according to any one of claims 1 to 3, comprising a nucleic acid sequence in which the 5'UTR is at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 99 and 101.

5. An RNA molecule according to any one of claims 1 to 4, comprising a nucleic acid sequence in which the 5'UTR is at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 99 and 101.

6. 5'UTR, Sequence ID 99 (5'UTR_BMD562) and Sequence ID 101 (5'UTR_BMD576) An RNA molecule according to any one of claims 1 to 5, comprising a nucleic acid sequence selected from the group consisting of the following.

7. The RNA molecule according to any one of claims 1 to 6, further comprising a 3' untranslated region (3'UTR).

8. The RNA molecule according to claim 7, wherein the 3'UTR comprises a nucleotide having the sequence described in SEQ ID NO: 103 (3'UTR_hHBB).

9. The RNA molecule according to any one of claims 1 to 8, wherein the FimH polypeptide encoded by the RNA molecule is its full length, cleaved form, fragment, or variant.

10. The RNA molecule according to any one of claims 1 to 9, wherein the FimH polypeptide encoded by the RNA molecule contains at least one mutation.

11. The RNA molecule according to any one of claims 1 to 10, wherein the FimH polypeptide encoded by the RNA molecule has at least 90%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NOs: 1 to 64.

12. The RNA molecule according to any one of claims 1 to 11, wherein the FimH polypeptide encoded by the RNA molecule has an amino acid sequence selected from SEQ ID NOs: 1 to 64.

13. The RNA molecule according to any one of claims 1 to 12, wherein the FimH polypeptide encoded by the RNA molecule is selected from the group consisting of FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), and FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), or immunogenic fragments thereof.

14. An RNA molecule according to any one of claims 1 to 13, wherein a FimH polypeptide encoded by the RNA molecule is fused to a C-terminal membrane targeting domain.

15. The RNA molecule according to any one of claims 1 to 14, wherein the C-terminal membrane targeting domain is DAFgpi or a variant thereof.

16. The RNA molecule according to claim 15, wherein DAFgpi is a variant comprising serine / glycine linker substitutions of eight DAF amino acid residues proximal to the ω-site serine by a serine / glycine linker having the amino acid sequence GSSGSGSS (SEQ ID NO: 94).

17. The RNA molecule according to any one of claims 14 to 16, wherein the FimH polypeptide encoded by the RNA molecule has an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NOs. 77, 79, 81, or 83.

18. The RNA molecule according to any one of claims 14 to 17, wherein the FimH polypeptide encoded by the RNA molecule is selected from the group consisting of SEQ ID NOs: 77, 79, 81, and 83.

19. The RNA molecule according to any one of claims 1 to 18, wherein the open reading frame is transcribed from a nucleic acid comprising a nucleotide sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the sequences of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO:

138.

20. The RNA molecule according to claim 19, wherein the open reading frame is transcribed from a nucleic acid containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO:

138.

21. The RNA molecule according to any one of claims 1 to 19, wherein the open reading frame includes a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the sequences described in SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO:

139.

22. The RNA molecule according to claim 21, wherein the open reading frame includes a nucleic acid sequence selected from the group consisting of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO:

139.

23. An RNA molecule according to any one of claims 1 to 22, further comprising a 5' cap portion or a 3' poly-A tail.

24. 5' The cap portion is m7G(5')ppp(5')(2'OMeA)pG or (m ２ ７，３’－Ｏ ) Gppp(m ２’－Ｏ An RNA molecule according to any one of claims 1 to 23, wherein it is ApG.

25. The RNA molecule according to claim 24, wherein the polyA tail contains a sequence having sequence number 92.

26. An RNA molecule according to any one of claims 1 to 25, comprising a nucleotide having the sequence described in SEQ ID NOs. 66-75, SEQ ID NOs. 82, SEQ ID NOs. 84, SEQ ID NOs. 86, SEQ ID NOs. 88, or SEQ ID NOs.

90.

27. The RNA molecule according to claim 26, which is transcribed from a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one sequence selected from sequence numbers 107-116 or sequence numbers 120-124.

28. The RNA molecule according to any one of claims 1 to 27, wherein the open reading frame contains at least 55%, 60%, 65%, 70%, or 75% of G / C content, or 50% to 75% or 55% to 70% or about 50% to 75% or 55% to 70%.

29. The RNA molecule according to any one of claims 1 to 28, wherein the encoded FimH polypeptide is localized in the cell membrane, localized in the Golgi, and / or secreted.

30. The RNA molecule according to any one of claims 1 to 29, wherein the RNA comprises at least one modified nucleotide.

31. The RNA molecule according to claim 30, wherein the modified nucleotide is pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, or 2'-O-methyluridine.

32. Modified nucleotides are pseudouridine (Ψ) or N １ - The RNA molecule according to claim 31, which is methylpseudridine (m1Ψ).

33. Each uridine in an RNA molecule is either pseudouridine (Ψ) or N １ - The RNA molecule according to claim 32, which is replaced with methylpseudridine (m1Ψ).

34. The RNA molecule according to any one of claims 1 to 33, wherein the RNA is mRNA.

35. The RNA molecule according to claim 34, wherein the RNA is modRNA.

36. A composition comprising an RNA molecule according to any one of claims 1 to 35, wherein the RNA molecule is formulated in lipid nanoparticles (RNA-LNP).

37. The composition according to claim 36, wherein the lipid nanoparticles comprise at least one of cationic lipids, PEG-modified lipids, neutral lipids, and steroids or steroid analogs.

38. The composition according to claim 37, wherein the cationic lipid is (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

39. PEG-modified lipids include glycol lipids containing PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG, PEG-modified diacylglycerol (PEG-DAG), e.g., 1-(monomethoxy- The composition according to claim 37 or 38, wherein the composition is polyethylene glycol-2,3-dimyristoylglycerol (PEG-DMG), PEG-modified phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), for example, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEG-modified ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, for example, co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate.

40. The composition according to claim 39, wherein the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

41. The neutral lipids are distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl -Phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (oleoyl The composition according to any one of claims 37 to 40, wherein the composition is phosphatidyethanolamine (SOPE) or 1,2-dieridoyl-sn-glycero-3-phosphoethanolamine (phophoethanolamine) (trans-DOPE).

42. The composition according to claim 41, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

43. The composition according to any one of claims 37 to 42, wherein the steroid or steroid analog is cholesterol.

44. A vaccine, the composition according to any one of claims 36 to 43.

45. A mutant FimH polypeptide having at least 80% identity to any one of the amino acid sequences described in SEQ ID NO: 77, SEQ ID NO: 81, or SEQ ID NO:

83.

46. The mutant FimH polypeptide according to claim 45, comprising an amino acid having the sequence described in SEQ ID NO: 81 or SEQ ID NO:

83.

47. A polynucleotide encoding a mutant FimH polypeptide having at least 80% identity to any one of the amino acid sequences described in SEQ ID NOs: 77, 81, or 83.

48. A polynucleotide encoding a mutant FimH polypeptide containing a nucleic acid having the sequence described in SEQ ID NO: 117, SEQ ID NO: 118, or SEQ ID NO:

139.

49. The polynucleotide according to claim 48, wherein the polynucleotide encoding the mutant FimH polypeptide is transcribed from a nucleic acid containing the nucleotide sequence described in SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO:

138.

50. (i) A method for inducing an immune response to extraenteric pathogenic Escherichia coli (E. coli) in a subject, or (ii) a method for inducing the production of opsonized phagocytic antibodies and / or neutralizing antibodies specific to extraenteric pathogenic Escherichia coli (E. coli), comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP and / or vaccine according to any one of claims 1 to 44.

51. The method according to claim 50, wherein the subject is at risk of developing a urinary tract infection.

52. The method according to claim 50, wherein the subject is at risk of developing bacteremia.

53. The method according to claim 50, wherein the subject is at risk of developing urinary tract sepsis.

54. The method according to claim 50, wherein the subject is at risk of developing cystitis.

55. Use of RNA molecules, RNA-LNPs and / or compositions according to any one of claims 1 to 44 in the manufacture of a pharmaceutical product for use in (i) inducing an immune response to extraenterally pathogenic Escherichia coli (E. coli) in a subject, or (ii) in inducing the production of opsonized phagocytic antibodies and / or neutralizing antibodies specific to extraenterally pathogenic Escherichia coli (E. coli) in a subject.

56. The use according to claim 55, wherein the infection, disease, or condition is a urinary tract infection.

57. The use described in claim 55, wherein the subject is at risk of developing bacteremia.

58. The use according to claim 55, wherein the subject is at risk of developing sepsis.

59. The use described in claim 55, wherein the subject is at risk of developing cystitis.

60. The method or use according to any one of claims 50 to 59, wherein the subject is under approximately one year old, approximately one year old or older, approximately five years old or older, approximately ten years old or older, approximately twenty years old or older, approximately thirty years old or older, approximately forty years old or older, approximately fifty years old or older, approximately sixty years old or older, approximately seventy years old or older, or older.

61. The method or use according to any one of claims 50 to 59, wherein the subject is approximately 50 years of age or older.

62. The method or use according to any one of claims 50 to 59, wherein the subject is a pregnant woman.

63. The method or use according to any one of claims 50 to 62, wherein an RNA molecule or composition is administered as a vaccine.

64. The method or use according to any one of claims 50 to 63, wherein the RNA molecule or composition is administered by intradermal or intramuscular injection.

65. The method or use according to any one of claims 50 to 64, wherein the subject is administered a single dose, two doses, three doses or more, and optionally a booster dose, of an RNA molecule, composition, or vaccine.

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