Method for stabilizing RNA

Abstract

The present invention relates to RNA molecules encoding Escherichia coli pilus H antigen (FimH). The present disclosure further relates to compositions comprising an RNA molecule (RNA-LNP) formulated in a lipid nanoparticle. The disclosure further relates to the use of the RNA molecules, RNA-LNPs and compositions for the prevention of E. coli infections, including urethral infections.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 508,060, filed June 14, 2023; U.S. Provisional Application No. 63 / 610,042, filed December 14, 2023; and U.S. Provisional Application No. 63 / 649,495, filed May 20, 2024. The entire contents of each of the foregoing applications are incorporated herein by reference. Technical Field

[0003] This invention relates to compositions and methods for the preparation, production, and therapeutic use of RNA vaccines, wherein the RNA vaccine comprises encoding one or more Escherichia coli strains (Escherichia coli). E. coli The fimbrial antigen is a polynucleotide molecule, such as fimbrial H antigen (FimH).

[0004] References to sequence lists

[0005] This application was submitted electronically via EFS-Web and includes a sequence list in .xml format submitted electronically. The .xml file contains a sequence list named "PC72992A Sequence Listing.xml", created on May 29, 2024, and with a file size of 378KB. The sequence list contained in this .xml file is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0006] One in five women will experience a urinary tract infection (UTI) in their lifetime, contributing significantly to morbidity and mortality and placing a burden on healthcare systems. While several different bacteria can cause UTIs, the most common cause (90 to 95% of cases) is Escherichia coli (E. coli). E. coli Escherichia coli ( E. coli These are Gram-negative bacteria that colonize the human gut microbiota or cause serious invasive diseases (Bonten, M. et al.). Clin infect dis , 2021. 72(7):1211-1219). Escherichia coli is one of the most common causes of bacteremia and UTI. Uropathogenic Escherichia coli (UPEC) is the most common pathogen causing 80 to 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 confined to the bladder, it is called cystitis. These bacterial infections can climb from the bladder to the kidneys and cause pyelonephritis. It is estimated that 50% of women will experience at least one symptomatic UTI in their lifetime (Terlizzi, ME, G. Gribaudo, and ME Maffei). Front microbiol (2017. 8: 1566). Children and the elderly are also at significant risk of developing these infections. UTI has a high incidence rate and a recurrence rate of 27% to 44%. With the increase in antibiotic resistance and pathogenic isolates, multidrug-resistant strains (such as Escherichia coli ST131) have emerged.

[0007] UPECs typically originate in the gut, then migrate to the urogenital tract by adhering to the host's urothelial cells, and rapidly replicate upon reaching the bladder (Flores-mireles, AL et al.). Nat rev microbiol , 2015.13(5): 269-284; and Klein, RD and SJ Hultgren. Nat rev microbiol , 2020. 18(4): 211-226). Pili adhesins facilitate adhesion and contain type 1 fimbriae, which bind to mannose-mediated glycoproteins expressed on the surface of host urinary epithelial cells. Type 1 fimbriae are highly conserved in clinical UPEC isolates and are composed of a group called fim The gene cluster encodes accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG), and an adhesin called FimH. FimH is essential for establishing bladder infection in mouse and pig UTI models (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 targeting FimH by mimicking the mannosylation receptor further validated the role of FimH in UTI and showed promise as a treatment in animal models (Cusumano, CK et al.). Sci transl med., 2011. 3(109): 109ra115). Furthermore, FimH is in a state of positive selection among Escherichia coli isolates of human cystitis (Chen, SL et al.). Proc natl acad sci U S A , 2009.106(52): 22439-44) and positively selected residues may affect the toxicity of mouse cystitis models (Schwartz, DJ et al.). Proc natl acad sci USA , 2013. 110: 15530-15537).

[0008] FimH consists of two domains, one of which is responsible for binding to the lectin-binding domain of mannose-modified glycoproteins. LD The fimH protein, along with the pilin domain, connects FimH to other structural subunits of the fimula (such as FimG) via a mechanism called donor strand exchange (Le Trong, I. et al.). J. Struct Biol ., 2010;172(3): 380-388). The fimbriae domain of FimH forms an incomplete immunoglobulin fold, creating grooves that provide binding sites for the N-terminal β-chain of FimG, while a strong intermolecular connection is formed between FimH and FimG. Although FimH LD It can be expressed in a soluble, stable form, but the full-length FimH is unstable when used alone (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), unless it forms a complex with the chaperone FimC, or is complementary to 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). The design and expression of full-length FimH molecules, which link FimG donor peptides to full-length FimH via a glycine-serine linker, have been previously described (PCT International Publication No. WO2021 / 084429, published on May 6, 2021) and have been designated as FimH-DSG.

[0009] Regarding FimHLD In terms of its ability to stimulate functional immunity, it is considered a poor immunogen. Some studies suggest that, despite the presence or absence of adjuvants, FimH... LD All of these can induce antibody titers, but functional neutralizing titers were only observed in the presence of adjuvants (PCT International Publication No. WO2021 / 084429, published on May 6, 2021). Studies suggest that locking FimH in an open conformation, which reduces affinity for mannoside ligands, can improve functional immunity (Kisiela, DI et al.). Proc Natl Acad Sci USA 110, 19089-19094 (2013)).

[0010] Although progress has been made in the development of UTI vaccines, no approved vaccines are currently available. Therefore, there is a need for immunogenic compositions of FimH antigens with reduced affinity for mannoside ligands and modified biochemical properties, resulting in improved functional immunity relative to wild-type FimH.

[0011] RNA technology (especially mRNA technology) is particularly advantageous as a vaccine or therapeutic platform. For effective RNA vaccines or therapies, maximizing protein expression is a key focus, enabling the production of the desired amount of protein or antigen from a minimal amount of RNA. However, mRNA-based therapies may face challenges including low production efficiency, short half-life of mRNA administered in circulation, and low translation efficiency. Therefore, there is a need for RNA compositions with improved stability and translation efficiency, including methods that improve protein expression and achieve high expression by optimizing the sequence and structure of the 5' untranslated region of mRNA. Summary of the Invention

[0012] This invention addresses an unmet need for modified immunogenic compositions, as provided herein, for Escherichia coli infection.

[0013] In one aspect, this disclosure provides immunogenic compositions and methods for preventing, treating, or improving an infection, disease, or condition in a subject, comprising administering an RNA molecule, such as an immunogenic RNA polynucleotide encoding an amino acid sequence, such as an immunogenic antigen, the RNA molecule comprising an *E. coli* FimH protein (“FimH”), an immunogenic variant thereof, or an immunogenic fragment of the FimH protein or an immunogenic variant thereof, such as an antigenic peptide or protein. Thus, the immunogenic antigen comprises a FimH protein epitope for inducing an immune response against FimH in the subject. The administration of the RNA polynucleotide encoding the immunogenic antigen provides (after expression of the polynucleotide by appropriate target cells) an antigen, such as an antibody and / or immune effector cells, for inducing, estimating, precipitating, and / or amplifying an immune response. In one aspect, the immune response to be induced according to this disclosure is both a B cell-mediated immune response (e.g., an antibody-mediated immune response) and a T cell-mediated immune response. In one aspect, the immune response is an anti-FimH immune response.

[0014] The immunogenic compositions described herein comprise RNA molecules containing RNA (as the active substance) that can be translated into one or more proteins in recipient cells. In addition to wild-type, codon-optimized, or mutant sequences encoding antigen sequences, the RNA molecules may contain one or more structural components optimized to maximize RNA efficacy for stability and translation efficiency (5' cap, 5' UTR, subgenomic promoter, 3' UTR, polyadenylated tail). In one aspect, the RNA molecule contains all of these components. The RNA molecules described herein may be complexed with lipids and / or proteins to produce RNA particles for administration (e.g., lipid nanoparticles (LNPs)). In one aspect, the RNA molecules described herein are complexed with lipids to produce RNA-lipid nanoparticles for administration (e.g., RNA-LNPs). In another aspect, the RNA molecules described herein are complexed with proteins for administration. In another aspect, the RNA molecules described herein are complexed with both lipids and proteins for administration. If different combinations of RNA molecules are used, the RNA molecules may be co-complexed with lipids and / or proteins or separately to produce RNA particles for administration.

[0015] This disclosure provides an RNA molecule and an RNA-LNP comprising at least one open reading frame (ORF) and a 5' untranslated region (5'UTR) encoding a FimH antigen, wherein the 5' UTR contains at least 90% identical nucleic acid sequence to the nucleic acid sequence shown in any one of SEQ ID NO: 95 to 101. In some aspects, the FimH antigen is a FimH polypeptide. In some aspects, the FimH polypeptide is its full length, truncated, fragment, or variant. In some aspects, the FimH polypeptide contains at least one mutation.

[0016] This disclosure provides an RNA molecule and RNA-LNP comprising at least one ORF encoding a FimH polypeptide, wherein the FimH polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1 to 64, 77, 79, 81, or 83. In some aspects, the FimH polypeptide has, at least has, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any of the amino acid sequences shown in SEQ ID NO: 1 to 64, 77, 79, 81, or 83. In some aspects, the FimH polypeptide consists of any of the amino acid sequences shown in SEQ ID NO: 1 to 64, 77, 79, 81, or 83.

[0017] On the other hand, this disclosure provides an RNA molecule comprising at least one ORF encoding a FimH polypeptide and an RNA-LNP, wherein the FimH polypeptide comprises FimH-DSG (SEQ ID NO: 59), a FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), a FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), an immunogenic fragment thereof, or any combination of two or more of the foregoing. In some aspects, the FimH polypeptide has, at least has, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any amino acid sequence of SEQ ID NO: 1 to 64, 77, 79, 81, or 83.

[0018] This disclosure provides an RNA molecule and an RNA-LNP comprising at least one ORF transcribed from at least one DNA nucleic acid. In some aspects, 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 aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence having, at least having, or having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any nucleic acid sequence of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence composed of any nucleic acid sequence of any one of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138.

[0019] This disclosure further provides an RNA molecule comprising at least one ORF containing an RNA nucleic acid sequence. In some aspects, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NO: 66 to SEQ ID NO: 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 90. In some aspects, the RNA molecule comprises a nucleic acid sequence having, at least having, or having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any nucleic acid sequence of any one of SEQ ID NO: 66 to SEQ ID NO: 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 90. In some aspects, the RNA molecule comprises a nucleic acid sequence consisting of any one of the nucleic acid sequences of any one of SEQ ID NO: 66 to SEQ ID NO: 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 90. In some aspects, the RNA molecule having any one of SEQ ID NO: 66 to SEQ ID NO: 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 90 contains at least one modified nucleotide. In some aspects, each uridine in any one of SEQ ID NO: 66 to SEQ ID NO: 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 90 is replaced by a modified nucleotide (e.g., modified RNA; modRNA). On one hand, the modified nucleotide is 1-methyl-3'-pseudouridine (also known as pseudouridine) (Ψ). On the other hand, the modified nucleotide is N. 1 -Methylpseudouridine (m1Ψ).

[0020] 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 aspects, the RNA molecule comprises a 5' untranslated region (5'-UTR). In some aspects, the 5' UTR comprises a sequence selected from any one of SEQ ID NO: 95 to SEQ ID NO: 102. In some aspects, 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 one of SEQ ID NO: 95 to SEQ ID NO: 102. In some aspects, the 5' UTR comprises a sequence consisting of any one of SEQ ID NO: 95 to SEQ ID NO: 102. On the other hand, the 5' UTR contains at least 92% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NO: 95, 98, 99, and 101. In some aspects, the 5' UTR contains at least 95% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NO: 95, 99, and 101. In some aspects, the 5' UTR contains at least 98% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NO: 99 and 101. In some aspects, the 5' UTR contains at least 99% identical nucleic acid sequences to the nucleic acid sequences selected from the group consisting of SEQ ID NO: 99 and 101. In some aspects, the 5' UTR contains nucleic acid sequences selected from the group consisting of:

[0021] SEQ ID NO: 99(5'UTR_BMD562); and

[0022] SEQ ID NO: 101 (5'UTR_BMD576).

[0023] In some aspects, the RNA molecule and RNA-LNP include a 3' untranslated region (3'-UTR). In some aspects, the 3' UTR contains a sequence selected from any one of SEQ ID NO: 103 to SEQ ID NO: 106. In some aspects, the 3' UTR contains a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any one of SEQ ID NO: 103 to SEQ ID NO: 106. In some aspects, the 3' UTR contains a sequence composed of any one of SEQ ID NO: 103 to SEQ ID NO: 106. In a preferred embodiment, the 3' UTR is represented by SEQ ID NO: 103.

[0024] This disclosure further provides RNA molecules comprising a 5' cap portion and RNA-LNPs. This disclosure further provides RNA molecules comprising a 3' polyadenylated tail and RNA-LNPs. In some aspects, the polyadenylated tail comprises a sequence having SEQ ID NO: 93 or SEQ ID NO: 140.

[0025] In some aspects, the RNA molecule includes a 5' UTR and a 3' UTR. In some aspects, the RNA molecule includes a 5' cap, a 5' UTR, and a 3' UTR. In some aspects, the RNA molecule includes a 5' cap, a 5' UTR, a 3' UTR, and a polyadenylated tail. In some aspects, the RNA molecule includes a 5' cap, a 3' UTR, and a polyadenylated tail. In some aspects, one, two, three, or more of the aforementioned components may be excluded from the RNA molecule. In some aspects, each uridine in any of the 5' UTR, 3' UTR, and polyadenylated tail is replaced by a modified base. In some aspects, the modified base is pseudouridine (Ψ). In another aspect, the modified base is N1-pseudouridine (m). 1 Ψ).

[0026] In some respects, the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG or (m2 7,3 ' -O )Gppp(m 2 ' -O )ApG.

[0027] In some respects, the length of the polyadenylate tail may contain +1 / -1 A.

[0028] This disclosure provides RNA molecules as described in Table 20. In some aspects, the RNA molecule comprises a 5' UTR of SEQ ID NO: 95, a FimHORF of SEQ ID NO: 119, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In other aspects, the RNA molecule comprises a 5' UTR of SEQ ID NO: 96, a FimHORF of SEQ ID NO: 119, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In yet another aspect, the RNA molecule comprises a 5' UTR of SEQ ID NO: 97, a FimHORF of SEQ ID NO: 119, a 3' UTR of SEQ ID NO: 104, and / or a polyadenylated tail of SEQ ID NO: 140. In another instance, the RNA molecule comprises the 5' UTR of SEQ ID NO: 98, the FimHORF of SEQ ID NO: 119, the 3' UTR of SEQ ID NO: 103, and / or the polyadenylated tail of SEQ ID NO: 140. In another instance, the RNA molecule comprises the 5' UTR of SEQ ID NO: 99, the FimHORF of SEQ ID NO: 119, the 3' UTR of SEQ ID NO: 103, and / or the polyadenylated tail of SEQ ID NO: 140. In yet another instance, the RNA molecule comprises the 5' UTR of SEQ ID NO: 100, the FimHORF of SEQ ID NO: 119, the 3' UTR of SEQ ID NO: 105, and / or the polyadenylated tail of SEQ ID NO: 140. In some respects, the RNA molecule comprises the 5' UTR of SEQ ID NO: 95, the FimHORF of SEQ ID NO: 117, the 3' UTR of SEQ ID NO: 103, and / or the polyadenylated tail of SEQ ID NO: 140. In other respects, the RNA molecule comprises the 5' UTR of SEQ ID NO: 102, the FimHORF of SEQ ID NO: 117, the 3' UTR of SEQ ID NO: 106, and / or the polyadenylated tail of SEQ ID NO: 140. In yet another respect, the RNA molecule comprises the 5' UTR of SEQ ID NO: 95, the FimHORF of SEQ ID NO: 118, the 3' UTR of SEQ ID NO: 103, and / or the polyadenylated tail of SEQ ID NO: 140.In another instance, the RNA molecule comprises a 5' UTR of SEQ ID NO: 102, a FimH ORF of SEQ ID NO: 119, a 3' UTR of SEQ ID NO: 106, and / or a polyadenylated tail of SEQ ID NO: 93. In another instance, the RNA molecule comprises a 5' UTR of SEQ ID NO: 95, a FimH ORF of SEQ ID NO: 139, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In yet another instance, the RNA molecule comprises a 5' UTR of SEQ ID NO: 99, a FimH ORF of SEQ ID NO: 118, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In some aspects, the RNA molecule comprises a 5' UTR of SEQ ID NO: 99, a FimH ORF of SEQ ID NO: 139, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In other aspects, the RNA molecule comprises a 5' UTR of SEQ ID NO: 101, a FimH ORF of SEQ ID NO: 118, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In yet another aspect, the RNA molecule comprises a 5' UTR of SEQ ID NO: 101, a FimH ORF of SEQ ID NO: 139, a 3' UTR of SEQ ID NO: 103, and / or a polyadenylated tail of SEQ ID NO: 140. In some aspects, the FimH ORF further comprises the stop codon described herein. In some aspects, the polyadenylated tail length may contain +1 / -1 Å or +2 / -2 Å. In some respects, the individual uridines in the RNA molecule are replaced by pseudouridines (Ψ). In other respects, the individual uridines in the RNA molecule are replaced by N1-methylpseudouridines (m). 1 Replace with Ψ).

[0029] This disclosure further provides an RNA molecule comprising at least one open reading frame derived from codon-optimized DNA. In some aspects, the G / C content of the open reading frame is at least, at most, exactly, or between (including or excluding) 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%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, is or about 50% to 75%, or is or about 55% to 70%. In some aspects, the G / C content is or about 58%, is or about 66%, or is or about 62%.

[0030] This disclosure further provides RNA molecules comprising at least one codon-optimized open reading frame. This disclosure further provides RNA molecules comprising stable RNA. This disclosure further provides RNA molecules comprising RNA having at least one modified nucleotide. In some respects, the modified nucleotides are pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, or 2'-O-methyluridine. In some respects, the modified nucleotide is pseudouridine (Ψ). In some respects, 1, 2, 3, 4, 5 or more of the aforementioned modified nucleotides can be excluded from the RNA molecule.

[0031] This disclosure further provides RNA molecules as messenger RNA (mRNA), which may be nucleoside-modified RNA (modRNA). In some respects, the RNA is mRNA. In other respects, the RNA is modRNA.

[0032] This disclosure further provides immunogenic compositions comprising the RNA molecules described herein. The RNA molecules may be formulated in, encapsulated in, compounded with, bound to, or adsorbed onto lipid nanoparticles (LNPs) in such immunogenic compositions (e.g., FimH RNA-LNP). In some aspects, the lipid nanoparticles comprise at least one of cationic lipids, polymer-conjugated lipids (such as PEG-lipids), and at least one structural lipid (e.g., neutral lipids and steroids or steroid analogs). In some aspects, one, two, three, or more of the aforementioned lipids may be excluded from the lipid nanoparticles.

[0033] In some respects, the lipid nanoparticles contain cationic lipids. In some respects, the cationic lipid is (4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0034] In some respects, lipid nanoparticles comprise polymer-conjugated lipids. In other respects, lipid nanoparticles comprise PEG-lipids, also known as polyethylene glycol-modified lipids. In some respects, PEG-lipids are PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide, and glycol lipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[((polyethylene glycol monomethyl ether)2000)carbamoyl]-1,2-dimyristoxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG, PEGylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol). PEG-2,3-dimyristate glyceryl ester (PEG-DMG), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate) (PEG-S-DMG), polyethylene glycol-modified ceramide (PEG-cer), or PEG carbamate dialkoxypropyl ester, such as comethoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate. In some aspects, the PEG-lipid is 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159).

[0035] In some aspects, the lipid nanoparticles contain at least one structural lipid, such as a neutral lipid. In some aspects, the neutral lipid is selected from distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimide). Aminomethyl cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and / or 1,2-dipentanoyl-sn-glycerol-3-phosphate ethanolamine (transDOPE). In some respects, the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0036] In some respects, lipid nanoparticles contain a second structural lipid, such as a steroid or steroid analogue. In some respects, the steroid or steroid analogue is cholesterol.

[0037] In some respects, the average diameter of lipid nanoparticles is from about 1 to about 500 nm, such as at least, at most, exactly, or between (including or excluding) 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, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm. Between any two of nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm or 500 nm.

[0038] In some aspects, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA polynucleotide encoding the FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, encapsulated in an LNP with a lipid composition comprising a cationic lipid at a concentration of 0.8 to 0.95 mg / mL, a polyethylene glycol-modified lipid at a concentration of 0.05 to 0.15 mg / mL, a first structural lipid at a concentration of 0.1 to 0.25 mg / mL, and a second structural lipid at a concentration of 0.3 to 0.45 mg / mL, and further comprising a buffer 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 aspects, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA molecule / polynucleotide encoding the FimH polypeptide as disclosed herein, at a concentration of at least, at most, exactly, or between (including or excluding) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably from 0.01 to about 0.09 mg / mL, to contain concentrations of about 0.8 to 0.95 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 between 0.05 and 0.15 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 polyethylene glycol-modified lipids at concentrations between 0.1 and 0.25 mg / mL. A lipid composition comprising a first structural lipid at a concentration of at least, at most, exactly, or between (including or excluding) 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 a second structural lipid at a concentration of 0.3 to about 0.45 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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) is encapsulated in an LNP.In some aspects, the liquid composition further comprises a buffer composition comprising a first buffer at a concentration of or about 0.1 to 0.3 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 a concentration of or about 1.25 to 1.4 mg / mL. (e.g., at least, at most, exactly, or between (including or excluding) 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) a second buffer and a stabilizer at a concentration of 95 to about 110 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg / mL). In some aspects, 1, 2, 3, 4, 5, or more of the foregoing components may be excluded from the liquid RNA-LNP composition. In some respects, concentrations of 1, 2, 3, 4, 5 or more of the aforementioned components may be excluded from the liquid RNA-LNP composition.

[0039] In certain aspects, the liquid RNA-LNP immunogenic composition comprises an RNA molecule / polynucleotide encoding the FimH polypeptide as disclosed herein, at a concentration of at least, at most, exactly, or between (including or excluding) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably from 0.01 to about 0.09 mg / mL, to include concentrations from about 0.8 to 0.95 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at concentrations between any two of the specified concentrations (mg / mL), 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159) at concentrations of 0.05 to about 0.15 mg / mL (e.g., at least, at most, exactly or between (including or excluding) 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 concentrations of 0.1 to about 0.25 mg / mL. (For example, at least, at most, exactly, or between (including or excluding) 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) of 1,2-distearatel-sn-glycerol-3-phosphocholine (DSPC) and concentrations of 0.3 to about 0.45 mg / mL (for example, at least, at most, exactly, or between (including or excluding) 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 lipid composition of cholesterol (between any two mg / mL) is encapsulated in an LNP.In some aspects, the liquid composition further comprises a Tris buffer composition comprising tris-hydroxymethylaminomethane at a concentration of or about 0.1 to 0.3 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 a concentration of or about 1.25 to 1.4 mg / mL. Tris hydrochloride (HCl) at a concentration 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 (e.g., at least, at most, exactly, or between 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg / mL) and sucrose at a concentration of 1 to about 110 mg / mL (e.g., at least, at most, exactly, or between 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg / mL). In some aspects, 1, 2, 3, 4, 5, or more of the foregoing components may be excluded from the liquid RNA-LNP composition. In some respects, concentrations of 1, 2, 3, 4, 5 or more of the aforementioned components may be excluded from the liquid RNA-LNP composition.

[0040] In some aspects, the liquid RNA-LNP immunogenic composition comprises an RNA polynucleotide encoding the FimH polypeptide disclosed herein, at a concentration of at least, at most, exactly, or between 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably from 0.01 to about 0.09 mg / mL, encapsulated in an LNP with a lipid composition of ALC-0315 at a concentration of 0.8 to 0.95 mg / mL, ALC-0159 at a concentration of 0.05 to 0.15 mg / mL, DSPC at a concentration of 0.1 to 0.25 mg / mL, and cholesterol at a concentration of 0.3 to 0.45 mg / mL, and further comprises a Tris buffer composition comprising tris(hydroxymethyl)aminomethane at a concentration of 0.1 to 0.3 mg / mL, Tris HCl at a concentration of 1.25 to 1.4 mg / mL, and HCl at a concentration of 95 to 110 mg / mL. The LNP contains approximately 5 to 15 mM Tris buffer (e.g., at least, at most, exactly, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mM, including or excluding) and approximately 200 to 400 mM sucrose (e.g., at least, at most, exactly, 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, including or excluding) at or above approximately 7.0 to 8.0 mM. (For example, a pH value of at least, at most, exactly, or between (including or excluding) 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0). In some aspects, 1, 2, 3, or more of the foregoing components may be excluded from the liquid RNA-LNP composition. In some aspects, the concentrations of 1, 2, 3, 4, 5, or more of the foregoing components may be excluded from the liquid RNA-LNP composition.

[0041] Specifically, the RNA-LNP immunogenic composition is a freeze-dried (reconstituted) RNA-LNP composition comprising an RNA polynucleotide encoding the FimH polypeptide disclosed herein, at a concentration of at least, at most, exactly, or between (including or excluding) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably from 0.01 to about 0.09 mg / mL, at a concentration of 0.8 to 0.95 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 with concentrations between 0.05 and 0.15 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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); polyethylene glycol-modified lipids with concentrations between 0.1 and 0.25 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 first structural lipids with concentrations between 0.3 and 0.45 mg / mL. A lipid composition of a second structural lipid (e.g., at least, at most, exactly, or between (including or excluding) 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) is encapsulated in an LNP.In some aspects, the freeze-drying composition further comprises a first buffer solution with a concentration of 0.01 and 0.15 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 a first buffer solution with a concentration of 0.5 and 0.65 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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). A second buffer solution of 35 to 50 mg / mL (e.g., at least, at most, exactly or between (including or excluding) 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / mL) and a salt of 5 to 15 mg / mL (e.g., at least, at most, exactly or between (including or excluding) 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL) for reconstitution. In certain aspects, the lyophilized composition is reconstituted in a carrier or diluent of 0.6 to 0.75 mL (e.g., at least, at most, exactly, or between (including or excluding) 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). The concentration of the lyophilized RNA-LNP composition is determined after reconstitution. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing components may be excluded from the lyophilized RNA-LNP composition. In some aspects, the concentrations of 1, 2, 3, 4, 5, or more of the foregoing components may be excluded from the lyophilized RNA-LNP composition.

[0042] In certain aspects, the freeze-dried (reconstructed) RNA-LNP composition comprises an RNA polynucleotide encoding the FimH polypeptide disclosed herein, at a concentration of at least, at most, exactly, or between (including or excluding) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably from 0.01 to 0.09 mg / mL, with ALC-0315 at a concentration of 0.8 to 0.95 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 a concentration of 0.05 to 0.15 mg / mL. ALC-0159 at concentrations 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 (e.g., at least, at most, exactly, or between (including or excluding) 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 to 0.45 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 lipid composition containing cholesterol at a concentration of at least, at most, exactly, or between (including or excluding) 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 is encapsulated in an LNP and further comprises a Tris buffer composition containing a concentration of 0.01 to about 0.15 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg / mL). Tris(hydroxymethyl)aminomethane at concentrations between 0.5 and 0.65 mg / mL (e.g., at least, at most, exactly, or between (including or excluding) 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 HCl at a concentration of 65 mg / mL, sucrose at a concentration of 35 to 50 mg / mL (e.g., at least, at most, exactly, or between any two of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / mL, including or excluding) and sodium chloride (NaCl) at a concentration of about 5 to 15 mg / mL (e.g., at least, at most, exactly, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL, including or excluding) for reconstitution. In a particular aspect, the freeze-dried composition is reconstituted in 0.6 to about 0.75 mL of sodium chloride (e.g., at least, at most, exactly, or between (including or excluding) 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). The concentration of the freeze-dried RNA-LNP composition is determined after reconstitution. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned components may be excluded from the freeze-dried RNA-LNP composition. In some aspects, the concentrations of 1, 2, 3, 4, 5, or more of the aforementioned components may be excluded from the freeze-dried RNA-LNP composition.

[0043] This disclosure provides RNA molecules, RNA-LNPs, and immunogenic compositions that can administer FimH RNA encapsulated in an LNP to a subject at a dose of at least, at most, exactly, or between (including or excluding) 1 µg, 15 µg, 30 µg, 45 µg, 60 µg, 75 µg, 90 µg, 100 µg, or higher. In some aspects, concentrations of 1, 2, 3, 4, 5, or more of the aforementioned FimH RNA encapsulated in the LNP may be excluded.

[0044] 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 twice (e.g., 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 can be administered twice, once on day 0 and once after 2 months. This disclosure further provides an RNA molecule, RNA-LNP, and immunogenic composition, which can be administered twice, once on day 0 and again after 6 months. This disclosure further provides an RNA molecule, RNA-LNP, and immunogenic composition, which can be administered 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more times. In some aspects, periodic booster doses at intervals of 1 to 5 years may be ideal for maintaining the protective level of the antibody. This disclosure further provides administration of at least one booster dose. In some aspects, dosing regimens 1, 2, 3, 4, 5, or more of the foregoing may be excluded.

[0045] This disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of the 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 manufacture of a medicament for inducing an immune response in a subject.

[0046] This disclosure provides a method for inducing an immune response in a subject, comprising administering to a subject an effective amount of the RNA molecule and / or RNA-LNP (which contains at least one open reading frame encoding the FimH polypeptide) or a composition thereof. This disclosure further provides the use of the RNA molecule and / or RNA-LNP or composition comprising at least one open reading frame encoding the FimH polypeptide described herein in the manufacture of a medicament for inducing an immune response in a subject.

[0047] This disclosure provides a method for inducing an immune response in a subject, comprising administering to a subject an effective amount of the RNA molecule and / or RNA-LNP (which contains at least one open reading frame of a polypeptide encoding a gene of interest) or a composition thereof described herein. This disclosure further provides the use of the RNA molecule and / or RNA-LNP or composition containing at least one open reading frame of a polypeptide encoding a gene of interest described herein in the manufacture of a medicament for inducing an immune response in a subject.

[0048] This disclosure provides methods for preventing, treating, or improving an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of the 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 manufacture of a medicament for preventing, treating, or improving an infection, disease, or condition in a subject. In some aspects, the infection or condition is associated with *Escherichia coli* FimH. In some aspects, the infection, disease, or condition is a urinary tract infection (UTI), urinary tract sepsis, cystitis, or pyelonephritis.

[0049] This disclosure provides methods for preventing, treating, or improving an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of the RNA molecule and / or RNA-LNP (which contains at least one open reading frame encoding a FimH polypeptide) or an immunogenic composition 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 a FimH polypeptide described herein in the manufacture of a medicament for preventing, treating, or improving an infection, disease, or condition in a subject. In some aspects, the infection, disease, or condition is associated with *Escherichia coli* FimH. In some aspects, the infection, disease, or condition is a urinary tract infection (UTI), urinary tract sepsis, cystitis, or pyelonephritis.

[0050] This disclosure further provides methods for preventing, treating, or improving an infection, disease, or condition in a subject, including administering to the subject an effective amount of an RNA molecule and / or RNA-LNP (which contains at least one open reading frame of a polypeptide encoding a gene of interest described herein) or an immunogenic composition. This disclosure further provides the use of RNA molecules and / or RNA-LNPs or immunogenic compositions containing at least one open reading frame of a polypeptide encoding a gene of interest described herein in the manufacture of a medicament for preventing, treating, or improving an infection, disease, or condition in a subject. In some aspects, the infection, disease, or condition is associated with a gene of interest.

[0051] In some respects, the subjects are at least, at most, exactly, or between (including or excluding) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old, or between 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or older. In some respects, the subjects' age is, at least, at most, less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. In some respects, the subjects' age is about 50 years old or older. In a further aspect, the subjects' ages ranged from 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 entire birth generation was included in the relevant immunization population. For example, this could be achieved by initiating an immunization program at any time from birth to 6 months, 6 months to 5 years, pregnant women (or women of childbearing age) whose infants are protected by passive antibody transfer, and subjects older than 50 years. In some aspects, RNA molecules and / or RNA-LNPs were not administered in age groups 1, 2, 3, 4, 5, or more of the aforementioned age groups.

[0052] In some implementations, the subjects are humans. In some specific implementations, the humans are children, such as infants. In some other specific implementations, the humans are women, especially pregnant women. In some respects, the subjects are immunocompetent. In some respects, the subjects are immunocompromised.

[0053] This disclosure provides for the methods or uses described herein, wherein RNA molecules, RNA-LNPs, and / or immunogenic compositions are administered as vaccines.

[0054] This disclosure provides for the methods or uses described herein, wherein RNA molecules, RNA-LNPs, and / or immunogenic compositions are administered via intradermal or intramuscular injection.

[0055] One embodiment of the present invention provides an Escherichia coli vaccine comprising: at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one FimH antigenic polypeptide or an immunogenic fragment thereof, formulated in lipid nanoparticles.

[0056] In one aspect of the E. coli vaccine, the RNA further comprises a 5' cap analog. In a preferred aspect, the 5' cap analog comprises m7G(5')ppp(5')(2'OMeA)pG or N 1 5'-methylpseuuridine-5'-triphosphate.

[0057] In another aspect of E. coli vaccines, the RNA further contains modified nucleotides.

[0058] In another aspect of the E. coli vaccine, at least one of the antigenic polypeptides 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 combination of two or more of the foregoing.

[0059] In another aspect of the 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 a triple mutant of FimH-DSG (G15A, G16A, V27A) (SEQ ID NO: 62); or c) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a triple mutant of FimHLD (G15A, G16A, V27A) (SEQ ID NO: 54).

[0060] In another aspect of the E. coli vaccine, the RNA molecule encoding the FimH polypeptide is fused with a C-terminal membrane-targeting domain.

[0061] In another aspect of the E. coli vaccine, the RNA encoding the FimH polypeptide is fused to a C-terminal membrane-targeting domain and they are separated by a linker. In a preferred aspect of the E. coli vaccine, the encoded linker has the amino acid sequence GSSGSGSS (SEQ ID NO: 94).

[0062] In another aspect of the E. coli vaccine, the C-terminal membrane targeting domain is a glycoprotein. In yet another aspect, the membrane targeting sequence is derived from a human DAF protein GPI sequence or a synthetic GPI sequence.

[0063] In another aspect of E. coli vaccines, FimH is secreted and lacks a C-terminal membrane-targeting domain.

[0064] In another aspect of E. coli vaccines, open reading frames encoded by RNA are codon-optimized.

[0065] In another aspect of E. coli vaccines, the vaccine further contains cationic lipids.

[0066] In another aspect of E. coli vaccines, the vaccine contains lipid nanoparticles that encompass RNA molecules.

[0067] In another aspect of the E. coli vaccine, the vaccine comprises a) lipid nanoparticles enclosing at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding FimH-DSG; b) lipid nanoparticles enclosing at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a triple mutant of FimH-DSG (G15A, G16A, V27A); or c) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a triple mutant of FimHLD (G15A, G16A, V27A) (SEQ ID NO: 54).

[0068] In another aspect of the E. coli vaccine, the lipid nanoparticles have a size of at least 40 nm. In yet another aspect of the E. coli vaccine, the lipid nanoparticles have a size of at most 180 nm.

[0069] In another aspect of the E. coli vaccine, at least 80% of the total RNA in the encapsulation composition is included.

[0070] In another aspect of the E. coli vaccine, the vaccine contains ALC-0315 (4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0071] In another aspect of the E. coli vaccine, the vaccine contains ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide).

[0072] In another aspect of the E. coli vaccine, the vaccine contains 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0073] In another aspect of E. coli vaccines, the RNA polynucleotides contain a 5' cap, a 5' UTR, a 3' UTR, and a polyadenylate tail.

[0074] In another aspect of the E. coli vaccine, each uridine is replaced by a modified base, wherein the modified base is a pseudouridine (Ψ) or N. 1 -Methyl-pseudouridine (m 1 Ψ).

[0075] In another aspect of the E. coli vaccine, the polyadenylate tail is 80 nucleotides in length.

[0076] In another aspect of the E. coli vaccine, the FimH peptide contains serine substitutions at positions N228 and N235.

[0077] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to further illustrate this disclosure and not to limit the scope of the claims. The language in the specification should not be construed as indicating that any unstated component is necessary in the practice of this disclosure.

[0078] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described using the language of a "use" claim, such as the "use" of any compound, composition, or reagent discussed herein, to achieve or practice the stated therapeutic, diagnostic, or physiological purpose or effect. Use of one or more compositions may be based on any of the methods described herein.

[0079] Several documents are referenced in the text of this disclosure. Each of these documents (including all patents, patent applications, scientific publications, manufacturers' specifications, user manuals, etc.) cited herein, whether above or below, is incorporated herein in its entirety by reference. Nothing herein should be construed as an admission that this disclosure is not authorized to precede such disclosure.

[0080] It is anticipated that any aspect discussed in this specification can be practiced with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0081] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed descriptions and specific examples given to illustrate particular aspects of this disclosure are provided only in an illustrative manner, as various changes and modifications within the spirit and scope of this disclosure will be apparent to those skilled in the art based on this detailed description.

[0082] Brief description of the attached figures

[0083] Figure 1A and Figure 1B The FimH gene variant of the present invention is described. Figure 1A The FimH tertiary structure shows the position of the glycine loop mutations G15A and G16A relative to the natural variant V27A. A 7-residue Gly-Ser linker separates the C-terminus of the fimbriae protein domain from the donor chain G (DsG) peptide. Figure 1B Showing the linear representation of FimH LD and FimH DSG The gene has mutations in the N-terminal IgK signal peptide ([SP]) condensate and fimbriae domains, as well as a G peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor. Legend:SP, mouse IgGκ signal peptide; FimH LD , FimH condensation protein domain; gpi, glycosylphosphatidylinositol anchor. Indicates the FimH amino acid substitutions introduced to prevent N-glycosylation (N7S, N70S, N228S, and N235S) or to stabilize conformations (G15A, G16A, V27A, also known as the triple mutant "TM"); G, stabilizing donor peptide added to the C-terminus of the full-length FimH protein.

[0084] Figure 2 The positive surface expression of FimH on the surface of Expi293 suspension cells was described by flow cytometry using the modRNA LNP of the present invention.

[0085] Figure 3A and Figure 3B Describes the neutralizing antibody titer induced by FimH modRNA LNP in the serum of PD2 (6-week-old) mice. The results of the comparison between BMD2 and WHO vectors are shown in... Figure 3A And summarized in Figure 3B This confirms FimH DSG modRNA expression of FimH LD Or secretory type FimH DSG The modRNA is more immunogenic. p The values ​​are derived from pairwise comparisons of log-transformed data using an unpaired t-test corrected with Welch (GraphPad Prism): p<0.0001 p<0.05, ns- not significant.

[0086] Figure 4A and Figure 4B This shows the FimH mentioned in this article. DSG - The GPI construct is equivalent to the baseline. Figure 4A Displays various full-length FimHs DGS -GPI modRNA LNP (containing Figure 3A and Figure 3B The functional valence caused by BMD2 (and the baseline group) shown in the figure is summarized in Figure 4B middle.

[0087] Figure 5A to Figure 5D The membrane-targeted FimH construct of the present invention demonstrates the induction of a strong Th1 response.

[0088] Figure 6 The membrane-targeted FimH construct of the present invention demonstrates induction of a strong CD8 T cell response.

[0089] Figure 7A andFigure 7B Describes the substitution of the GPI anchor peptide with the Gly-Ser linker (SEQ ID NO: 94). Figure 7A The processing of the N-terminal signal peptide (black box) and C-terminal GPI domain in the endoplasmic reticulum (ER) was visualized, followed by covalent attachment of a glycolipid anchor to the ω-site serine residue where the C-terminal polypeptide (white box) is cleaved by GPI transaminases. Description taken from Kinoshita T. 2020. Open Biol 10:190290. Figure 7B Showing the substitution of eight DAF amino acid residues near the ω-site serine with the serine / glycine linker. mlgK refers to the mouse IgK signal peptide. Not plotted to scale.

[0090] Figure 8A and Figure 8B The substitution of the DAF GPI peptide with the GlySer linker did not affect FimH expression on the surface of transfected Expi293 cells. modRNA was transfected into Expi293 cells, and FimH surface expression was detected by flow cytometry using FimH mAb 926. Figure 8A The average fluorescence intensity (MFI) of the surface staining is displayed. Figure 8B The display shows the use of interpolation method EC. 50 The percentage of positive staining for titer. Titer was determined using a 4-parameter sigmoid curve fitting (GraphPad Prism).

[0091] Figure 9A and Figure 9B Describe the neutralizing titer at the PD2 (6-week) time point. The neutralizing titer of *E. coli* was assessed two weeks after mice received a second dose of 1 µg modRNALNP (results are shown in...). Figure 9A And integrated into Figure 9B ). As pointed out p The values ​​are derived from pairwise comparisons of the log-transformed data using an unpaired t-test corrected by Welch (GraphPad Prism).

[0092] Figure 10A to Figure 10D Describes antigen-specific T cell responses in spleen cells from vaccinated mice. Results show membrane-targeted FimH DSG The candidate can induce a strong Th1 response. p The values ​​are derived from pairwise comparisons of log-transformed data using an unpaired t-test corrected with Welch (GraphPad Prism): p<0.0001 p<0.05 p<0.05.

[0093] Figure 11Describes antigen-specific T cell responses in spleen cells from vaccinated mice, showing membrane-targeted FimH. DSG The candidate induced a strong CD8+ T cell response. p The values ​​are derived from pairwise comparisons of log-transformed data using an unpaired t-test corrected with Welch (GraphPad Prism): p<0.0001 p<0.005 p<0.05, ns- not significant.

[0094] Figure 12A to Figure 12D The results showed that the FimH-DSG gpi anchor modRNA and the FimH-DSG secretory modRNA were immunogenic in non-human primates. Figure 12A Describe the protocol for immunization, blood collection, and challenge (n=1 experiment, 9 NHPs per group). Figure 12B to 12D The NHP titers of immunized placebo (round), FimH-DSG V27A G15A G16A (subunit protein) with LiNA-2 adjuvant (square), FimH-DSG gpi anchor modRNA (triangle) and FimH-DSG secreted modRNA (inverted triangle) are shown at weeks 0, 4 and 14. Figure 12B Describes the neutralizing titer (Log IC50) of yeast mannan in a neutralization assay at time points before vaccination and after the second and third doses. 50 ). Figure 12C Describe serum anti-FimH IgG titers measured after the second and third doses. Each symbol represents an individual animal. Error bars represent the geometric mean of the 95% confidence interval (CI). Dashed lines indicate the lower limit of quantitation (LLOQ) of the analysis. Figure 12D Describe the urinary anti-FimH IgG titers measured after the second and third doses.

[0095] Figure 13A to Figure 13B Bacteriuria was reduced in NHPs immunized with FimH modRNA and FimH subunit proteins. Bacteriuria was quantified by qPCR over 28 days. Figure 13A NHP was administered at weeks 0, 4, and 14 using placebo (round), FimH subunit protein (square), FimH gpi-anchored modRNA (triangle), and FimH secreted modRNA (inverted triangle). At week 19, 10 mg / L was injected via intravesical catheter. 8UPEC isolate PFEEC0578, colony-forming units (CFU). Urine samples were collected via bladder catheterization on a designated post-infection day. Urine samples were also used to assess viable colonies (…). Figure 13B The symbols represent individual animals. The error bars represent the geometric mean of the 95% CI. The ratio represents the number of NHPs (100 bacteria) in the urine sample relative to the total number of challenged animals. The dashed line represents the lower limit of quantitation (LLOQ).

[0096] Figure 14 This shows a reduction in pro-inflammatory biomarkers in NHPs vaccinated with FimH-DSG G15A G16A V27A and FimH modRNA. The concentration of interleukin-8 (IL-8) in urine is shown within 28 days post-infection. Individual symbols represent individual animals. Error bars represent the geometric mean of the 95% CI. The dashed line indicates the lower limit of quantitation (LLOQ).

[0097] Figure 15A to Figure 15C Displaying FimH-induced CD4 + T-cell response. The vaccination program is described in... Figure 12A PBMCs obtained at week 0 (pre-prime) and week 16 (day 7 after the third booster of FimH gpi modRNA, FimH secreted modRNA, and FimH subunit protein) were stimulated with FimH peptide, and CD4 counts were assessed by flow cytometry. + and CD8 + Cytokines produced by T cells. Showing production of TNFα (… Figure 15A ), IFN-γ ( Figure 15B ) and IL-2 ( Figure 15C CD4 + Percentage of T cells. No CD8 cell elicited after immunization one week following the third booster dose after peptide stimulation. + T cell response (data not shown).

[0098] Sequence identifier

[0099] SEQ ID NO: 1 represents wild-type Escherichia coli FimH LD The amino acid sequence of (FimHLD_WT).

[0100] SEQ ID NO: 2 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G65A_V27A.

[0101] SEQ ID NO: 3 represents the amino acid sequence of the mutant Escherichia coli FimHLD_F1I.

[0102] SEQ ID NO: 4 represents the amino acid sequence of the mutant Escherichia coli FimHLD_F1L.

[0103] SEQ ID NO: 5 represents the amino acid sequence of the mutant Escherichia coli FimHLD_F1V.

[0104] SEQ ID NO: 6 represents the amino acid sequence of the mutant Escherichia coli FimHLD_F1M.

[0105] SEQ ID NO: 7 represents the amino acid sequence of the mutant Escherichia coli FimHLD_F1Y.

[0106] SEQ ID NO: 8 represents the amino acid sequence of the mutant Escherichia coli FimHLD_F1W.

[0107] SEQ ID NO: 9 represents the amino acid sequence of the mutant Escherichia coli FimHLD_Q133K.

[0108] SEQ ID NO: 10 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15A.

[0109] SEQ ID NO: 11 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15P.

[0110] SEQ ID NO: 12 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G16A.

[0111] SEQ ID NO: 13 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G16P.

[0112] SEQ ID NO: 14 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15A_G16A.

[0113] SEQ ID NO: 15 represents the amino acid sequence of the mutant Escherichia coli FimHLD_R60P.

[0114] SEQ ID NO: 16 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G65A.

[0115] SEQ ID NO: 17 represents the amino acid sequence of the mutant Escherichia coli FimHLD_P12C_A18C.

[0116] SEQ ID NO: 18 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G14C_F144C.

[0117] SEQ ID NO: 19 represents the amino acid sequence of the mutant Escherichia coli FimHLD_P26C_V35C.

[0118] SEQ ID NO: 20 represents the amino acid sequence of the mutant Escherichia coli FimHLD_P26C_V154C.

[0119] SEQ ID NO: 21 represents the amino acid sequence of the mutant Escherichia coli FimHLD_P26C_V156C.

[0120] SEQ ID NO: 22 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V27C_L34C.

[0121] SEQ ID NO: 23 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V28C_N33C.

[0122] SEQ ID NO: 24 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V28C_P157C.

[0123] SEQ ID NO: 25 represents the amino acid sequence of the mutant Escherichia coli FimHLD_Q32C_Y108C.

[0124] SEQ ID NO: 26 represents the amino acid sequence of the mutant Escherichia coli FimHLD_N33C_L109C.

[0125] SEQ ID NO: 27 represents the amino acid sequence of the mutant Escherichia coli FimHLD_N33C_P157C.

[0126] SEQ ID NO: 28 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V35C_L107C.

[0127] SEQ ID NO: 29 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V35C_L109C.

[0128] SEQ ID NO: 30 represents the amino acid sequence of the mutant Escherichia coli FimHLD_S62C_T86C.

[0129] SEQ ID NO: 31 represents the amino acid sequence of the mutant Escherichia coli FimHLD_S62C_L129C.

[0130] SEQ ID NO: 32 represents the amino acid sequence of the mutant Escherichia coli FimHLD_Y64C_L68C.

[0131] SEQ ID NO: 33 represents the amino acid sequence of the mutant Escherichia coli FimHLD_Y64C_A127C.

[0132] SEQ ID NO: 34 represents the amino acid sequence of the mutant Escherichia coli FimHLD_L68C_F71C.

[0133] SEQ ID NO: 35 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V112C_T158C.

[0134] SEQ ID NO: 36 represents the amino acid sequence of the mutant Escherichia coli FimHLD_S113C_G116C.

[0135] SEQ ID NO: 37 represents the amino acid sequence of the mutant Escherichia coli FimHLD_S113C_T158C.

[0136] SEQ ID NO: 38 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V118C_V156C.

[0137] SEQ ID NO: 39 represents the amino acid sequence of the mutant Escherichia coli FimHLD_A119C_V155C.

[0138] SEQ ID NO: 40 represents the amino acid sequence of the mutant Escherichia coli FimHLD_L34N_V27A.

[0139] SEQ ID NO: 41 represents the amino acid sequence of the mutant Escherichia coli FimHLD_L34S_V27A.

[0140] SEQ ID NO: 42 represents the amino acid sequence of the mutant Escherichia coli FimHLD_L34T_V27A.

[0141] SEQ ID NO: 43 represents the amino acid sequence of the mutant Escherichia coli FimHLD_A119N_V27A.

[0142] SEQ ID NO: 44 represents the amino acid sequence of the mutant Escherichia coli FimHLD_A119S_V27A.

[0143] SEQ ID NO: 45 represents the amino acid sequence of the mutant Escherichia coli FimHLD_A119T_V27A.

[0144] SEQ ID NO: 46 represents the amino acid sequence of the mutant Escherichia coli FimH-DSG_A115V.

[0145] SEQ ID NO: 47 represents the amino acid sequence of the mutant Escherichia coli FimH-DSG_V163I.

[0146] SEQ ID NO: 48 represents the amino acid sequence of the mutant Escherichia coli FimH-DSG_V185I.

[0147] SEQ ID NO: 49 represents the amino acid sequence of the mutant Escherichia coli FimH-DSG_DSG_V3I.

[0148] SEQ ID NO: 50 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15A_V27A.

[0149] SEQ ID NO: 51 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G16A_V27A.

[0150] SEQ ID NO: 52 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15P_V27A.

[0151] SEQ ID NO: 53 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G16P_V27A.

[0152] SEQ ID NO: 54 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15A_G16A_V27A.

[0153] SEQ ID NO: 55 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V27A_R60P.

[0154] SEQ ID NO: 56 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G65A_V27A.

[0155] SEQ ID NO: 57 represents the amino acid sequence of the mutant Escherichia coli FimHLD_V27A_Q133K.

[0156] SEQ ID NO: 58 represents the amino acid sequence of the mutant Escherichia coli FimHLD_G15A_G16A_V27A_Q133K.

[0157] SEQ ID NO: 59 represents the amino acid sequence of the full-length FimH of wild-type Escherichia coli, which includes the donor chain FimG peptide (FimH-DSG_WT) linked by a linker.

[0158] SEQ ID NO: 60 represents the amino acid sequence of the mutant Escherichia coli FimH-DSG_V27A.

[0159] SEQ ID NO: 61 represents the amino acid sequence of the mutant Escherichia coli FimH-DSG_G15A_V27A.

[0160] SEQ ID NO: 62 represents the mutant Escherichia coli FimH DSG The amino acid sequence of _G15A_G16A_V27A.

[0161] SEQ ID NO: 63 represents the mutant Escherichia coli FimH DSG The amino acid sequence of _V27A_Q133K.

[0162] SEQ ID NO: 64 represents the mutant Escherichia coli FimH DSG The amino acid sequence of _G15A_G16A_V27A_Q133K.

[0163] SEQ ID NO: 65 represents the amino acid sequence of the mouse Igκ signal peptide sequence.

[0164] SEQ ID NO: 66 represents BMD2 / FimH DSG -GPI / hHBB_80pA nucleic acid sequence.

[0165] SEQ ID NO: 67 represents BMD70 / FimH DSG- The nucleic acid sequence of GPI / hHBB_80pA.

[0166] SEQ ID NO: 68 represents BMD91 / FimH DSG- The nucleic acid sequence of GPI / CYP2E1_80pA.

[0167] SEQ ID NO: 69 represents BMD105 / FimH DSG- The nucleic acid sequence of GPI / hHBB_80pA.

[0168] SEQ ID NO: 70 represents BMD562 / FimH DSG- The nucleic acid sequence of GPI / hHBB_80pA.

[0169] SEQ ID NO: 71 represents BMD3 / FimH DSG- The nucleic acid sequence of GPI / hHBB-AES_80pA.

[0170] SEQ ID NO: 72 represents BMD2 / FimHLD- The nucleic acid sequence of GPI / hHBB_80pA.

[0171] SEQ ID NO: 73 represents WHO / FimH LD- The nucleic acid sequence of GPI / WHO_80pA.

[0172] SEQ ID NO: 74 represents BMD2 / FimH DSG- The nucleic acid sequence of Sec / hHBB_80pA.

[0173] SEQ ID NO: 75 represents WHO / FimH DSG- Nucleic acid sequence of GPI / WHO_30L70.

[0174] SEQ ID NO: 76 represents FimH LD -CtDAFGPI nucleic acid sequence.

[0175] SEQ ID NO: 77 represents the FimH shown in SEQ ID NO: 76. LD The amino acid sequence of -CtDAFGPI.

[0176] SEQ ID NO: 78 represents FimH DSG Secretory nucleic acid sequences.

[0177] SEQ ID NO: 79 represents the FimH shown in SEQ ID NO: 78. DSG Secretory type, SEQ ID NO: 84, BMD562 / FimH DSG- Sec / hHBB_80pA and BMD576 / FimH shown in SEQ ID NO: 88 DSG- The amino acid sequence of Sec / hHBB_80pA.

[0178] SEQ ID NO: 80 represents FimH DSG -CtDAFGPI nucleic acid sequence.

[0179] SEQ ID NO: 81 represents the FimH shown in SEQ ID NO: 80. DSG The amino acid sequence of -CtDAFGPI.

[0180] SEQ ID NO: 82 represents BMD2 / FimH DSG- The nucleic acid sequence of SerGlyGPI / hHBB_80pA.

[0181] SEQ ID NO: 83 represents BMD2 / FimH shown in SEQ ID NO: 82. DSG- SerGlyGPI / hHBB_80pA, SEQ ID NO: 86, shows BMD562 / FimH DSG- SerGlyGPI / hHBB_80pA and BMD576 / FimH shown in SEQ ID NO: 90 DSG- The amino acid sequence of SerGlyGPI / hHBB_80pA.

[0182] SEQ ID NO: 84 represents BMD562 / FimH DSG- The nucleic acid sequence of Sec / hHBB_80pA.

[0183] SEQ ID NO: 86 represents BMD562 / FimH DSG- The nucleic acid sequence of SerGlyGPI / hHBB_80pA.

[0184] SEQ ID NO: 88 represents BMD576 / FimH DSG- The nucleic acid sequence of Sec / hHBB_80pA.

[0185] SEQ ID NO: 90 represents BMD576 / FimH DSG- The nucleic acid sequence of SerGlyGPI / hHBB_80pA.

[0186] SEQ ID NO: 92 represents the nucleic acid sequence of the 80A polyadenylate tail.

[0187] SEQ ID NO: 93 represents the nucleic acid sequence of a split polyadenylated tail, which is called the "30L70" polyadenylated tail.

[0188] SEQ ID NO: 94 represents the amino acid sequence in the DAF GPI anchor that replaces the glycine-serine linker of 8 amino acids.

[0189] SEQ ID NO: 95 represents the nucleic acid sequence of 5' UTR_BMD2.

[0190] SEQ ID NO: 96 represents the nucleic acid sequence of 5' UTR_BMD70.

[0191] SEQ ID NO: 97 represents the nucleic acid sequence of 5' UTR_BMD91.

[0192] SEQ ID NO: 98 represents the nucleic acid sequence of 5'UTR_BMD105.

[0193] SEQ ID NO: 99 represents the nucleic acid sequence of 5'UTR_BMD562.

[0194] SEQ ID NO: 100 represents the nucleic acid sequence of 5'UTR_BMD3.

[0195] SEQ ID NO: 101 represents the nucleic acid sequence of 5' UTR_BMD576.

[0196] SEQ ID NO: 102 represents the nucleic acid sequence of 5' UTR_WHO.

[0197] SEQ ID NO: 103 represents the nucleic acid sequence of 3' UTR_hHBB.

[0198] SEQ ID NO: 104 represents the nucleic acid sequence of 3' UTR_CYP2E1.

[0199] SEQ ID NO: 105 represents the nucleic acid sequence of 3' UTR_hHBB-AES.

[0200] SEQ ID NO: 106 represents the nucleic acid sequence of 3' UTR_WHO.

[0201] Specific embodiments of the present invention

[0202] This invention can be more readily understood by referring to the following detailed description of the inventive embodiments and the examples contained herein. It should be understood that the invention is not limited to specific manufacturing methods, which may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0203] The exemplary embodiments of the invention (E) provided herein include:

[0204] E1. An RNA molecule comprising at least one open reading frame (ORF) and a 5' untranslated region (5' UTR), the ORF encoding a fimH antigen (FimH) polypeptide, wherein the 5' UTR comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence shown in any one of SEQ ID NO: 95 to 101.

[0205] E2. An RNA molecule as described in embodiment E1, wherein the 5' UTR contains at least 92% identical nucleic acid sequence to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 95, 98, 99 and 101.

[0206] E3. An RNA molecule as described in any one of embodiments E1 to E2, wherein the 5' UTR contains a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 95, 99 and 101.

[0207] E4. An RNA molecule as described in any one of embodiments E1 to E3, wherein the 5' UTR contains a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 99 and 101.

[0208] E5. An RNA molecule as described in any one of embodiments E1 to E4, wherein the 5' UTR contains a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 99 and 101.

[0209] E6. An RNA molecule according to any one of embodiments E1 to E5, wherein the 5' UTR contains a nucleic acid sequence selected from the group consisting of:

[0210] SEQ ID NO: 99 (5'UTR_BMD562); and

[0211] SEQ ID NO: 101 (5'UTR_BMD576).

[0212] In one aspect of embodiment E6, the 5' UTR comprises a nucleic acid sequence as shown in SEQ ID NO: 99. In another aspect of embodiment E6, the 5' UTR comprises a nucleic acid sequence as shown in SEQ ID NO: 101.

[0213] E7. An RNA molecule as described in any of embodiments E1 to E6, wherein the RNA molecule further comprises a 3' untranslated region (3' UTR).

[0214] E8. An RNA molecule as described in embodiment E7, wherein the 3' UTR contains nucleotides having the sequence shown in SEQ ID NO: 103 (3'UTR_hHBB).

[0215] E9. An RNA molecule as described in any of embodiments E1 to E8, wherein the FimH polypeptide encoded by the RNA molecule is its full length, truncated, fragment, or variant.

[0216] E10. An RNA molecule as described in any of embodiments E1 to E9, wherein the FimH polypeptide encoded by the RNA molecule contains at least one mutation.

[0217] E11. An RNA molecule as described in 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 NO: 1 to 64.

[0218] E12. An RNA molecule as described in 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 NO: 1 to 64.

[0219] E13. An RNA molecule as described in 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 an immunogenic fragment thereof.

[0220] E14. An RNA molecule as described in any of embodiments E1 to E13, wherein the FimH polypeptide encoded by the RNA molecule is fused to a C-terminal membrane targeting domain.

[0221] E15. An RNA molecule as described in any of embodiments E1 to E14, wherein the C-terminal membrane targeting domain is DAFgpi or a variant thereof.

[0222] E16. An RNA molecule as described in embodiment E15, wherein the DAFgpi is a variant comprising a serine / glycine linker having a serine / glycine linker having the amino acid sequence GSSGSGSS (SEQ ID NO: 94) replacing 8 DAF amino acid residues near the ω site serine.

[0223] E17. An RNA molecule as described in 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 shown in SEQ ID NO: 77, 79, 81, or 83.

[0224] E18. An RNA molecule as described in 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 NO: 77, 79, 81 and 83.

[0225] In one aspect of embodiment E18, the FimH polypeptide encoded by an RNA molecule is represented by SEQ ID NO: 77. In another aspect of embodiment E18, the FimH polypeptide encoded by an RNA molecule is represented by SEQ ID NO: 79. In another aspect of embodiment E18, the FimH polypeptide encoded by an RNA molecule is represented by SEQ ID NO: 81. In a further aspect of embodiment E18, the FimH polypeptide encoded by an RNA molecule is represented by SEQ ID NO: 83.

[0226] E19. An RNA molecule as described in any 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 of the sequences of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 138.

[0227] E20. An RNA molecule as described in 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.

[0228] In one aspect of embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 76. In another aspect of embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 78. In another aspect of embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 80. In a further aspect of embodiment E20, the open reading frame is transcribed from a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 138.

[0229] E21. An RNA molecule as described in any 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 of the sequences shown in SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 139.

[0230] E22. An RNA molecule as described in embodiment 21, wherein the open reading frame comprises 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.

[0231] In one aspect of embodiment E22, the open reading frame comprises the nucleic acid sequence shown in SEQ ID NO: 117. In another aspect of embodiment E22, the open reading frame comprises the nucleic acid sequence shown in SEQ ID NO: 118. In another aspect of embodiment E22, the open reading frame comprises the nucleic acid sequence shown in SEQ ID NO: 119. In a further aspect of embodiment E22, the open reading frame comprises the nucleic acid sequence shown in SEQ ID NO: 139.

[0232] E23. An RNA molecule as described in any of embodiments E1 to E22, wherein the RNA molecule further comprises a 5' cap portion or a 3' polyadenylate tail.

[0233] E24. An RNA molecule as described in any of embodiments E1 to E23, wherein the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG or (m2 7,3 ' -O )Gppp(m 2 ' -O )ApG.

[0234] In one aspect of implementation scheme E24, the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG. In another aspect of implementation scheme E24, the 5' cap portion is (m2 7,3 ' -O )Gppp(m 2 ' -O )ApG.

[0235] E25. An RNA molecule as described in embodiment E24, wherein the polyadenylated tail comprises a sequence having SEQ ID NO: 93 or SEQ ID NO: 140.

[0236] E26. An RNA molecule as described in any one of embodiments E1 to E25, wherein the RNA molecule comprises nucleotides having the sequences shown in SEQ ID NO: 66 to 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 90.

[0237] In one aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 66. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 67. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 68. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 69. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 70. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 71. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 72. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 73. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 74. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 75. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 82. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 84. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 86. In another aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 88. In a further aspect of embodiment E26, the RNA molecule comprises a nucleotide having the sequence shown in SEQ ID NO: 90.

[0238] E27. An RNA molecule as described in embodiment E26, wherein the RNA molecule is 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 NO: 107 to 116 or SEQ ID NO: 120 to 124.

[0239] In one aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in any one of SEQ ID NO: 107 to 116 or SEQ ID NO: 120 to 124. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 107. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 108. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 109. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 110. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 111. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 112. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 113. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 114. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 115. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 116. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 120. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 121. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 122. In another aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 123. In a further aspect of embodiment E27, the RNA molecule is transcribed from a nucleic acid having the sequence shown in SEQ ID NO: 124.

[0240] E28. An RNA molecule as described in any of embodiments E1 to E27, wherein the open reading frame contains at least 55%, 60%, 65%, 70%, or 75% G / C content, or about 50% to 75% or 55% to 70% G / C content.

[0241] E29. An RNA molecule as described in any of embodiments E1 to E28, wherein the encoded FimH polypeptide is located in the cell membrane, in the Golgi apparatus, and / or secreted.

[0242] E30. An RNA molecule as described in any of embodiments E1 to E29, wherein the RNA contains at least one modified nucleotide.

[0243] E31. An RNA molecule as described in embodiment E30, wherein the modified nucleotide is pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, or 2'-O-methyluridine.

[0244] E32. An RNA molecule as described in implementation scheme E31, wherein the modified nucleotide is pseudouridine (Ψ) or N. 1 -Methylpseudouridine (m1Ψ).

[0245] E33. An RNA molecule as described in embodiment E32, wherein each uridine in the RNA molecule is converted to pseudouridine (Ψ) or N. 1 -Methylpseudouridine (m1Ψ) substitution.

[0246] E34. An RNA molecule as described in any of embodiments E1 to E33, wherein the RNA is mRNA.

[0247] E35. An RNA molecule as described in implementation scheme E34, wherein the RNA is a modRNA.

[0248] 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).

[0249] E37. The composition of embodiment E36, wherein the lipid nanoparticles comprise at least one of cationic lipids, PEGylated lipids, neutral lipids, and steroids or steroid analogs.

[0250] E38. The composition of embodiment E37, wherein the cationic lipid is (4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0251] E39. The composition of embodiment E37 or E38, wherein the polyethylene glycol-modified lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide, or a glycol lipid comprising: PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[((polyethylene glycol monomethyl ether)2000)carbamoyl]-1,2-dimyristoxypropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG. Polyethylene glycol diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristate glyceryl ester (PEG-DMG), polyethylene glycol phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate) succinate (PEG-S-DMG), polyethylene glycol ceramide (PEG-cer), or PEG carbamate dialkoxypropyl ester, such as comethoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(o>-methoxy(polyethoxy)ethyl)carbamate.

[0252] E40. The composition of embodiment E39, wherein the polyethylene glycol-modified lipid is 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159).

[0253] E41. A composition according to any one of embodiments E37 to E40, wherein the neutral lipid is distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine. Alkylamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-dipentanoyl-sn-glycerol-3-phosphoethanolamine (transDOPE).

[0254] E42. The composition of embodiment E41, wherein the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

[0255] E43. A composition according to any one of embodiments E37 to E42, wherein the steroid or steroid analogue is cholesterol.

[0256] E44. A composition according to any one of embodiments E36 to E43, wherein the composition is a vaccine.

[0257] E45. A mutant FimH polypeptide comprising at least 80% identity with any one of the amino acid sequences shown in SEQ ID NO: 77, SEQ ID NO: 81 or SEQ ID NO: 83.

[0258] E46. The mutant FimH polypeptide of embodiment E45, wherein the mutant FimH polypeptide comprises amino acids having the sequence shown in SEQ ID NO: 81 or SEQ ID NO: 83.

[0259] E47. A polynucleotide encoding a mutant FimH polypeptide comprising at least 80% identity with any one of the amino acid sequences shown in SEQ ID NO: 77, SEQ ID NO: 81 or SEQ ID NO: 83.

[0260] E48. A polynucleotide encoding a mutant FimH polypeptide comprising a nucleic acid having the sequence shown in SEQ ID NO: 117, SEQ ID NO: 118 or SEQ ID NO: 139.

[0261] E49. The polynucleotide of embodiment E47, wherein the polynucleotide encoding the mutant FimH polypeptide is transcribed from a nucleic acid containing the nucleotide sequence shown in SEQ ID NO:76, SEQ ID NO:78 or SEQ ID NO:138.

[0262] E50. A method for (i) inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli or (ii) inducing a subject to produce opsonization and / or neutralizing antibodies against extraintestinal pathogenic Escherichia coli, wherein the method comprises administering to the subject an effective amount of an RNA molecule, RNA-LNP, and / or vaccine as described in any one of embodiments E1 to E44.

[0263] E51. The method of implementation E50, wherein the subject is at risk of developing a urinary tract infection.

[0264] E52. The method of implementation E50, wherein the subject is at risk of developing bacteremia.

[0265] E53. The method of implementation E50, wherein the subject is at risk of developing urinary tract sepsis.

[0266] E54. The method of implementation E50, wherein the subject is at risk of developing cystitis.

[0267] E55. Use of an RNA molecule, RNA-LNP and / or composition as described in any one of embodiments E1 to E44 for the manufacture of a medicament for (i) inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli or (ii) inducing a subject to produce opsonization and / or neutralizing antibodies against extraintestinal pathogenic Escherichia coli.

[0268] E56. As in the implementation of E55, where the infection, disease, or condition is a urinary tract infection.

[0269] E57. As in implementation scheme E55, wherein the subject is at risk of developing bacteremia.

[0270] E58. As in implementation scheme E55, wherein the subject is at risk of developing sepsis.

[0271] E59. As in implementation scheme E55, where the subject is at risk of developing cystitis.

[0272] E60. The method or use of any of E50 to E59, wherein the subject is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older.

[0273] E61. The method or use as described in any of E50 to E59, wherein the subject is about 50 years of age or older.

[0274] E62. The method or use as described in any of E50 to E59, wherein the subject is a pregnant woman.

[0275] E63. The method or use as described in any of embodiments E50 to E62, wherein the RNA molecule or composition is administered as a vaccine.

[0276] E64. The method or use as described in any of embodiments E50 to E63, wherein the RNA molecule or composition is administered via intradermal or intramuscular injection.

[0277] E65. The method or use as described in any of E50 to E64, wherein the subject is administered a single, two, three or more doses of an RNA molecule, composition or vaccine, and optionally a booster dose.

[0278] The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter described.

[0279] All references cited in this article, including patent applications, patent publications, and UniProtKB accession numbers, are incorporated herein by reference as if each individual reference were specifically and individually indicated as being incorporated herein by reference in its entirety.

[0280] I. Defining an instance

[0281] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0282] In this application, the terms “about,” “approximately,” and “substantially” are used according to their simple and common meaning in the fields of cell biology and molecular biology to indicate a deviation of ±10% from the attached value. Therefore, in any disclosed aspect, these terms may be replaced with “within [percentage] of the specified content.” In a non-limiting aspect, percentages include 0.1%, 0.5%, 1%, 5%, and 10%.

[0283] References to numerical ranges in this document are intended only as a shorthand for referring to each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually referenced herein.

[0284] The word “a” or “a kind” used, when used with the term “comprising”, can mean “a / a kind”, but it can also be composed of the meanings of “one or more / one or more kinds”, “at least one / at least one kind”, and “one or more than one / a kind or more kinds”.

[0285] 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” is an inclusive form of “or”.

[0286] The phrase “substantially all” is defined as “at least 95%”; if substantially all members of a group have a particular property, then at least 95% of the members of the group have that property. In some respects, substantially all means that any one, at least one, or any two of the following—95%, 96%, 97%, 98%, 99%, or 100%—have that property.

[0287] The composition and its method of use may “comprising,” “consistent with,” or “composed of” any component or step disclosed throughout the specification. Throughout the specification (unless the context requires otherwise), the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “containing”) are used. The terms “consisting of” and “containing” are inclusive or open-ended and will be understood to mean including the specified steps or components or groups of steps or components, but not excluding any other steps or components or groups of steps or components. It is anticipated that aspects described herein in the context of the term “consisting of” may also be implemented in the context of the terms “consisting of” or “substantially consisting of”. Compositions and methods “substantially consisting of” any of the disclosed ingredients or steps will limit the scope of the claims to specific materials or steps that do not substantially affect the basic and novel characteristics of the claimed disclosure. The term “consisting of” (and any form of “consisting of,” such as “consistof” and “consists of”) means including (and limited to) anything following the phrase “consisting of”. The phrase “consisting of” indicates that the listed components are necessary or mandatory, and no other components may be present.

[0288] The use of terms such as “one / an aspect,” “specific aspect,” “related aspect,” “a certain aspect,” “additional aspect,” or “further aspect,” or combinations thereof throughout this specification, means that the specific feature, structure, or characteristic associated with that aspect is included in at least one aspect of this disclosure. Therefore, the aforementioned phrases appearing throughout this specification do not necessarily all refer to the same aspect. Furthermore, a specific feature, structure, or characteristic may be combined in any suitable manner in one or more aspects.

[0289] The terms “inhibit,” “reduce,” or “lower,” or any variations thereof, encompass 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 a desired result. The terms “enhance,” “promote,” or “increase,” or any variations thereof, encompass 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 a desired result or the manufacture of a protein or molecule.

[0290] As used herein, the terms “reference,” “standard,” or “control” describe a value being compared to. For example, comparing a reagent, subject, population, sample, or value of interest to a reference, standard, or control reagent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and / or determined substantially simultaneously with the test or determination of interest on the reagent, subject, population, sample, or value of interest, and / or may be determined or characterized under comparable conditions or settings.

[0291] The term "isolated" means a nucleic acid or polypeptide that is substantially free of its original source of cellular material, bacterial material, viral material, or culture medium (when manufactured using recombinant DNA technology), or chemical precursors or other chemicals (when chemically synthesized). Furthermore, an isolated compound means a compound that can be administered to a subject as an isolated compound; in other words, a compound cannot be simply considered "isolated" if it is attached to a column or embedded in an agarose gel. Additionally, "isolated nucleic acid fragments" or "isolated peptides" are nucleic acid or protein fragments that are not naturally occurring as fragments and / or are atypical in their functional state and / or have been altered or removed from their natural state through artificial intervention. For example, DNA naturally present in living animals is not "isolated," but synthetic DNA or DNA partially or completely separated from its natural state coexisting material is "isolated." Isolated nucleic acids can exist in substantially purified forms or in non-native environments, such as, for example, cells in which nucleic acids have been delivered.

[0292] The term "nucleic acid" as used in this article refers to molecules containing nucleic acid components, also known as DNA or RNA molecules. It is used interchangeably with the term "polynucleotide." Nucleic acid molecules are polymers containing nucleotide monomers or composed of nucleotide monomers covalently linked to each other by phosphodiester bonds along a sugar / phosphate backbone. Nucleic acids also encompass modified nucleic acid molecules, such as DNA or RNA molecules with modified bases, modified sugars, or modified backbones. Nucleic acids can exist in various forms, such as: incorporated sequences or isolated segments and recombinant vectors of polynucleotides encoding polypeptides (such as one or both chains of an antigen or antibody, or fragments, derivatives, mutant proteins, or variants thereof); polynucleotides sufficient to serve as hybridization probes, PCR primers or sequencing primers for recognizing, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; and antisense nucleic acids used to suppress the expression of the polynucleotides, mRNA, modRNA, and complementary sequences described above. Nucleic acids may encode epitopes that antibodies can bind to.

[0293] The term "epitope" refers to a portion specifically recognized by components of an immunoglobulin (e.g., antibody or receptor). In some aspects, an epitope on an antigen consists of a plurality of chemical atoms or chemical groups. In some aspects, the surface of such chemical atoms or groups is exposed when the antigen adopts an associated three-dimensional configuration. In some aspects, when the antigen adopts this configuration, such chemical atoms or groups are physically close to each other in space. In some aspects, when the antigen adopts an alternative configuration (e.g., linearization), at least some of such chemical atoms or groups are physically separated from each other.

[0294] Nucleic acids can be single-stranded or double-stranded and may contain RNA and / or DNA nucleotides and their artificial variants (e.g., peptide nucleic acids). In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for polypeptide purification, delivery, secretion, post-translational modification, or therapeutic benefits such as targeting or efficacy. Tags or other heterologous polypeptides may be added to the sequence of a modified coding polypeptide, where "heterologous" means a polypeptide that is not the same as the modified polypeptide.

[0295] The term "polynucleotide" refers to a nucleic acid molecule that can be recombinant or isolated from the total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, granules, bacteriophages, viruses, etc. In some respects, a polynucleotide contains a regulatory sequence substantially separate from its naturally occurring gene or protein-coding sequence. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Other coding or non-coding sequences may (but are not required to) be present within the polynucleotide.

[0296] In some aspects, there exist polynucleotide variants that are substantially identical to the sequences disclosed herein; those comprising, using the methods described herein (e.g., BLAST analysis using standard parameters), sequence identity equal to, at least, at most, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher of the polynucleotide sequences provided herein. In some aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90% identity in its full-length sequence with the amino acid sequence described herein; or a nucleotide sequence complementary to the isolated polynucleotide. In some aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 95% identity in its full-length sequence with the amino acid sequence described herein; or a nucleotide sequence complementary to the isolated polynucleotide.

[0297] Regardless of the length of the coding sequence itself, nucleic acid fragments can bind to other nucleic acid sequences, such as promoters, polyadenylated nucleotide signals, additional restriction enzyme sites, multiple cloning sites, and other coding fragments, causing their overall length to vary depending on the context. Nucleic acids can be of arbitrary length. For example, the nucleic acid length can be equal to 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, at least one, at most one, or between any two, and / or may contain one or more additional sequences, such as regulatory sequences, and / or may be a portion of a larger nucleic acid, such as a vector. It should be considered that nucleic acid fragments of virtually any length can be used, with the total length limited by the ease of preparation and use as outlined in the guidelines for recombinant nucleic acid procedures.

[0298] In this regard, the term "gene" is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (containing any sequence required for proper transcription, post-translational modification, or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid fragments that express (or are suitable for expression) proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or part of a polypeptide may contain a continuous nucleic acid sequence encoding all or part of such a polypeptide. It should also be considered that a particular polypeptide may be encoded by nucleic acids containing variations in slightly different nucleic acid sequences, but still encoding the same or substantially similar polypeptides.

[0299] As used herein, the term “expression” of a nucleic acid sequence means the production of any gene product from the nucleic acid sequence. In some respects, the gene product may be a transcript. In some respects, the gene product may be a polypeptide. In some respects, the expression of a nucleic acid sequence involves one or more of the following: (1) the production of an RNA template (e.g., from a DNA sequence) ， (1) Transcription; (2) RNA transcript processing (e.g., splicing, editing, etc.); (3) Translation of RNA into polypeptides or proteins; and / or (4) Post-translational modifications of polypeptides or proteins.

[0300] Generally, the term "engineered" refers to aspects that are artificially manipulated. For example, a polynucleotide is considered "engineered" when two or more sequences that are not connected in sequence in nature are artificially manipulated to be directly linked to each other in an engineered polynucleotide and / or when a particular residue in a polynucleotide is not naturally present and / or is artificially linked to an entity or group that is not connected to it in nature.

[0301] As used herein, the term "DNA" refers to a nucleic acid molecule containing nucleotides, such as deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate, which consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and are polymerized through a characteristic backbone structure. Typically, the backbone structure is formed by a phosphodiester bond between the sugar moiety (e.g., deoxyribose) of the nucleotide of the first adjacent monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers (e.g., the sequence of bases linked to the sugar / phosphate backbone) is called the DNA sequence. DNA can be single-stranded or double-stranded. In double-stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, such as through A / T base pairing and G / C base pairing. DNA may contain all (or most) of the deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-furanose ribosyl group. Without any restrictions, DNA can encompass double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinant DNA, and modified DNA.

[0302] As used herein, the term "RNA" refers to a nucleic acid molecule containing nucleotides, such as adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers, linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (e.g., ribose) of the first adjacent monomer and the phosphate moiety of the second adjacent monomer. RNA can be obtained through transcription of a DNA sequence (e.g., within a cell). In eukaryotic cells, transcription typically occurs in the nucleus or mitochondria. In vivo, transcription of DNA produces immature RNA, which is processed into messenger RNA (mRNA). The processing of immature RNA (e.g., in eukaryotes) involves various post-transcriptional modifications (such as splicing, 5' capping, polyadenylation, and release from the nucleus or mitochondria). Mature messenger RNA is processed to provide a nucleotide sequence that can be translated into an amino acid sequence of peptides or proteins. Mature mRNA may contain a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a polyadenylation tail sequence. RNA may contain all or most of the ribonucleotide residues. As used herein, the term "ribonucleotide" means a nucleotide lacking a hydroxyl group at the 2' position of the β-D-furanose ribosyl group. In one aspect, RNA can be messenger RNA (mRNA) associated with RNA transcripts encoding peptides or proteins. As is known to those skilled in the art, mRNA typically contains a 5' untranslated region (5' UTR), a polypeptide-coding region, and a 3' untranslated region (3' UTR). Without any limitation, RNA can encompass double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinant RNA, and modified RNA (modRNA).

[0303] "Isolated RNA" is defined as an RNA molecule that can be recombinant or isolated from total genomic nucleic acids. Isolated RNA molecules or proteins may exist in substantially purified form or in non-native environments, such as, for example, host cells.

[0304] Compared to naturally occurring RNA, "modified RNA" or "modRNA" means an RNA molecule that has at least one addition, deletion, substitution, and / or alteration of one or more nucleotides. This alteration may mean the addition of non-nucleotide material to an internal RNA nucleotide or to the 5' and / or 3' end of the RNA. In one aspect, this modRNA contains at least one modified nucleotide, such as a change in the bases of a nucleotide. For example, the modified nucleotide may replace one or more uridine and / or cytidine nucleotides. For example, these substitutions may occur in every uridine and / or cytidine in the RNA sequence, or may occur only in selected uridine and / or cytidine nucleotides. Such alteration of standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxyribonucleotides. For example, at least one uridine nucleotide in the RNA sequence may be replaced by N1-methylpseudouridine. Other such altered nucleotides are known to those skilled in the art. Such modified RNA molecules are considered analogs of naturally occurring RNA. In some aspects, RNA is produced by transcription in vitro using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In some respects, RNA can be replicon RNA (replicon), especially self-replicating RNA or self-amplifying RNA (saRNA).

[0305] As envisioned herein, without any limitations, RNA can be used as a therapeutic modality for treating and / or preventing several conditions in mammals, including humans. The methods described herein include administering the RNA described herein to mammals, such as humans. For example, in one aspect, such methods of using RNA include an RNA vaccine encoding an antigen to induce strong neutralizing antibodies and accompanying / concurrent T-cell responses to achieve protective immunization. In some aspects, a minimal vaccine dose is administered to induce strong neutralizing antibodies and accompanying / concurrent T-cell responses to achieve protective immunization. In one aspect, the administered RNA is in vitro transcribed RNA. For example, such RNA may be used to encode at least one antigen intended to elicit 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 aspect, the pathogen is a peptide or protein antigen derived from *Escherichia coli* FimH. Conditions and / or diseases that can be treated with the 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*.

[0306] As used herein, “preventing” when used in connection with the occurrence of a disease, condition, and / or illness means reducing the risk of developing a disease, condition, and / or illness and / or delaying the onset of one or more characteristics or symptoms of a disease, condition, or illness. Prevention is considered complete when the occurrence of a disease, condition, or illness has been delayed for a predetermined period of time.

[0307] As will be understood from the context, the “risk” of a disease, condition, and / or illness means the likelihood that a particular individual will develop a disease, condition, and / or illness. In some respects, risk is expressed as a percentage. In some respects, risk is, at least, or at most, from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100%. In some respects, risk is expressed as risk relative to the risk associated with a reference sample or a group of reference samples. In some respects, the reference sample or a group of reference samples has a known risk of a disease, condition, illness, and / or event. In some respects, the reference sample or a group of reference samples is from individuals comparable to the particular individual. In some respects, risk may reflect one or more genetic attributes, such as those that predispose an individual to develop (or not develop) a particular disease, condition, and / or illness. In some respects, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes. Susceptible to: Individuals who are "susceptible to" a disease, condition, and / or illness have a higher risk of developing that disease, condition, and / or illness than the general population. In some respects, individuals who are susceptible to a disease, condition, and / or illness may never be diagnosed with that disease, condition, and / or illness. In some respects, individuals who are susceptible to a disease, condition, and / or illness may exhibit symptoms of that disease, condition, and / or illness. In some respects, individuals who are susceptible to a disease, condition, and / or illness may not exhibit symptoms of that disease, condition, and / or illness. In some respects, individuals who are susceptible to a disease, condition, and / or illness will develop that disease, condition, and / or illness. In some respects, individuals who are susceptible to a disease, condition, and / or illness will not develop that disease, condition, and / or illness.

[0308] As used herein, the terms “protein,” “polypeptide,” or “peptide” are synonyms and refer to a polymer of amino acid monomers, such as a molecule containing at least two amino acid residues. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, heterologs, homologs, fragments and other equivalents, variants, and analogs of the foregoing. Polypeptides can be a single molecule or a complex of multiple molecules, such as dimers, trimers, or tetramers. Proteins comprise one or more peptides or polypeptides and can fold into a three-dimensional form, which may be necessary for the protein to perform its biological function.

[0309] As used herein, the terms “wild-type” or “WT” or “native” refer to the endogenous form of a molecule that is naturally present in an organism. In some aspects, the wild-type form of a protein or polypeptide is used; however, in other aspects of this disclosure, a modified protein or polypeptide is used to generate an immune response. The terms described above are used interchangeably.

[0310] "Modified protein" or "modified polypeptide" or "variant" means a protein or polypeptide whose chemical structure (especially its amino acid sequence) has been altered relative to the wild-type protein or polypeptide. In some respects, the modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have several activities or functions). This is particularly taken into consideration that modified / variant proteins or polypeptides can be altered, but in other respects (such as immunogenicity) can retain wild-type activity or function. Proteins specifically mentioned herein generally refer to native (wild-type) or recombinant (modified) proteins. Proteins can be directly isolated from their native organism, produced by recombinant DNA / exogenous expression, produced by solid-phase peptide synthesis (SPPS), or by other in vitro methods. In certain respects, this refers to isolated nucleic acid fragments and recombinant vectors having a nucleic acid sequence incorporating a polypeptide (e.g., an antigen or a fragment thereof). The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule manipulated in vitro or a replication product of such a molecule.

[0311] Regarding amino acid sequences (peptides or proteins), the term "fragment" refers to a portion of an amino acid sequence, such as a shortened N-terminus and / or C-terminus of the amino acid sequence. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame (OPF) lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated OPF lacking the 5' end, provided that the truncated OPF contains a start codon that initiates translation. A fragment of an amino acid sequence contains, 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 polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has sequence identity with a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence derived therefrom, having at least, at most, exactly, or between 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%.

[0312] In one aspect, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 70% sequence identity with a derived polypeptide, DNA nucleic acid, or RNA nucleic acid sequence. In another aspect, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 80% sequence identity with a derived polypeptide, DNA nucleic acid, or RNA nucleic acid sequence. In another aspect, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 85% sequence identity with a derived polypeptide, DNA nucleic acid, or RNA nucleic acid sequence. In another aspect, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 90% sequence identity with a derived polypeptide, DNA nucleic acid, or RNA nucleic acid sequence. In another aspect, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 95% sequence identity with a derived polypeptide, DNA nucleic acid, or RNA nucleic acid sequence. In another aspect, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 97% sequence identity with a derived polypeptide, DNA nucleic acid, or RNA nucleic acid sequence. On the one hand, a polypeptide fragment, DNA nucleic acid, or RNA nucleic acid sequence means a sequence that has at least 99% sequence identity with the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived.

[0313] As used herein in the context of molecules, such as nucleic acids, proteins, or small molecules, the term "variant" means a molecule that exhibits significant structural identity with a reference molecule but is structurally different from it, for example, having or lacking one or more chemical motifs or being at the level of one or more chemical motifs compared to the reference molecule. In some respects, a variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of its structural identity with the reference molecule. As those skilled in the art will understand, any biological or chemical reference molecule has a certain characteristic structural component. A variant is defined as a different molecule that shares one or more of this characteristic structural component but differs from the reference molecule in at least one respect. In some respects, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid due to one or more differences in the amino acid or nucleotide sequence and / or one or more differences in chemical motifs (e.g., carbohydrates, lipids, phosphate groups), wherein the chemical motif is a covalent component of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some aspects, the variant polypeptide or nucleic acid exhibits overall sequence identity with the reference polypeptide or nucleic acid of at least, at most, exactly, or between 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some aspects, the variant polypeptide or nucleic acid does not share at least one characteristic sequence component with the reference polypeptide or nucleic acid. In some aspects, the reference polypeptide or nucleic acid has one or more biological activities. In some aspects, the variant polypeptide or nucleic acid has one or more biological activities of the reference polypeptide or nucleic acid. In some aspects, the variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some aspects, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some aspects, a polypeptide or nucleic acid of interest is considered a “variant” of the reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference polypeptide or nucleic acid but with a small amount of sequence variation at a specific position. Preferably, the variant polypeptide or nucleic acid sequence has at least one modification, such as one to about 20 modifications, compared to the reference polypeptide or nucleic acid sequence. In one respect, the variant polypeptide or nucleic acid sequence has 1 to approximately 10 modifications compared to the reference polypeptide or nucleic acid sequence. In another respect, the variant polypeptide or nucleic acid sequence has 1 to approximately 5 modifications compared to the reference polypeptide or nucleic acid sequence. In yet another respect, the variant polypeptide or nucleic acid sequence has 1 to approximately 4 modifications compared to the reference polypeptide or nucleic acid sequence. Typically, compared to the reference, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of residues in the variant are replaced, inserted, or deleted.Typically, compared to a reference, variant peptides or nucleic acids contain a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (e.g., residues involved in specific biological activities). In some aspects, compared to a reference, variant peptides or nucleic acids contain about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue. In some aspects, compared to a reference, variant peptides or nucleic acids contain fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and often fewer than about 5, about 4, about 3, or about 2 additions or deletions. In some aspects, compared to a reference, variant peptides or nucleic acids contain no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions, and in some aspects, no additions or deletions are present.

[0314] In some respects, the reference polypeptide or nucleic acid is a naturally occurring "wild-type," "WT," or "native" sequence containing allelic variations. Wild-type polypeptide or nucleic acid sequences have unmodified sequences. For the purposes of this disclosure, a "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. A "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, splicing variants, post-translational modification variants, conformations, isomers, allelic variants, species variants, and species homologs, especially those that occur naturally. The term "variant" specifically includes fragments of amino acid or nucleic acid sequences.

[0315] Mutation can introduce alterations into nucleic acids, thereby causing changes in the amino acid sequence of the encoded polypeptide (e.g., an antigen, antibody, or antibody derivative). Mutations can be introduced using any technique known in the art. On one hand, site-directed mutagenesis, for example, is used to alter one or more specific amino acid residues. On the other hand, random mutagenesis, for example, is used to alter one or more randomly selected residues. In some aspects, regardless of how they are generated, mutant polypeptides can be expressed and screened for desired properties.

[0316] Mutations can be introduced into nucleic acids without significantly altering the biological activity of the encoded polypeptide. For example, nucleotide substitutions can be made, resulting in the substitution of amino acid residues at non-essential amino acid sites. Alternatively, one or more mutations can be introduced to selectively alter the biological activity of the polypeptide encoded by the nucleic acid. For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative alterations include increasing, decreasing, or eliminating activity. Examples of qualitative alterations include changing the antigen specificity of an antibody.

[0317] "Sequence similarity" indicates the percentage of amino acids that are the same or represent conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are the same between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are the same between the sequences.

[0318] The terms "%identical," "% identity," or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may (but are not necessarily) be randomly distributed across the entire length of the sequences to be compared. Comparison of two sequences is typically performed by comparing sequences relative to fragments or a "window of comparison" after best alignment to identify local regions of the corresponding sequences. Best alignments for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, AdsApp. Math. 2, 482; the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or computer programs using these algorithms (Genetic Computing Group, University of Wisconsin Genetic Software Packages GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA). In some respects, the percentage of identity between two sequences is determined using the BLASTN or BLASTP algorithm, which is available on the National Center for Biotechnology Information (NCBI) website.

[0319] The identity percentage is obtained by determining the number of identical positions in the sequences to be 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.

[0320] In some respects, the degree of similarity or identity is given for regions that are at least, at most, exactly, or between approximately 50%, 60%, 70%, 80%, 90%, or 100% of the full length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for nucleotides that are at least, at most, exactly, or between approximately 100, 120, 140, 160, 180, or 200, and in some respects, for consecutive nucleotides. In other respects, the degree of similarity or identity is given for the full length of the reference sequence.

[0321] Homologous amino acid sequences may exhibit at least, at most, exactly, or between 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% amino acid residue identity. In one aspect, homologous amino acid sequences exhibit at least 95% amino acid residue identity. In another aspect, homologous amino acid sequences exhibit at least 98% amino acid residue identity. In yet another aspect, homologous amino acid sequences exhibit at least 99% amino acid residue identity.

[0322] Fragments or variants of amino acid sequences (peptides or proteins) can be “functional fragments” or “functional variants.” The terms “functional fragment” or “functional variant” of an amino acid sequence refer to any fragment or variant exhibiting one or more functional properties that are the same as or similar to the functional properties of the amino acid sequence from which it is derived, e.g., functionally identical. With respect to an antigen or antigenic sequence, a specific function is one or more immunogenic activities expressed by the amino acid sequence from which the fragment or variant is derived. As used herein, the terms “functional fragment” or “functional variant” specifically mean a variant molecule or sequence that contains one or more altered amino acid sequences compared to the parent molecule or sequence, yet still achieves one or more functions of the parent molecule or sequence, e.g., inducing an immune response. In one respect, modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the properties of the molecule or sequence. The terms “mutant” of wild-type E. coli FimH protein, “mutant” of E. coli FimH protein, “mutant of E. coli FimH protein”, or “modified E. coli FimH protein” refer to a polypeptide that exhibits an introduced mutation relative to wild-type FimH protein and is immunogenic to wild-type FimH protein.

[0323] The amino acid sequence “derived from” the specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence derived from the specific amino acid sequence has the same, substantially the same, or homologous amino acid sequences to the specific sequence or fragments thereof. The amino acid sequence derived from the specific amino acid sequence may be a variant of the specific sequence or fragments thereof. For example, as will be understood by those skilled in the art, antigens applicable herein may be modified such that their sequence differs from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0324] In this disclosure, vector means nucleic acid molecule, such as artificial nucleic acid molecule. Vectors can be used to incorporate nucleic acid sequences, such as nucleic acid sequences containing open reading frames. Vectors include (but are not limited to) storage vectors, expression vectors, cloning vectors, and transfer vectors. Vectors can be RNA vectors or DNA vectors. In some aspects, vectors are DNA molecules. In some aspects, vectors are plasmid vectors. In some aspects, vectors are viral vectors. Generally, expression vectors will contain the desired coding sequence and other suitable sequences required for the expression of the coding sequence operatively linked in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Cloning vectors are typically used to engineer and amplify a specific desired fragment (generally a DNA fragment) and may lack the functional sequences required for the expression of the desired fragment.

[0325] As used herein, the term "pharmaceutical composition" means an active agent formulated together with one or more pharmaceutically acceptable carriers. Pharmaceutical compositions may be immunogenic compositions. In some aspects, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to the relevant population, shows a statistically significant likelihood of achieving the intended therapeutic effect. In some aspects, pharmaceutical compositions may be specifically formulated as, for example, a sterile solution or suspension or a sustained-release formulation for parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection.

[0326] As used herein, the term "vaccination" means the administration of an immunogenic composition intended to produce an immune response, for example, against a disease-associated (e.g., pathogenic) agent (e.g., bacteria). In some aspects, vaccination may be administered before, during, and / or after exposure to a disease-associated agent, and in some aspects, before, during, and / or shortly after exposure to the agent. In some aspects, vaccination involves multiple administrations of a vaccine composition at appropriate time intervals. In some aspects, vaccination produces an immune response to an infectious agent. In some aspects, vaccination produces an immune response to a tumor; in some such aspects, vaccination is "personalized" because it is partially or completely targeted at epitopes of a tumor identified in a particular individual (e.g., it may be or may include one or more neoepitopes).

[0327] An immune response refers to a humoral response, a cellular response, or both humoral and cellular responses within an organism. Immune responses can be measured by analysis, which includes (but is not limited to) analyses measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, analyses measuring T cell activation or proliferation, and / or measurements measuring the regulation of the activity or expression of one or more cytokines.

[0328] As used herein, the term "combination therapy" refers to those situations where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some respects, two or more regimens may be administered simultaneously; in some respects, such regimens may be administered sequentially (e.g., all "agents" of the first regimen are administered before any agent of the second regimen); in some respects, such agents are administered in an overlapping dosing regimen. In some respects, "administering" combination therapy may involve administering one or more agents or methods to a subject who receives other agents or methods in the combination. For clarity, combination therapy does not require the individual agents to be administered together in a single composition (or even simultaneously), although in other respects, 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 part of a single chemical complex or covalent entity).

[0329] Those skilled in the art will understand that the term "dosing regimen" can be used to refer to groups of unit doses (usually more than one group) administered individually to a subject, typically separated by a period of time. In some aspects, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some aspects, a dosing regimen comprises a plurality of doses, each separated from the others by time. In some aspects, the individual doses are separated from each other by a time period of equal length; in some aspects, a dosing regimen comprises a plurality of doses and at least two different time periods separating the individual doses. In some aspects, all doses within a dosing regimen are of the same unit dose. In some aspects, the different doses within a dosing regimen have different amounts. In some aspects, a dosing regimen comprises a first dose of a first dose, followed by one or more additional doses of a second dose different from the first dose. In some aspects, a dosing regimen comprises a first dose of a first dose, followed by one or more additional doses of a second dose identical to the first dose. In some aspects, when administered in a relevant population (e.g., a therapeutic dosing regimen), the dosing regimen is associated with a desired or beneficial outcome.

[0330] II. Escherichia coli fimbrial antigen H (FimH)

[0331] As used herein, the term “FimH antigenic polypeptide” includes FimH polypeptides or immunogenic mutants thereof, including (but not limited to) FimH polypeptides as shown in SEQ ID NO: 1 to 64, 77, 79, 81 or 83.

[0332] As used herein, the term "E. coli polypeptide" includes any E. coli polypeptide. In a preferred embodiment, the E. coli polypeptide is a fimbrial antigen. In a preferred embodiment, the E. coli fimbrial antigen is FimH.

[0333] The FimH antigenic polypeptide is described in PCT International Publication No. WO2022 / 137078, which is incorporated herein by reference in its entirety.

[0334] Embodiments of this disclosure provide RNA (e.g., mRNA) vaccines comprising polynucleotides encoding Escherichia coli FimH antigens. These E. coli FimH RNA vaccines, as provided herein, can be used to induce balanced immune responses, including cellular and humoral immunity.

[0335] Some embodiments provide vaccines and pharmaceutically acceptable loads or excipients comprising one or more RNA polynucleotides having an open reading frame encoding the FimH protein, formulated in cationic lipid nanoparticles. In some embodiments, the FimH protein is selected from FimH-DSG, FimH-DSG triple mutants (G15A, G16A, V27A), or FimH LD Triple mutants (G15A, G16A, V27A).

[0336] As used herein, when the term "TM" is used in conjunction with an antigen, it should refer to a triple mutant, especially FimH with mutations at amino acid positions G15A, G16A, and V27A. LD Or a triple mutant of the FimH-DSG peptide. Therefore, the terms "FimH-DSG triple mutant (G15A, G16A, V27A)" and "FimH-DSG™" are used interchangeably. Furthermore, the term "FimH..." LD Triple mutants (G15A, G16A, V27A) and "FimH" LD "TM" can be used interchangeably.

[0337] As used in this article, the abbreviation "Ct" should refer to the C-terminal domain of a polypeptide or polynucleotide.

[0338] Some implementations provide methods for preventing or treating Escherichia coli infection, including administering any of the vaccines described herein to a subject. In some implementations, the antigen-specific immune response includes a T-cell response. In some implementations, the antigen-specific immune response includes a B-cell response. In some implementations, the antigen-specific immune response includes both a T-cell response and a B-cell response. In some implementations, the method of generating an antigen-specific immune response involves the single administration of a vaccine. In some implementations, the vaccine is administered to the subject via intradermal, intramuscular, subcutaneous, intranasal, or oral administration.

[0339] In some implementations, RNA (e.g., mRNA) polynucleotides or portions thereof may encode one or more polypeptides or fragments thereof of Escherichia coli FimH as antigens.

[0340] III. RNA molecules

[0341] In some respects, the RNA molecules described herein are coding RNA molecules. Coding RNA comprises functional RNA molecules that can be translated into peptides or polypeptides. In some respects, coding RNA molecules comprise at least one open reading frame (ORF) encoding at least one peptide or polypeptide. An open reading frame comprises a codon sequence that can be translated into a peptide or protein. Coding RNA molecules may contain one (monocistronic) ORF, two (bicistronic) ORFs, or more (multicistronic) ORFs, which may be codon sequences that can be translated into a polypeptide or protein of interest.

[0342] Several mRNA vaccine platforms are available in existing technologies. The basic structure of in vitro transcribed (IVT) mRNA is quite similar to that of "mature" eukaryotic mRNA, consisting of (i) an open reading frame (ORF) encoding the protein, flanked by (ii) 5' and 3' untranslated regions (UTRs), and (iii) a 5' cap structure and (iv) a 3' polyadenylated tail at the ends. These non-coding structural features play important roles in the pharmacology of mRNA and can be individually optimized to regulate mRNA stability, translation efficiency, and immunogenicity.

[0343] By incorporating modified nucleosides, mRNA transcripts known as "nucleoside-modified mRNA" or "modRNA" can be generated, exhibiting reduced immunostimulatory activity and thus improved safety profiles. Furthermore, modified nucleosides allow for the design of mRNA vaccines with significantly enhanced stability and translational capabilities, as they can avoid the direct antibacterial pathway induced by IFN-type degradation and programmed to inhibit invading mRNA. For example, replacing uridine with pseudouridine in in vitro transcribed (IVT) mRNA reduces the activity of 2'-5'-oligoadenylate synthetase, which regulates the cleavage of mRNA by RNase L. Additionally, lower activity of protein kinase R, an enzyme involved in inhibiting mRNA translation, was measured.

[0344] Besides incorporating modified nucleotides, other methods have been shown to increase the translational capacity and stability of mRNA. One example is the development of "sequence-engineered mRNA." Here, mRNA expression can be strongly increased through sequence optimization of the mRNA's ORF and UTR, for example, by enriching GC content or by selecting the UTR of naturally long-lived mRNA molecules.

[0345] In addition, several modifications are applied to the terminal structures of mRNA. Anti-reverse cap (ARCA) modification ensures proper cap orientation at the 5' end, resulting in a nearly intact mRNA fragment that can effectively bind to the ribosome. Other cap modifications (such as phosphate thioester cap analogs) can further improve affinity for eukaryotic translation initiation factor 4E and increase resistance to RNA decapping complexes.

[0346] Conversely, by modifying its structure, the efficacy of mRNA in promoting innate immune responses can be further improved, but translational ability may be impaired. Stabilizing mRNA with a phosphate thioester backbone or by precipitating it with the cationic protein protamine can reduce antigen expression but result in stronger immunostimulatory capacity.

[0347] In one aspect, the present invention relates to immunogenic compositions comprising an mRNA molecule containing one or more polypeptides or fragments thereof encoding *Escherichia coli* FimH as an antigen. In some embodiments, the mRNA molecule comprises nucleoside-modified mRNA. The RNA molecule may encode one or more polypeptides of interest, such as antigens, or more than one antigen, such as two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. Alternatively, or additionally, an RNA molecule may also encode more than one polypeptide of interest, such as antigens, such as bicistronic or tricistronic RNA molecules encoding different or the same antigens.

[0348] The sequence of an RNA molecule can be codon-optimized or deoptimized for expression in a desired host (such as human cells). In some aspects, the genes of interest described herein (e.g., antigens) are encoded by coding sequences that have been codon-optimized and / or have increased levels of guanosine / cytidine (G / C) compared to wild-type coding sequences. In some aspects, one or more regions of the coding sequence have been codon-optimized and / or have increased G / C levels compared to corresponding regions of the wild-type coding sequence. In some aspects, codon optimization and / or increased G / C levels do not alter the sequence encoding the amino acid sequence.

[0349] The term "codon optimization" is understood by those skilled in the art to refer to codon changes in the coding region of a nucleic acid molecule to reflect typical codon usage in the host organism, without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, in some aspects, coding regions are codon-optimized for optimal expression in subjects to be treated with the RNA polynucleotides described herein. Codon optimization is based on findings of translation efficiency determined by the varying frequencies of tRNA molecules occurring in cells. Therefore, the sequences of these RNAs can be modified to allow the insertion of codons available for frequently occurring tRNA molecules to replace "rare codons."

[0350] In some respects, the G / C content of the coding region of RNA (e.g., the sequence of the 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, wherein in some respects, the amino acid sequence encoded by the RNA is unmodified compared to the amino acid sequence encoded by the wild-type RNA. RNA sequence modification is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of its mRNA. Sequences with increased G (guanosine) / C (cytidine) content are more stable than those with increased A (adenosine) / U (uridine) content. Given the fact that several codons encode the same amino acid (also known as degeneration of the genetic code), the most stable and advantageous codons (also known as alternative codon usage) can be determined. Depending on the amino acid encoded by the RNA, there are many possibilities for RNA sequence modification compared to its wild-type sequence. Specifically, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U, or contain lower amounts of A and / or U nucleotides. Therefore, in some respects, the G / C content of the coding region of the RNA described herein is increased by at least, at most, exactly, or between 10%, 20%, 30%, 40%, 50%, 55%, or even more than that of the coding region of wild-type RNA.

[0351] In some respects, RNA molecules contain approximately 20 to approximately 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500). 0, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).

[0352] In some respects, RNA molecules possess at least, at most, exactly, or between about 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, 680 0, 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, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000 nucleotides.

[0353] In some respects, RNA molecules contain at least 100 nucleotides. For example, in some respects, the length of RNA is between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; and between 7,000 and 25,000 nucleotides. In some respects, RNA molecules possess at least, at most, exactly, or between about 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, 2 050, 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, 61 00, 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, 81008150, 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, 101 00, 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, 1 Nucleotides between any two of the following values: 3450, 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.

[0354] The mRNAs used in this disclosure typically comprise a first region encoding a linked nucleotide of interest (e.g., a coding region), a first flanking region located at the 5' end (e.g., 5'-UTR) of the first region, a second flanking region located at the 3' end (e.g., 3'-UTR) of the first region, at least one 5'-cap region, and a 3'-stabilizing region. In some embodiments, the mRNAs of the present invention further comprise a polyadenylated region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the mRNAs of the present invention may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some embodiments, the mRNAs of the present invention may comprise a 5' cap structure, a chain-terminating nucleotide, a stem loop, a polyadenylated sequence, and / or a polyadenylated signal. Any region of the nucleic acid may contain one or more alternative components (e.g., alternative nucleotides). For example, the 3'-stable region may contain alternative nucleosides (such as L-nucleoside, reverse thymine, or 2'-O-methylnucleoside) and / or coding regions, and the 5'-UTR, 3'-UTR, or cap region may contain alternative nucleosides such as 5-substituted uridine (e.g., 5-methoxyuridine), 1-substituted pseudouridine (e.g., 1-methyl-pseudouridine), and / or 5-substituted cytidine (e.g., 5-methyl-cytidine).

[0355] In some embodiments, the RNA disclosed herein includes the following components in the 5' to 3' orientation: a 5' cap containing the 5' cap disclosed herein; a 5' untranslated region containing a cap proximal sequence (5' UTR), a sequence encoding a payload (e.g., E. coli FimH protein); a 3' untranslated region (3' UTR); and a polyadenylate sequence.

[0356] In some embodiments, the LNP comprises one or more RNAs, and optionally one or more RNAs, lipids, and amounts thereof to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of the nitrogen atoms of one or more lipids to the number of phosphate groups in the RNA. Generally, a lower N:P ratio is preferred. One or more RNAs, lipids, and amounts thereof may be selected to provide an N:P ratio of about 2:1 to about 30:1, such as 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 some 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 can be approximately 5.0:1, approximately 5.5:1, approximately 6.0:1, approximately 6.5:1, or approximately 7.0:1.

[0357] A. Modified nucleobases

[0358] In this disclosure, RNA molecules may contain modified nucleobases, which may be incorporated into modified nucleosides and nucleotides. In some aspects, RNA molecules may contain one or more modified nucleotides. Naturally occurring nucleotide modifications are known in the art.

[0359] The mRNA of the present invention may contain one or more naturally occurring components, including standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one embodiment, the nucleotides comprising all or substantially all of (a) a 5'-UTR, (b) an open reading frame (ORF), (c) a 3'-UTR, (d) a polyadenylate tail, and any combination thereof (a, b, c, d) above comprise naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine).

[0360] As described herein, the mRNA of the present invention may comprise one or more alternative components that impart useful properties, including those that increase stability and / or substantially induce an innate immune response that introduces polynucleotides into cells. For example, modRNAs may exhibit reduced degradation in cells in which modRNAs are introduced, relative to their corresponding unaltered mRNAs. These alternatives may enhance protein production efficiency, intracellular polynucleotide retention and / or cell-contact activity, and have reduced immunogenicity.

[0361] The mRNA of the present invention may comprise one or more modified (e.g., altered or substituted) nucleotides, nucleoside nucleotides, or combinations thereof. mRNAs useful in LNPs may contain any useful modifications or alterations, such as linkages between nucleotides, sugars, or nucleosides (e.g., linkage to a phosphate group / linkage to a phosphodiester / linkage to a phosphodiester backbone). In some embodiments, alterations (e.g., one or more alterations) are present in each of the nucleotides, sugars, and nucleoside linkages. Alterations according to this disclosure may be changes to ribonucleic acid (RNA), such as replacing the 2'-OH of the furanyl ribosyl ring with 2'-H, threonine nucleic acid (TNA), glycerol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or mixtures thereof.

[0362] The mRNA of the present invention may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or all of A, G, U, C) may or may not be uniformly altered in the mRNA or in a given predetermined sequence region. In some instances, all nucleotides X in the mRNA (or in a given sequence region) are altered, wherein X may be any one or a combination of nucleotides A, G, U, C, such as 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.

[0363] Different sugar alterations and / or nucleotide linkages (e.g., skeletal structures) can occur at various positions within the polynucleotide. Those skilled in the art will understand that nucleotide analogs or other alterations can be located at any position on the polynucleotide without substantially reducing its function. The alteration can also be a 5'- or 3'-end alteration. In some embodiments, the polynucleotide includes a 3'-end alteration. The polynucleotide may contain about 1% to about 100% of alternative nucleotides (related to the overall nucleotide content, or to one or more types of nucleotides, such as one or more of A, G, U, and C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 1...). 00%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%. It should be understood that any remaining percentage is due to the presence of standard nucleotides (e.g., A, G, U, or C).

[0364] The polynucleotide may contain at least 0 and at most 100% of alternative nucleotides, or any intermediate percentage, such as at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, or at least 90% of alternative nucleotides. For example, the 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 an alternative uracil (e.g., 5-substituted uracil). The alternative uracil may be replaced by a compound having a single unique structure, or by a plurality of compounds having different structures (e.g., 2, 3, 4, or more unique structures). In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the polynucleotide is replaced with an alternative cytosine (e.g., 5-substituted cytosine). The alternative cytosine can be replaced by a compound having a single unique structure, or by a plurality of compounds having different structures (e.g., 2, 3, 4, or more unique structures).

[0365] In some instances, nucleic acids do not substantially induce an innate immune response in cells that have introduced polynucleotides (e.g., mRNA). Characteristics of induction of an innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-1, MDA5, etc.), and / or 3) termination or reduction of protein translation.

[0366] In some embodiments, the mRNA contains one or more alternative nucleosides or nucleotides. Alternative nucleosides and nucleotides may contain alternative nucleobases. The nucleobases of the nucleic acid are organic bases, such as purines or pyrimidines or derivatives thereof. Nucleobases may be standard bases (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleobases may be altered or completely replaced to provide polynucleotide molecules with enhanced properties, such as enhanced stability, such as resistance to nucleases. Non-standard or modified bases may contain, for example, one or more substitutions or modifications, including (but not limited to) alkyl, aryl, halogen, sideoxy, hydroxyl, alkoxy, and / or thiosubstituted groups; one or more fused or open rings; oxidation; and / or reduction.

[0367] In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having alternative uracils include pseudouridine (ψ), pyridine-4-ketoribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, and 2-thio-uracil (s). 2 U), 4-thiouracil (s) 4U), 4-thiopseudouridine (s4ψ), 2-thiopseudouridine (s2ψ), 5-hydroxyuracil (ho) 5 U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m 3 U), 5-methoxy-uracil (mo) 5 U), uracil 5-oxyacetic acid (cmo) 5 U), methyl 5-oxyacetate of uracil (mcmo) 5 U), 5-carboxymethyl-uracil (cm) 5 U), 1-carboxymethyl-pseuuridine, 5-carboxyhydroxymethyl-uracil (chm) 5 U), 5-carboxymethyl-uracil methyl ester (mchm) 5 U), 5-methoxycarbonylmethyl-uracil (mcm) 5 U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm) 5 s 2 U), 5-aminomethyl-2-thiouracil (nmVu), 5-methylaminomethyluracil (mnm) 5 U), 5-methylaminomethyl-2-thiouracil (mnmVu), 5-methylaminomethyl-2-selenouracil (mnm) 5 se 2 U), 5-carbamoylmethyluracil (ncm) 5 U), 5-Carboxymethylaminomethyl-uracil (cmnm) 5 U), 5-carboxymethylaminomethyl-2-thio-uracil (cmnmVu), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-tauronic acid methyl-uracil (xm) 5 U), 1-Taurate methyl-pseuuridine, 5-Taurate methyl-2-thio-uracil (xm) 5 s 2 U), 1-Tauratemethyl-4-thio-pseuuridine, 5-methyl-uracil (m 5 U, such as those containing nucleobase deoxythymidine), 1-methyl-pseuuridine (mV), 5-methyl-2-thio-uracil (mV) 5 s 2U), 1-methyl-4-thio-pseuuridine (ms4ψ), 4-thio-1-methyl-pseuuridine, 3-methyl-pseuuridine (m 2-Thio-1-methyl-pseuuridine, 1-methyl-1-deazo-pseuuridine, 2-thio-1-methyl-1-deazo-pseuuridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydrouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseuuridine, 4-methoxy-2-thio-pseuuridine, Nl-methyl-pseuuridine, 3-(3-amino-3-carboxypropyl)uracil (acpU), 1-methyl-3-(3-amino-3-carboxypropyl)pseuuridine (acp) ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2'-O-dimethyluridine (m5Um), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mem) Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (mUm), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)uracil, 5-(carbamoylhydroxymethyl)uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propenylamino)]uracil. Pseudouridine is an example of a modified nucleoside, which is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

[0368] In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytosine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), and 5-halo-cytosine (e.g., 5-iodo-cytosine). 5-Hydroxymethyl-cytosine (hm5C), 1-methyl-pseudo-cytosine, pyrrolo-cytosine, pyrrolo-pseudo-cytosine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudo-cytosine, 4-thio-1-methyl-pseudo-cytosine, 4-thio-1-methyl-1-deazo-pseudo-cytosine, 1-methyl-1-deazo-pseudo-cytosine, zebralin (zebu larine), 5-aza-za-zabraline, 5-methyl-zabraline, 5-aza-2-thio-zabraline, 2-thio-zabraline, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudo-cytosine, 4-methoxy-1-methyl-pseudo-cytosine, lysine (k2C), 5,2'-O-dimethyl-cytosine (m5Cm), N4-acetyl 2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine.

[0369] In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deadenine, 7-deadenine-8-aza-adenine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diamino-purine, 7-deadenine-8-aza-2,6-diamino-purine, and 1-methyl-1-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-glycylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6A) t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxyvalinecarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxyvalinecarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenine (m6Am), N6,N6,2'-O-trimethyl-adenine (m62Am), 1,2'-O-dimethyl-adenine (ml) Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentazonatyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0370] In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methyl-wyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxy-wyosine (o2yW), hydroxy-wyosine (OHyW), and incompletely modified hydroxy-wyosine (OHyW). ), 7-denitroguanine, queuosine (Q), epoxyqueuosine (oQ), galactosylqueuosine (galQ), mannosylqueuosine (manQ), 7-cyano-7-denitroguanine (preQ0), 7-aminomethyl-7-denitroguanine (preQ1), archaeosine (G+), 7-denitro-8-aza-guanine, 6-thio-guanine, 6-thio-7-denitroguanine, 6-thio-7-denitro-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (mlG), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22) G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-sideoxy-guanine, 7-methyl-8-sideoxy-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m 2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (mlGm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl-guanine.

[0371] The alternative nucleobases of a nucleotide can be purines, pyrimidines, purines, or pyrimidine analogs independently. For example, the nucleobases can be substitutes for adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase may also include, for example, naturally occurring bases and synthetic derivatives, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine and other alkyl derivatives, 2-propyl of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen (e.g., 8-bromo), 8-amino, 8-thiol, 8-Thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine, deadenine, 7-deadenine, 3-deadenine; deadenine, 7-deadenine, 3-deadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deadenine, imidazo[4,5-d]pyrazine, thiazo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; or 1,3,5-triazine. When abbreviations A, G, C, T, or U are used to describe nucleotides, each letter indicates a representative base and / or its derivative. For example, A contains adenine or an adenine analogue, such as 7-deadenine.

[0372] In some respects, RNA molecules contain nucleic acid sequences having at least one uridine replaced by a pseudouridine. In other respects, RNA molecules contain nucleic acid sequences having at least, at most, exactly, or between 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% of their constituent nucleic acid sequences. Urate substituted with pseudouridine at any two of the following percentages: 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%. In some respects, RNA molecules contain all the nucleic acid sequences in which uridine is replaced by pseudouridine.

[0373] B. 5' cap

[0374] mRNA can contain a 5' cap. The 5'-cap structure of polynucleotides is involved in nuclear export and increases polynucleotide stability, and it binds to mRNA cap-binding protein (CBP), which is responsible for the stability of polynucleotides in the cell and translational capacity by associating CBP with polyadenylate-binding protein to form mature circular mRNA material. The cap further assists in the removal of 5'-proximal introns during mRNA splicing.

[0375] Endogenous polynucleotide molecules can be capped at the 5' end, thereby creating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the sense nucleotide transcribed at the 5' end of the polynucleotide. This 5'-guanosine cap can then be methylated to produce an N7-methyl-guanosine residue. The ribose of the terminal and / or anteterminal transcribed nucleotides of the polynucleotide may also be 2'-O-methylated. 5'-uncapping via hydrolysis and cleavage of the guanosine cap structure can target polynucleotide molecules such as mRNA for degradation.

[0376] Modification of the polynucleotide can produce a non-hydrolyzable cap structure that prevents decapping, thus increasing the polynucleotide's half-life. Since cap structure hydrolysis requires the cleavage of the 5'-ppp-5' phosphodiester bond, alternative nucleotides can be used during the capping reaction. For example, vaccinia capping enzymes from New England Biolabs (Ipswich, MA) can be used with α-thioguanosine nucleotides according to the manufacturer's instructions to generate phosphothiodiester bonds in the 5'-ppp-5' cap.

[0377] Other alternative guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, can be used. Other modifications include (but are not limited to) 2'-O-methylation of the ribose of the polynucleotide at the 2'-hydroxyl group of the sugar and / or the ribose of the 5'-anti-terminal nucleotide (as mentioned above). Several different 5'-cap structures can be used to generate the 5'-cap of the mRNA molecule.

[0378] Cap analogs (also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs) have a chemical structure different from that of a natural (e.g., endogenous, wild-type, or physiological) 5'-cap while retaining cap function. Cap analogs can be chemically (e.g., non-enzymatically) or enzymatically synthesized and / or linked to a polynucleotide. For example, the cap of an anti-reverse cap analog (ARCA) contains two guanosines linked by 5'-5'-triphosphate groups, one of which contains an N7-methyl group and a 3'-O-methyl group (e.g., N7,'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7G-3'mppp-G, which can be equivalently named 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unchanged guanosine is replaced by a 5'-terminal nucleotide linked to a capped polynucleotide (e.g., mRNA). N7- and 3'-O-methylated guanosine provides the terminal portion of a capped polynucleotide (e.g., mRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (e.g., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0379] 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 phophoroselenoate group at different phosphate ester positions, such as the dinucleotide cap analog described in U.S. Patent No. 8,519,110, the cap structure of which is incorporated herein by reference.

[0380] Alternatively, the cap analogue may be an N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogue known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogues include N7-(4-chlorophenoxyethyl)-G(5)ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5)ppp(5')G cap analogues (see, for example, the various cap analogues and methods of synthesizing cap analogues described in Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574; the cap structures in that literature are incorporated herein by reference). In other cases, the cap analogue suitable for use in the polynucleotides of this disclosure is a 4-chloro / bromophenoxyethyl analogue.

[0381] Although cap analogs allow polynucleotides to be capped during in vitro transcription, up to 20% of transcripts remain uncapped. This, along with the structural differences between cap analogs and the endogenous 5' cap structure of polynucleotides produced through endogenous cellular transcription mechanisms, may lead to reduced translational capacity and decreased cell stability.

[0382] Alternative polynucleotides can also be post-transcriptionally capped using enzymes to produce more realistic 5'-cap structures. As used herein, the phrase "more realistic" means that a feature structurally or functionally closely reflects or mimics an endogenous or wild-type feature. That is, a "more realistic" feature better represents endogenous, wild-type, natural, or physiological cellular function and / or structure compared to prior art synthetic features or analogs, or surpasses the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more realistic 5'-cap structures that can be used in the polynucleotides of this disclosure are those that, among other things, have enhanced binding to cap-binding proteins, increased half-life, reduced 5' endonuclease sensitivity, and / or reduced 5' decapping compared to synthetic 5'-cap structures known in the art (or wild-type, natural, or physiological 5'-cap structures). For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can generate a standard 5'-5'-triphosphate bond between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, wherein the cap guanosine is N7-methylated and the 5'-terminal nucleotide of the polynucleotide is 2'-O-methyl. This structure is referred to as the Capl structure. Compared to other 5' cap analogs known in the art, this cap produces higher translational efficiency, cell stability, and reduced activation of pro-inflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')N1mpNp (Cap 1), 7mG(5')-ppp(5')N1mpN2mp (Cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (Cap 4).

[0383] A further cap structure includes N having the structure shown below. 1 -Methylpseudouridine-5'-triphosphate (also known as N-methylpseudouridine-5'-triphosphate) 1 -Methylpseudouridine-5'-triphosphate, N 1 meΨTP、m 1 ΨTP, 1-methyl-pseudouridine phosphoramidite or N 1 5'-Methyl-pseudouridine-5'-triphosphate; TriLink Biotechnologies):

[0384] .

[0385] Because alternative polynucleotides can be capped post-transcriptionally, and because this method is more efficient, nearly 100% of the mRNA can be capped. This contrasts with the approximately 80% capping rate when cap analogs are ligated to polynucleotides during in vitro transcription.

[0386] The 5'-cap may contain an endogenous cap or a cap analogue. The 5'-cap may contain a guanosine analogue. Available guanosine analogues include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-side-oxy-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the polynucleotide contains a modified 5'-cap. Modifications to the 5'-cap can increase the stability of the polynucleotide, increase its half-life, and increase its translation efficiency. A modified 5'-cap may contain (but is not limited to) one or more of the following modifications: modification at the 2'-position and / or 3'-position of capped guanosine triphosphate (GTP), substitution of the sugar epoxide (which produces a carbocyclic ring) by a methylene moiety (CH2), modification of the triphosphate bridge portion of the cap structure, or modification of the nucleobase (G) portion.

[0387] C. Untranslated Region (UTR)

[0388] The 5' UTR is a regulatory region located at the 5' end of the open reading frame of a protein. It is transcribed into mRNA but not translated into an amino acid sequence or a corresponding region of RNA polynucleotides (such as mRNA molecules). Untranslated regions (UTRs) can exist at the 5' (upstream) end of the open reading frame (5' UTR) and / or the 3' (downstream) end of the open reading frame (3' UTR).

[0389] In some respects, UTRs are derived from naturally abundant mRNAs in specific tissues (e.g., lymphoid tissues) where mRNA expression is targeted. In other respects, UTRs enhance protein synthesis. Without being bound by mechanistic or theoretical constraints, UTRs can increase protein synthesis by increasing the time mRNA is retained on the translation polysome (messenger stability) and / or the rate at which ribosomes initiate translation on the messenger (messenger translation efficiency). Therefore, UTR sequences can be used to prolong protein synthesis using tissue-specific methods.

[0390] In some respects, the 5' UTR and 3' UTR sequences are derived computationally. In some respects, the 5' UTR and 3' UTR sequences are derived from mRNAs naturally abundant in tissues. Tissues may be, for example, the liver, stem cells, or lymphoid tissue. Lymphoid tissue may contain, 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 respects, the 5' UTR and 3' UTR sequences are derived from alphavirus. In some respects, the 5' UTR and 3' UTR sequences are derived from wild-type alphavirus.

[0391] In some respects, the RNAs disclosed herein contain a 5' UTR. The 5' UTR (if present) is located at the 5' end and initiates transcription upstream of the start codon in the protein-coding region. The 5' UTR (if present) is located downstream of the 5' cap, such as directly adjacent to it. The 5' UTR may contain various regulatory components, such as 5' cap structures, stem-loop structures, and intraribosome initiation sites (IRES), which play a role in the control of translation initiation.

[0392] In some respects, the 5' UTR disclosed herein includes a cap proximal sequence, as disclosed herein. In some respects, the cap proximal sequence includes a sequence adjacent to the 5' cap. In some respects, the cap proximal sequence includes nucleotides at positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0393] In some aspects, the cap structure comprises one or more polynucleotides of a proximal cap sequence. In some aspects, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1) of an RNA polynucleotide. In some aspects, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some aspects, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of an RNA polynucleotide.

[0394] Those skilled in the art will understand upon reading this disclosure that in some aspects one or more residues of the cap proximal sequence (e.g., one or more residues +1, +2, +3, +4, and / or +5) may be included in the RNA by virtue of being contained within a cap entity (e.g., a Cap 1 structure, etc.); or, in some aspects, at least some residues of the cap proximal sequence may be added enzymatically (e.g., by a polymerase such as T7 polymerase). For example, in some exemplary aspects, (m2) is used... 7,3 ' -O )Gppp(m 2 ' -O The ApG cap, with +1 and +2 residues forming the cap's (m2) 7,3 ' -O A and G residues are added, while +3, +4 and +5 residues are added by polymerase (e.g., T7 polymerase).

[0395] In some aspects, the proximal cap sequence contains N1 and / or N2 of the cap structure, where N1 and N2 are arbitrary nucleotides such as A, C, G, or U. In some aspects, N1 is A. In some aspects, N1 is C. In some aspects, N1 is G. In some aspects, N1 is U. In some aspects, N2 is A. In some aspects, N2 is C. In some aspects, N2 is G. In some aspects, N2 is U. In some aspects, the proximal cap sequence contains N1 and N2 and NN3, N4, and N5 of the cap structure, where N1 to N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some aspects, N1, N2, N3, N4, or N5 are arbitrary nucleotides such as A, C, G, or U. In some aspects, N1N2 contains any of the following: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, or UU. In some respects, 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.

[0396] In some aspects, the proximal cap sequence comprises N1 and N2 of the cap structure, and the sequence comprises: A3A4X5 (SEQ ID NO:167; where X5 is A, G, C, or U), wherein N1 and N2 are each independently selected from: A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, X5 is selected from A, C, G, or U. In some aspects, X5 is A. In some aspects, X5 is C. In some aspects, X5 is G. In some aspects, X5 is U.

[0397] In some aspects, the proximal cap sequence comprises N1 and N2 of the cap structure, and the sequence comprises: C3A4X5 (SEQ ID NO:168; where X5 is A, G, C, or U), wherein N1 and N2 are each independently selected from: A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, X5 is selected from A, C, G, or U. In some aspects, X5 is A. In some aspects, X5 is C. In some aspects, X5 is G. In some aspects, X5 is U.

[0398] In some aspects, the proximal cap sequence comprises N1 and N2 of the cap structure, and the sequence comprises: X3Y4X5 (SEQ ID NO:169; wherein X3 or X5 is independently selected from A, G, C, or U; and Y4 is not C). In some aspects, N1 and N2 are independently selected from A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, X3 and X5 are independently selected from A, C, G, or U. In some aspects, X3 and / or X5 is A. In some aspects, X3 and / or X5 is C. In some aspects, X3 and / or X5 is G. In some aspects, X3 and / or X5 is U. In some aspects, Y4 is C. In other aspects, Y4 is not C. In some aspects, Y4 is A. In some aspects, Y4 is G. In other aspects, Y4 is not G. In some aspects, Y4 is U.

[0399] In some aspects, the proximal cap sequence comprises N1 and N2 of the cap structure, and the sequence comprises: A3C4A5 (SEQ ID NO: 170). In some aspects, N1 and N2 are each independently selected from: A, C, G, or U. In some aspects, N1 is A and N2 is G.

[0400] In some aspects, the proximal cap sequence comprises N1 and N2 of the cap structure, and the sequence comprises: A3U4G5 (SEQ ID NO:171). In some aspects, N1 and N2 are each independently selected from: A, C, G, or U. In some aspects, N1 is A and N2 is G.

[0401] The 5'-UTR can be a flanking region of mRNA. The 5'-UTR can be homologous or heterologous to coding regions found in polynucleotides. A flanking region may contain multiple 5'-UTRs, which can be the same or different sequences. Any portion of the flanking region (including but not limited to) can be codon-optimized, and any portion can independently contain one or more different structural or chemical alterations before and / or after codon optimization.

[0402] To alter one or more properties of mRNA, a 5' UTR heterologous to the coding region of the mRNA can be engineered. The mRNA can then be administered to cells, tissues, or organisms, and results such as protein content, localization, and / or half-life can be measured to assess the potential beneficial effects of the heterologous 5' UTR on mRNA production. Variants of the 5' UTR can be utilized, in which one or more nucleotides, including A, T, C, or G, are added or removed from the end. The 5' UTR can also be codon-optimized or altered in any of the ways described herein.

[0403] In some aspects, the RNA molecule contains a 5' untranslated region (5'-UTR). In some aspects, the 5' UTR contains a sequence selected from any one of SEQ ID NO: 95 to SEQ ID NO: 102. In some aspects, the 5' UTR contains a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any one of SEQ ID NO: 95 to SEQ ID NO: 102. In some aspects, the 5' UTR contains a sequence selected from any one of SEQ ID NO: 95 to SEQ ID NO: 102. In some aspects, the 5' UTR contains a sequence consisting of any one of SEQ ID NO: 95 to SEQ ID NO: 102.

[0404] In some respects, the RNA disclosed herein contains a 3' UTR. The 3' UTR (if present) is located downstream of the open reading frame of a protein-coding sequence, such as downstream of a stop codon in a protein-coding region. The 3' UTR is typically the mRNA portion located between the protein-coding sequence and the polyadenylated tail. Therefore, in some respects, the 3' UTR (if present) is upstream of the polyadenylated sequence, such as directly adjacent to it. The 3' UTR can be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.

[0405] The 3' UTR may also contain components not encoded in the template, from which the RNA is translated but added post-transcriptionally during maturation, such as a polyadenylated tail. The 3' UTR of mRNA is not translated into an amino acid sequence. In some respects, the RNAs disclosed herein contain a 3' UTR that includes an F component and / or an I component. In some respects, the 3' UTR or its proximal sequence contains a restriction site. In some respects, the restriction site is a BamHI site. In some respects, the restriction site is an Xhol site.

[0406] In some aspects, RNA molecules and RNA-LNPs contain a 3' untranslated region (3'-UTR). In some aspects, the 3' UTR contains a sequence selected from any one of SEQ ID NO: 103 to SEQ ID NO: 106. In some aspects, the 3' UTR contains a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with any one of SEQ ID NO: 103 to SEQ ID NO: 106. In some aspects, the 3' UTR contains a sequence composed of any one of SEQ ID NO: 103 to SEQ ID NO: 106.

[0407] mRNA may include stem loops, such as (but not limited to) histone stem loops. A stem loop may be a nucleotide sequence of about 25 or about 26 nucleotides in length. The histone stem loop may be located at the 3' end relative to the coding region (e.g., at the 3' end of the coding region). As a non-limiting example, the stem loop may be located at the 3' end of the polynucleotide described herein. In some cases, the mRNA contains more than one stem loop (e.g., two stem loops). The stem loop may be located in the second-terminal region of the polynucleotide. As 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 a histone stem loop may be stabilized by adding a 3'-stabilizing region (e.g., a 3'-stabilizing region containing at least one chain-terminating nucleoside). Without wishing to be theoretically limited, adding at least one chain-terminating nucleoside may slow down the degradation of the polynucleotide and thus increase its half-life. In other cases, mRNAs containing histone stem-loops can be stabilized by modifying the 3'-region of the polynucleotide to prevent and / or inhibit the addition of oligonucleotides (U). In still other cases, mRNAs containing histone stem-loops can be stabilized by adding oligonucleotides terminated with 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 cases, the mRNAs of this disclosure may contain a histone stem-loop, a polyadenylated region, and / or a 5'-cap structure. The histone stem-loop may precede and / or follow the polyadenylated region. The polynucleotide containing the histone stem-loop and polynucleotide region sequence may include the chain-terminating nucleosides described herein. In other cases, the polynucleotides of this disclosure may contain 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 conserved stem-loop region may contain the miR sequence described herein. As a non-limiting example, the stem-loop region may contain a seed sequence of the miR sequence described herein. In another non-limiting example, the stem-loop region may contain a miR-122 seed sequence.

[0408] mRNA may contain at least one histone stem-loop and polyadenylated region or polyadenylated signal. In some cases, the polynucleotide encoding the histone stem-loop and polyadenylated region or polyadenylated signal may encode a pathogen antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and polyadenylated region or polyadenylated signal may encode a therapeutic protein. In some cases, the polynucleotide encoding the histone stem-loop and polyadenylated region or polyadenylated signal may encode a tumor antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and polyadenylated region or polyadenylated signal may encode an allergen antigen or an autoimmune autoantigen.

[0409] 5' cap

[0410] In some embodiments, the RNA molecule described herein includes a 5' cap. In some embodiments, the 5'-cap portion is a natural 5'-cap.

[0411] A “natural 5’-cap” is defined as a cap comprising 7-methylguanosine linked to the 5’ end of an mRNA molecule via a 5’ to 5’ triphosphate bond. In some embodiments, the 5’ cap portion is a 5’-cap analogue. In some embodiments, the 5’ end of the RNA is capped with a modified ribonucleotide of the structure m7G(5’)ppp(5’)N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered post-transcriptionally (e.g., post-transcriptional capping), where “N” is any ribonucleotide. In some embodiments, the 5’ end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction post-transcriptional RNA. In some embodiments, capping is performed after purification of the RNA molecule (e.g., tangential flow filtration). An exemplary enzymatic reaction for capping may involve using a vaccinia virus capping enzyme (VCE) comprising an mRNA triphosphatase, guanylate transferase, and guanine-7-methyltransferase, which catalyzes the N7-monomethylation of the cap 0 structure. The cap 0 structure helps maintain the stability and translational efficiency of the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2'-O-methyltransferase leading to the production of the cap 1 structure (m7Gppp[m2'-O]N), which may further increase translational efficiency. In some embodiments, the RNA molecule may be enzymatically capped at the 5' end using vaccinia guanylate transferase, guanosine triphosphate, and S-adenosyl-L-methionine to produce the cap 0 structure. The reverse 7-methylguanosine cap is added via a 5'-to-5' triphosphate bridge. Alternatively, the cap 1 structure may be produced using a 2'-O-methyltransferase with vaccinia guanylate transferase, wherein the 2'OH group in the penultimate nucleotide, in addition to the cap 0 structure, is methylated. S-adenosyl-L-methionine (SAM) is used as a cofactor for methyltransferases. Non-limiting examples of 5' cap structures are those that, among other things, exhibit enhanced binding of the cap-binding polypeptide, increased half-life, decreased 5' endonuclease sensitivity, and / or reduced 5' decapping compared to synthetic 5' cap structures known in the art (or compared to wild-type, natural, or physiological 5' cap structures). For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can produce a standard 5'-5'-triphosphate bond between the 5'-terminal nucleotide of mRNA and the guanine cap nucleotide, wherein the cap guanine contains N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl group. This structure is referred to as the Cap1 structure. Compared to other 5' cap analog structures known in the art, this cap produces higher translational efficiency and cellular stability, as well as reduced activation of pro-inflammatory cytokines. The cap structure includes (but is not limited to) 7mG(5')ppp(5')N,pN2p(cap 0) and 7mG(5')ppp(5')N1mpNp (cap 1).Cap 0 is an N7-methylguanosine linked to a 5' nucleotide via a 5'-to-5' triphosphate bond, commonly referred to as m7G cap or m7Gppp. In cells, the cap 0 structure assists in providing efficient translation of cap-carrying mRNA. Additional methylation at the 2'O position of the starting nucleotide produces Cap 1, or m7GpppNm-, where Nm represents any nucleotide with 2'O methylation. In some embodiments, the 5' cap comprises a cap analog, for example, a guanine analog. Exemplary guanine analogs include (but are not limited to) inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-dezo-guanosine, 8-sideoxy-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the capped region may comprise a single cap or a series of nucleotides forming a cap. In this embodiment, the length of the capped region can be equal to, at least, at most, or in any of the following 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or between any two, or at least 2, or 10, or fewer nucleotides. In some embodiments, the cap is absent. In some implementations, the first and second operating regions may be equal to any one, at least one, at most one, or between any two of 3 to 40 nucleotides, such as 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 nucleotides in length, and may contain one or more signal sequences and / or restriction sequences in addition to the start codon and / or stop codon.

[0412] In some implementations, the 5' cap is presented as Formula I:

[0413]

[0414] Where R 1 and R 2 Each is independently H or Me, and B 1 and B 2 Each is independently guanine, adenine, or uracil. In some implementations, B... 1 and B 2 R is a naturally occurring base. In some implementations, R... 1 It is methyl and R 2 It is hydrogen. In some implementations, B 1 It is guanine. In some implementations, B 1 It is adenine. In some implementations, B 2 It is adenine. In some implementations, B2 It is uracil. In some implementations, B 2 It is uracil and the molecule contains B 2 At least 5% of the total population of downstream uracil nucleotides are modified or replaced with non-natural nucleotides.

[0415] In some embodiments, the nucleotide immediately downstream of the 5' cap (in the 5'-to-3' direction) contains guanine. In some embodiments, B... 1 It is adenine and B 2 It is uracil. In some implementations, B 1 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 It is hydrogen. In some instances, the RNA does not contain a 5' cap. In some instances, 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 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 For hydrogen; this embodiment corresponds to CleanCap AU and includes B 2 =Uracil, and optionally replace B 2 Downstream uracil nucleotides have been shown to improve RNA functionality in some embodiments. 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 equine encephalitis virus. In some embodiments, the alphavirus is Mliki forest virus.

[0416] In some implementations, the nucleotide immediately downstream of the 5' cap (from 5' to 3') contains guanine, B. 1 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 The uridine nucleotides in the molecule are hydrogen; at least 50% of the total population of uridine nucleotides in the molecule are replaced by N1-methylpseudouridine, and substantially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B... 1 It is adenine, B 2 For uracil, R 1It is methyl and R 2 The uridine nucleotides in the molecule are hydrogen; at least 50% of the total population of uridine nucleotides in the molecule are replaced by methoxyuridine, and substantially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B 1 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 The uridine nucleotides in the molecule are hydrogen; at least 50% of the total population of uridine nucleotides in the molecule are replaced by 5-methyluridine, and substantially all cytosine nucleotides in the molecule are replaced by 5-methylcytosine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B 1 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 The uridine nucleotides in the molecule are hydrogen-rich; approximately 50% of the uridine nucleotides are replaced by 5-methoxyuridine and approximately 50% by N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 The uridine nucleotides in the molecule are hydrogen-rich; approximately 75% of the uridine nucleotides are replaced by 5-methoxyuridine and approximately 25% by N1-methylpseudouridine. In some embodiments, the nucleotide immediately downstream of the 5' cap (5' to 3') contains guanine, B... 1 It is adenine, B 2 For uracil, R 1 It is methyl and R 2 It is hydrogen; almost all uridine nucleotides in the molecule are replaced by about 25% 5-methoxyuridine and about 75% N1-methylpseuuridine.

[0417] In some embodiments, the 5' end cap is 7mG(5')ppp(5')NlmpNp. In some preferred embodiments, the 5' cap comprises:

[0418] .

[0419] In some implementations, the 5' cap contains CLEANCAP® reagent AG (3'OMe), m7(3'OMeG)(5')ppp(5')(2'OMeA)pG for co-transcriptional capping of mRNA.

[0420] .

[0421] In another embodiment, the 5' cap comprises CLEANCAP® AU for self-amplified mRNA, CLEANCAP® reagent AG (3' OMe) for co-transcriptional capping of mRNA, and m7G(5')ppp(5')(2'OMeA)pU.

[0422] .

[0423] D. Open Reading Frame (ORF)

[0424] The 5' and 3' UTRs can be operatively linked to an open reading frame (ORF), which can be a codon sequence capable of being translated into a polypeptide of interest. An open reading frame can be a sequence of several DNA or RNA nucleotide triplets that can be translated into a peptide or protein. An ORF may begin with a start codon at its 5' end and a subsequent region (e.g., a combination of three consecutive nucleotides typically encoding the amino acid methionine (ATG or AUG)), the length of which is typically a multiple of 3 nucleotides. An open reading frame may terminate 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 aspects, an open reading frame may terminate with one, two, three, four, or more stop codons, as known in the art. An open reading frame may be standalone or may be incorporated into a longer nucleic acid sequence, such as in a vector or mRNA. An open reading frame may also be referred to as a "(protein) coding region" or "coding sequence."

[0425] As stated in this article, RNA molecules may contain one (monocistronic), two (bisicstronic), or more (polycistronic) open reading frames.

[0426] This disclosure provides an RNA molecule comprising at least one open reading frame encoding the *E. coli* FimH polypeptide described herein. In some aspects, the RNA molecule comprises at least one open reading frame encoding the *E. coli* FimH protein described herein.

[0427] E. Genes of interest

[0428] The RNA molecules described herein may contain genes of interest. Genes of interest encode polypeptides of interest. Non-limiting examples of polypeptides of interest include, for example, biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, protoplasmic membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane-binding polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting groups, those polypeptides encoded by the human genome that have not yet identified therapeutic indications but are of practical use in the field of research and discovery, or combinations thereof. The sequence of a particular gene of interest can be readily identified by those skilled in the art using public or private databases, such as GENBANK®.

[0429] In some respects, RNA molecules contain the coding region of a gene of interest. In some respects, the gene of interest is or contains an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some respects, the antigenic polypeptide contains one epitope of an antigen. In some respects, the antigenic polypeptide contains multiple distinct epitopes of an antigen. In some respects, antigenic polypeptides containing multiple distinct epitopes of an antigen are multi-epitope. In some respects, antigenic polypeptides include: antigenic polypeptides derived from allergens, viral antigenic polypeptides, bacterial antigenic polypeptides, fungal antigenic polypeptides, parasitic antigenic polypeptides, antigenic polypeptides derived from infectious agents, antigenic polypeptides derived from pathogens, tumor antigenic polypeptides, or autoantigenic polypeptides.

[0430] The term "antigen" means a substance that can be recognized by the immune system (e.g., through the adaptive immune system) and is capable of evoking an antigen-specific immune response (e.g., as part of an adaptive immune response through the formation of antibodies and / or antigen-specific T cells). Antigens may be or may include peptides or proteins that are presented to T cells via the MHC. Antigens may be translation products of provided nucleic acid molecules, such as RNA molecules containing at least one coding sequence as described herein. Furthermore, fragments, variants, and derivatives of antigens (such as peptides or proteins) contain at least one epitope understood as an antigen.

[0431] In some respects, RNA encoding a gene of interest (e.g., an antigen) is expressed in the cells of a subject treated to provide the gene of interest (e.g., an antigen). In some respects, the RNA is temporarily expressed in the cells of the subject. In some respects, the gene of interest (e.g., an antigen) is expressed on the cell surface. In some respects, the gene of interest (e.g., an antigen) is expressed and presented in the context of MHC. In some respects, the gene of interest (e.g., an antigen) is expressed in the extracellular space, such as when the antigen is secreted.

[0432] In some respects, RNA molecules contain coding regions for genes of interest (e.g., antigens). In some respects, RNA molecules contain coding regions for genes of interest (e.g., antigens) derived from pathogens associated with infectious diseases. In some respects, RNA molecules contain coding regions for genes of interest (e.g., antigens) derived from *Escherichia coli* fimbrial antigens (FimH).

[0433] In some aspects, the RNA polynucleotides described herein, or compositions or medical preparations containing them, comprise the nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides comprise a sequence having at least 80% identity with the nucleotide sequences described herein. In some aspects, the RNA polynucleotides comprise a sequence encoding a polypeptide having at least 80% identity with the polypeptide sequence described herein. In some aspects, the RNA polynucleotides described herein, or compositions or medical preparations containing them, are transcribed from a DNA template. In some aspects, the DNA template used for transcribing the RNA polynucleotides described herein comprises a sequence complementary to the RNA polynucleotide. In some aspects, the gene of interest described herein, encoded by the RNA polynucleotides described herein, comprises the nucleotide sequences described herein. In some aspects, the RNA polynucleotide encodes a polypeptide having at least 80% identity with the polypeptide sequence described herein. In some aspects, the polypeptide described herein, encoded by the RNA polynucleotide transcribed from a DNA template, comprises a sequence complementary to the RNA polynucleotide.

[0434] In some respects, the RNA molecule encodes a FimH protein containing a sequence of any one of SEQ ID NO: 1 to 64, 77, 79, 81 or 83 or a fragment or variant thereof.

[0435] In some respects, the RNA molecule encodes a synthetic Escherichia coli FimH protein, or a fragment or variant thereof, from a nucleic acid sequence comprising any one of SEQ ID NO: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 66 to SEQ ID NO: 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 90.

[0436] F. Polyadenylate tail

[0437] In some respects, the RNA molecules disclosed herein contain a polyadenylated nucleotide (poly-A) sequence, as disclosed herein. In some respects, the polyadenylated nucleotide sequence is downstream of the 3' UTR, e.g., adjacent to the 3' UTR. "Polyadenylated nucleotide tail" or "polyadenylated nucleotide sequence" refers to a continuous adenine residue that can be attached to the 3' end of an RNA molecule. The polyadenylated nucleotide sequence is known to those skilled in the art and may follow the 3' UTR in the RNA molecule described herein. The polyadenylated nucleotide tail can increase the half-life of the RNA molecule.

[0438] mRNA may contain a polyadenylated sequence and / or a polyadenylation signal. The polyadenylated sequence may consist entirely or mostly of adenine nucleotides or analogs or derivatives thereof. The polyadenylated sequence may be located at the tail adjacent to the 3' untranslated region of the nucleic acid. During RNA processing, a long chain of adenosine nucleotides (the polyadenylated region) is typically added to the messenger RNA (mRNA) molecule to increase molecular stability. Immediately following transcription, the 3' end of the transcript is cleaved to a free 3'-hydroxyl group. Then, a polyadenylated polymerase adds the adenosine nucleotide chain to the RNA. This process (called polyadenylation) adds a polyadenylated region between 100 and 250 residues in length. The unique length of the polyadenylated region can provide certain advantages for the alternative polynucleotides of this disclosure. Generally, the length of the polyadenylated region of this disclosure is at least 30 nucleotides. In another embodiment, the length of the polyadenylated region is at least 35 nucleotides. 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 cases, the length of the polyadenylated region on the alternative polynucleotide molecule described herein may be 80 nucleotides, 120 nucleotides, or 160 nucleotides. In other cases, the length of the polyadenylated region on the alternative polynucleotide molecule described herein may be 20, 30, 40, 80, 100, 120, 140, or 160 nucleotides. In some cases, the length of the polyadenylated region is designed relative 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 specific feature or region of the alternative polynucleotide (such as mRNA), or the length of the final product expressed by the alternative polynucleotide. The length of the polyadenylated region can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater than any feature of the alternative polynucleotide (e.g., the mRNA portion containing the polyadenylated region). The polyadenylated region can also be designed as part of the alternative polynucleotide to which it belongs. In this context, the polyadenylated region can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the total length of the polyadenylated region.

[0439] In some cases, engineered binding sites and / or mRNA conjugation targeting polyadenylate-binding proteins (PABPs) can be used to enhance expression. The engineered binding site can be a sensor sequence that serves as a ligand binding site for the local microenvironment of the mRNA. As a non-limiting example, the mRNA may contain at least one engineered binding site to alter the binding affinity of PABPs and their analogues. The incorporation of at least one engineered binding site can increase the binding affinity of PABPs and their analogues.

[0440] Additionally, multiple different mRNAs can be linked together at the 3' end of the polyadenylated region (PAB) using alternative nucleotides at the 3' end. Transfection experiments can be performed in relevant cell lines, and protein yield can be analyzed by ELISA at 12, 24, 48, 72 hours, and day 7 post-transfection. As a non-limiting example, transfection experiments can be used to assess the effect of adding at least one engineered binding site on the binding affinity of PABP or its analogues. In some cases, the PAB can be used to regulate translation initiation. While not wishing to be limited by theory, the PAB recruits PABP, which in turn interacts with the translation initiation complex, and can therefore be essential for protein synthesis. In some cases, the PAB in this disclosure can also be used to prevent 3'-5' exonuclease digestion. In some cases, the mRNA may contain a polyadenylated G-quartet. The G-quartet is a cyclic hydrogen-bonded array of four guanosine nucleotides, which can be formed from G-rich sequences in both DNA and RNA. In this embodiment, the G-tetramer is incorporated at the end of the polyadenylated nucleotide region. The stability, protein yield, and other parameters, including half-life, of the resulting mRNA can be analyzed at various time points. It has been found that the polyadenylated nucleotide-G tetramer results in a protein yield equal to at least 75% of the protein yield observed with the 120-nucleotide polyadenylated nucleotide region alone. In some cases, the mRNA may contain a polyadenylated nucleotide region and can be stabilized by adding a 3'-stabilizing region. mRNA with a polyadenylated nucleotide region may further include a 5'-cap structure. In other cases, the mRNA may contain a polyadenylated nucleotide-G tetramer. mRNA with a polyadenylated nucleotide-G tetramer may further include a 5'-cap structure. In some cases, the 3'-stabilizing region used to stabilize the mRNA may contain either a polyadenylated nucleotide region or a polyadenylated nucleotide-G tetramer. In other cases, the 3'-stabilizing region used in this disclosure may contain a chain-terminating nucleoside, such as 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymidine, 2',3'-dideoxynucleosides (such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymidine), 2'-deoxynucleoside, or O-methylnucleoside. In other cases, mRNA containing a polyadenylate region or a polyadenylate-G tetrad may be stabilized by making alterations to the 3'-region of the polynucleotide that prevent and / or inhibit the addition of oligonucleotides (U).In other cases, mRNA containing a polyadenylate region or a polyadenylate-G tetrad can be stabilized by adding an oligonucleotide terminated with 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.

[0441] In one aspect, the RNA disclosed herein comprises a polyadenylated tail containing a sequence having at least, at most, exactly, or between 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 93. In another aspect, the polyadenylated tail comprises the sequence of SEQ ID NO: 93.

[0442] IV. RNA transcription

[0443] In some respects, the RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of this disclosure, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins.

[0444] According to this disclosure, "transcription" includes "in vitro transcription" or "IVT," which refers to the process of transcription occurring in vitro in a non-cellular system to produce synthetic RNA products for various applications, including, for example, the manufacture of proteins or polypeptides. Cloning vectors can be used for transcript production. Cloning vectors are generally referred to as transcription vectors and are encompassed by the term "vector" according to the invention. Depending on a specific aspect, the RNA used is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription from a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid (especially cDNA) and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0445] Synthetic IVT RNA products can be translated in vitro or directly introduced into the cell to which they will be translated. The terms “expression” or “translation,” as opposed to RNA, refer to processing within the ribosomes of the cell, whereby the mRNA chain guides the assembly of amino acid sequences to create peptides or proteins. Such synthetic RNA products include, but are not limited to, mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribosomes, aptamers, guide RNA molecules (e.g., CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, etc. IVT reactions typically utilize the DNA template (e.g., linear DNA template), ribonucleotides (e.g., unmodified or modified ribonucleotide triphosphates), and a suitable RNA polymerase as described and / or utilized herein.

[0446] In some respects, mRNA is produced by transcription in vitro using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In other respects, the RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription using a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acids (especially cDNA) and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0447] In some respects, the starting materials for IVT may include a linear DNA template, nucleotides, RNase inhibitors, pyrophosphate hydrolases, and / or T7 RNA polymerase. In some respects, IVT treatment is carried out in a bioreactor. The bioreactor may include a mixer. In some respects, nucleotides may be added to the bioreactor via IVT treatment.

[0448] In some aspects, one or more post-IVT reagents are added to the IVT mixture containing RNA in a bioreactor after IVT treatment. Exemplary post-IVT reagents may contain DNase I, configured to digest a linearized DNA template, and proteinase K, configured to digest DNase I and T7 RNA polymerase. In some aspects, the post-IVT reagents and the mixture are cultured in a bioreactor after IVT. In some respects, the bioreactor may contain at least, at most, exactly, or between 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 more liters of IVT mixture. IVT mixtures may have RNA concentrations of at least, at most, exactly, or between 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 more.

[0449] In some respects, IVT mixtures may contain residual spermidine, residual DNA, residual protein, peptide, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+, Na+, Ca2+), RNA fragments, residual nucleotides, free phosphates, or any combination thereof.

[0450] In some respects, at least a portion of the IVT mixture is filtered. The IVT mixture can be filtered by ultrafiltration and / or dialysis to remove at least some impurities from the IVT mixture and / or alter the buffer solution of at least a portion of the IVT mixture to produce a concentrated RNA solution as a osmotic residue.

[0451] In some respects, "ultrafiltration" and "dialysis filtration" refer to membrane filtration processes. Ultrafiltration typically uses membranes with pore sizes of at least, at most, exactly, or between 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. In some respects, ultrafiltration membranes are often classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO can be at least, at most, exactly, or between 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 The values ​​range from 100 kDa to any two of 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, and 10000 kDa. Technicians will understand that filter membranes can be constructed from various suitable materials, including, for example, polymers, cellulose, ceramics, etc., depending on the application. In some respects, membrane filtration may be more ideal for large-volume purification processes.

[0452] In some respects, IVT mixtures for ultrafiltration and dialysis filtration of purified RNA may include (1) direct flow filtration (DFF), also known as “cake” filtration, which applies a feed flow perpendicular to the membrane surface and attempts to allow 100% of the fluid to pass through the membrane, and / or (2) tangential flow filtration (TFF), also known as sweep filtration, in which the feed flow passes parallel to the membrane surface, a portion of which passes through the membrane (permeate) while the remainder (residue) is retained and / or recycled back to the feed tank.

[0453] In some aspects, filtration of the IVT mixture is performed via TFF, which includes an ultrafiltration step, a first dialysis filtration step, and a second dialysis filtration step. In some aspects, the first dialysis filtration step is performed in the presence of ammonium sulfate. The first dialysis filtration step can be configured to remove the majority of impurities from the IVT mixture. In some aspects, the second dialysis filtration step is performed without ammonium sulfate. The second dialysis filtration step can be configured to transfer RNA into a DS buffer formulation.

[0454] A suitable filtration membrane with an appropriate MWCO can be selected for ultrafiltration in TFF processing. The MWCO of the TFF membrane determines which solutes can pass through the membrane into the filtrate and which are retained in the osmate. The MWCO of the TFF membrane can be selected such that substantially all solutes of interest (e.g., the desired synthetic RNA material) are retained in the osmate, while undesired components (e.g., excess ribonucleotides, small nucleic acid fragments such as digested or hydrolyzed DNA templates, peptide fragments such as proteins and / or other impurities) pass through into the filtrate. In some aspects, the osmate containing the desired synthetic RNA material can be recycled to the feed vessel for refiltration in the next cycle. In some aspects, the TFF membrane may have an MWCO of at least, at most, exactly, or between any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or higher. In some aspects, the TFF membrane may have a MWCO of at least, at most, exactly, or between 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or higher. In some aspects, the TFF membrane may have an MWCO of about 250 to 350 kDa. In some aspects, the TFF membrane (e.g., a cellulose-based membrane) may have an MWCO of about 30 to 300 kDa; in some aspects, about 50 to 300 kDa, about 100 to 300 kDa, or about 200 to 300 kDa.

[0455] Dialysis filtration can be performed discontinuously or continuously. For example, in continuous dialysis filtration, the dialysis filtration solution can be added to the sample feed container at the same rate as the filtrate production. In this way, the volume in the sample container remains constant, but small molecules that can freely permeate across the membrane (e.g., salts, solvents, etc.) are removed. Using solvent removal as an example, each increase in dialysis filtration volume (DV) further reduces the solvent concentration. In discontinuous dialysis filtration, the solution is first diluted and then concentrated back to the initial volume. This process is then repeated until the remaining small molecules (e.g., salts, solvents, etc.) in the container reach the desired concentration. Each increase in dialysis filtration volume (DV) further reduces the concentration of small molecules (e.g., solvents). Continuous dialysis filtration typically requires a minimum volume for a given reduction in the number of molecules to be filtered. In other words, discontinuous dialysis filtration allows for rapid changes in osmotic conditions, such as pH, salt content, etc. In some aspects, the first dialysis filtration step is performed with a dialysis volume equal to or greater than any two of 2, 3, 4, 5, 6, 7, 8, 9, or 10 dialysis volumes. In some aspects, the second dialysis filtration step is performed with a dialysis volume equal to or greater than any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dialysis volumes. In some aspects, the first dialysis filtration step is performed with 5 dialysis volumes, and the second dialysis filtration step is performed with 10 dialysis volumes.

[0456] In some respects, for ultrafiltration and / or dialysis filtration, the IVT mixture is filtered at a rate equal to or higher than any two of the following: at least, at most, exactly, or between 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. Concentrated RNA solutions may contain at least, at most, exactly, or between 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg / mL of single-stranded RNA.

[0457] In some respects, the bioburden of the concentrated RNA solution obtained by filtration can also be reduced. Filtration for reducing bioburden can be performed using one or more filters. The one or more filters may contain pore sizes of at least, at most, exactly, 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.

[0458] As an example, reducing bioburden may include emptying the urate tank containing urate obtained from ultrafiltration and / or dialysis filtration to obtain urate. Reducing bioburden may include flushing the ultrafiltration and / or dialysis filtration system with a wash buffer solution to obtain a wash tank solution containing residual RNA remaining in the filtration system. The urate may be filtered to obtain filtered urate. The wash tank solution may be filtered using a first 0.2 µm filter to obtain a filtered wash tank solution. The urate may be filtered using either a first 0.2 µm filter or another 0.2 µm filter.

[0459] The filtered wash pool solution and the filtered effluent can be combined to form a combined pool solution. The combined pool solution can be filtered using a second 0.2 µm filter to obtain a filtered combined pool solution, which is further filtered using a third 0.2 µm filter to produce an RNA product solution.

[0460] V. RNA encapsulation

[0461] RNA in an RNA product solution can be encapsulated, and the RNA solution may further contain at least one encapsulation agent. In one aspect, the encapsulation agent comprises lipids, lipid nanoparticles (LNPs), lipid complexes, polymer particles, polymeric complexes, and monolithic delivery systems, as well as combinations thereof.

[0462] Lipid nanoparticles may contain a lipid component and one or more additional components, such as therapeutics and / or prophylactic agents. LNPs may be designed for one or more specific applications or targets. The components of an LNP may be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, specific formulations of an LNP may be selected for a specific application or target based on, for example, the efficacy and toxicity of a specific combination of components. The efficacy and tolerability of an LNP formulation may be affected by the stability of the formulation.

[0463] Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and / or preventative agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof within a mammal.

[0464] The physicochemical properties of lipid nanoparticles can be altered to increase selectivity for specific bodily targets. For example, particle size can be adjusted based on the fenestration size of different organs. Therapeutic and / or prophylactic agents contained in the LNP can also be selected based on desired delivery targets or multiple targets. For example, therapeutic and / or prophylactic agents can be selected for specific signs, conditions, diseases, or ailments and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In some embodiments, the LNP may contain mRNA encoding a polypeptide of interest that can be translated intracellularly to produce a polypeptide of interest. This composition can be programmed for specific delivery to a specific organ. In some embodiments, the composition can be programmed for specific delivery to a mammalian liver. In some embodiments, the composition can be programmed for specific delivery to a lymph node. In some embodiments, the composition can be programmed for specific delivery to a mammalian spleen.

[0465] In one aspect, the encapsulating agent is a lipid, and the resulting RNA is encapsulated in lipid nanoparticles (LNPs). Unbound by any particular theory, it is believed that cationic or cationically ionizable lipids or lipid-like materials and / or cationic polymers are incorporated with nucleic acids to form aggregates, and this aggregation results in colloidally stable particles. The lipids can be naturally occurring or synthetic. However, lipids are generally biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, glycerol phosphates, steroids, terpenes, lysolipids, glycosphingolipids, glucose lipolipids, sulfatids, lipids of fatty acids with ether and ester linkages, and polymerizable lipids, as well as combinations thereof. Lipids are water-insoluble substances and can be extracted with organic solvents. Compounds other than those specifically described herein are understood by those skilled in the art to be lipids and are encompassed by the compositions and methods of this disclosure. Lipid components and non-lipids can be covalently or non-covalently attached to each other.

[0466] In some respects, LNPs can be designed to protect RNA molecules (e.g., mRNA) from extracellular RNases and / or can be engineered for systemic delivery of RNA to target cells. In some respects, such LNPs may be particularly useful for delivering RNA molecules (e.g., mRNA, modRNA) when the RNA molecule is administered intravenously to a subject in need. In some respects, such LNPs may be particularly useful for delivering RNA molecules (e.g., mRNA) when the RNA molecule is administered intramuscularly to a subject in need.

[0467] In one aspect, the concentration of RNA in the RNA solution is <1 mg / mL. In another aspect, the concentration of RNA is at least about 0.05 mg / mL. In another aspect, the concentration of RNA is at least about 0.5 mg / mL. In another aspect, the concentration of RNA is at least about 1 mg / mL. In another aspect, the concentration of RNA is about 0.05 mg / mL to about 0.5 mg / mL. In another aspect, the concentration of RNA is at least 10 mg / mL. In another aspect, the concentration of RNA is at least 50 mg / mL. In some aspects, the concentration of RNA is at least, at most, exactly, or between any two of about 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, or more.

[0468] This disclosure provides RNA solutions and lipid preparation mixtures or compositions thereof, comprising, encapsulating therein and / or formulating with one or more lipids to form lipid nanoparticles (LNPs), liposomes, lipid complexes and / or nanoliposomes, encoding at least one RNA such as an antigen (e.g., E. coli FimH protein). In some aspects, the composition comprises lipid nanoparticles.

[0469] Lipid nanoparticles, or LNPs, refer to any form of particle produced when cationic lipids are combined with, optionally, one or more other lipids, such as in an aqueous environment and / or in the presence of RNA. In some aspects, lipid nanoparticles are contained in formulations that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA, modRNA), to target sites of interest (e.g., cells, tissues, organs, tumors, etc.). In some aspects, the lipid nanoparticles of this disclosure contain nucleic acids. Such lipid nanoparticles typically comprise cationic lipids and one or more excipients, such as one or more neutral lipids, charged lipids, steroids, polymer-conjugated lipids, or combinations thereof. In some aspects, active agents or therapeutic agents, such as nucleic acids (e.g., mRNA, modRNA), may be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space surrounded by some or all of the lipid portions of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms (e.g., adverse immune responses). Nucleic acids (e.g., mRNA, modRNA) or portions thereof may also be associated with or complexed with lipid nanoparticles. Lipid nanoparticles may contain any lipid capable of forming particles, to which nucleic acids may be attached, or in which one or more nucleic acids may be encapsulated.

[0470] In some respects, the provided RNA molecules (e.g., mRNA, modRNA) can be prepared together with LNPs. In other respects, the average diameter of the lipid nanoparticles can be from about 1 to 500 nm. In some aspects, the average diameter of the lipid nanoparticles is 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, about 70 nm to about 80 nm, or at least, at most, exactly or between 30 nm, 35 nm, 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. It must be between two values ​​in nm and be substantially non-toxic. The term "average diameter" refers to the average hydrodynamic diameter of a particle as measured by dynamic laser scattering (DLS) and data analysis using the so-called cumulant algorithm, which provides a so-called Z-mean with a length dimension and a dimensionless polydispersity index (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the particle's "average diameter," "diameter," "size," or "average size" are used synonymously with the value of the Z-mean.

[0471] The LNP described in this article can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or smaller. For example, LNP can represent a polydispersity index that is at least, at most, exactly, or between any two of 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. In some respects, the polydispersity index is based on dynamic light scattering measurements calculated by so-called cumulative analysis, as mentioned in the definition of "mean diameter". Under certain prerequisites, it can be regarded as a measurement of the overall size distribution of nanoparticles.

[0472] Lipid nanoparticles (LNPs) can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of LNPs. The zeta potential can be measured using dynamic light scattering or potentiometry (e.g., potentiometric titration). Particle size can also be determined using dynamic light scattering. Multiple properties of LNPs, such as particle size, polydispersity index, and zeta potential, can be measured using instruments such as the Zetasizer Nano ZS (Malvin Instruments Ltd., Malvin, Worcestershire, UK).

[0473] The average size of an LNP can range from tens to hundreds of nm, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 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 can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average size of the LNP can be about 70 nm to about 100 nm. In a particular embodiment, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0474] LNPs can be relatively homogeneous. A polydispersity index can be used to indicate the isomorphism of the LNP, such as the particle size distribution of lipid nanoparticles. Small polydispersity indices (e.g., less than 0.3) typically indicate a narrow particle size distribution. LNPs can have polydispersity indices from about 0 to about 0.25, such as 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 the LNP can be from about 0.10 to about 0.20.

[0475] The zeta potential of LNPs can be used to indicate the electrokinetic potential of a composition. For example, the zeta potential can describe the surface charge of the LNP. Lipid nanoparticles with relatively low charges (positive or negative) are generally desirable because substances with higher charges may engage in undesirable interactions with cells, tissues, and other components in the body. In some implementations, the zeta potential of the LNP may be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0476] In some aspects, when nucleic acids (e.g., RNA molecules) are present in the provided LNP, they resist degradation by nucleases in aqueous solution. In some aspects, the LNP is a lipid nanoparticle targeted at the liver. In some aspects, the LNP is a cationic lipid nanoparticle comprising one or more cationic lipids (e.g., those described herein). In some aspects, the cationic LNP may comprise at least one cationic lipid, at least one polymerically conjugated lipid, and at least one accessory lipid (e.g., at least one neutral lipid).

[0477] In some aspects, the RNA solution and lipid preparation mixture or composition thereof may have, have at least, or have at least, at most, exactly or between about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about The percentages of 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 any two of these 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 described herein or known to those skilled in the art.

[0478] The LNPs described herein can be prepared using a wide range of methods, which may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer, and mixing the colloid with nucleic acids to obtain nucleic acid particles. As used herein, the term "colloid" refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be referred to as a colloid or a colloidal suspension. Sometimes the term "colloid" refers only to the particles in the mixture and not to the entire suspension.

[0479] To prepare colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer used herein, this document applies methods conventionally used for the preparation of liposome vesicles, with appropriate modifications. The most commonly used methods for preparing liposome vesicles share the following basic stages: (i) dissolving the lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media). In membrane hydration, the lipids are first dissolved in a suitable organic solvent and dried at the bottom of a flask to produce a thin film. The resulting lipid film is hydrated using a suitable aqueous media to produce a liposome dispersion. Additional size reduction steps may also be included.

[0480] Reverse-phase evaporation is an alternative method for membrane hydration in the preparation of liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. The mixture is then subjected to simple ultrasonic agitation to homogenize the system. Removal of the organic phase under reduced pressure produces an emulsion gel, which subsequently becomes a liposome suspension.

[0481] The term "ethanol injection technique" refers to a 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 vesicles, and liposomes. Generally, the RNA-lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, in some aspects, such a colloidal liposome dispersion is formed as follows: an ethanol solution containing lipids (such as cationic lipids and other lipids) is injected into an aqueous solution under stirring. In some aspects, the RNA-lipid complex particles described herein can be obtained without an extrusion step.

[0482] The term "extrusion" or "extruded" refers to the production of particles with a fixed cross-sectional profile. Specifically, it means reducing the particle size, thereby forcing the particles through a filter with defined pores.

[0483] Other methods with organic solvent-free properties can also be used to prepare colloids according to this disclosure.

[0484] In some aspects, LNP-encapsulated RNA can be produced by rapidly mixing the RNA solution (e.g., RNA product solution) described herein and the lipid formulation described herein (containing, in an organic solvent, at least one cationic lipid and optionally one or more other lipid components) under conditions that induce a rapid change in the solubility of the lipid components (which drives the lipids to self-assemble into LNP form). In some aspects, suitable buffers comprise tris(hydroxymethyl)aminomethane (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 acidification buffer may be at least half a pH scale smaller 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 larger than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, the properties of the cationic lipid are chosen such that the nascent form of the particle occurs through association with a nucleic acid backbone (e.g., RNA) with an opposite charge. In this way, particles are formed around the nucleic acid, which, for example, in some respects, leads to a higher encapsulation efficiency than that achieved when there is no interaction between the nucleic acid and at least one lipid component.

[0485] The encapsulation efficiency of the therapeutic and / or preventative agents describes the amount of therapeutic and / or preventative agents that are encapsulated or otherwise associated with LNPs after preparation, relative to the initially provided amount. Encapsulation efficiency is expected to be high (e.g., close to 100%). For example, encapsulation efficiency can be measured by comparing the amount of therapeutic and / or preventative agents in a solution containing lipid nanoparticles before and after the lipid nanoparticles are disrupted with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or preventative agents (e.g., RNA) in solution. For the lipid nanoparticles described herein, the encapsulation efficiency of the therapeutic and / or preventative 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 encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%.

[0486] LNPs may optionally contain one or more coatings. For example, LNPs may be formulated into capsules, films, or coated tablets. Capsules, films, or tablets containing the compositions described herein may have any available size, tensile strength, hardness, or density.

[0487] Formulations comprising amphiphilic polymers and lipid nanoparticles can be formulated, wholly or partially, into pharmaceutical compositions. Pharmaceutical compositions 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 (which may comprise one or more different therapeutic and / or preventative agents). Pharmaceutical compositions may further comprise one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein. General guidance for the formulation and manufacture of pharmaceutical compositions and reagents is available, for example, in Remington's *The Science and Practice of Pharmacy*, 21st Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjuvants may be used in any pharmaceutical composition unless any conventional excipient or adjuvant is incompatible with one or more components of the LNP or one or more amphiphilic polymers in the formulation of this disclosure. If the excipient or auxiliary component binds to the component or amphiphilic polymer and causes any undesirable biological effects or other harmful effects, it may be incompatible with the component or amphiphilic polymer of the formulation's LNP.

[0488] In some embodiments, one or more excipients or adjuvants may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising LNP. For example, one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90%, or higher as is commonly found in pharmaceuticals. In some embodiments, the pharmaceutically acceptable purity of the excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipient is approved for human or veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient conforms to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmaceutical Committee. The relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients, and / or any additives in the pharmaceutical composition according to this disclosure may vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route of administration of the composition to be administered. For example, a pharmaceutical composition may contain one or more lipid nanoparticles in amounts between 0.1% and 100% (wt wt). As another example, a pharmaceutical composition may contain one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / v) in amounts between 0.1% and 15% (wt / vol).

[0489] In some embodiments, the lipid nanoparticles and / or pharmaceutical compositions of this disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at temperatures of 4°C or lower, such as between about -150°C and about 0°C, or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C). For example, pharmaceutical compositions comprising one or more amphiphilic polymers and one or more lipid nanoparticles are in the form of a solution or a solid (e.g., via freeze-drying) and are refrigerated for use. For storage and / or transportation, for example, at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In some embodiments, this disclosure also relates to methods for increasing the stability of lipid nanoparticles by adding an effective amount of an amphiphilic polymer and by storing the lipid nanoparticles and / or their pharmaceutical compositions at temperatures of 4°C or lower (such as temperatures between about -150°C and about 0°C or between about -80°C and about -20°C, e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C).

[0490] The chemical properties of the LNPs, LNP suspensions, freeze-dried LNP compositions, or LNP formulations disclosed herein can be characterized by a variety of methods. In some embodiments, mRNA integrity can be examined using electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reverse phase liquid chromatography).

[0491] In some embodiments, the LNP, LNP suspension, freeze-dried LNP composition or LNP formulation of this disclosure has an LNP integrity of about 20% or higher, about 25% or higher, about 30% or higher, about 35% or higher, about 40% or higher, about 45% or higher, about 50% or higher, about 55% or higher, about 60% or higher, about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher or about 99% or higher.

[0492] In some embodiments, the LNP integrity of the LNP, LNP suspension, freeze-dried LNP composition, or LNP formulation of this disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or higher than that of LNP, LNP suspension, freeze-dried LNP composition, or LNP formulation manufactured by equivalent methods. The latter is approximately 1 or more, approximately 2 or more, approximately 3 or more, approximately 4 or more, approximately 5 or more, approximately 10 or more, approximately 20 or more, approximately 30 or more, approximately 40 or more, approximately 50 or more, approximately 100 or more, approximately 200 or more, approximately 300 or more, approximately 400 or more, approximately 500 or more, approximately 1000 or more, approximately 2000 or more, approximately 3000 or more, approximately 4000 or more, approximately 5000 or more, or approximately 10000 or more.

[0493] In some embodiments, the Txo% of the LNP, LNP suspension, freeze-dried LNP composition or LNP formulation disclosed herein is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, or about 120 months or longer.

[0494] In some embodiments, the Txo% of the LNP, LNP suspension, freeze-dried LNP composition or LNP formulation of this disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, and the former is about 1 time or more, about 2 times or more, about 3 times or more, about 4 times or more, or about 5 times or more of the latter.

[0495] In some embodiments, the T1 / 2 of the LNP, LNP suspension, freeze-dried LNP composition or LNP formulation disclosed herein is about 12 months or longer, about 15 months or longer, about 18 months or longer, about 21 months or longer, about 24 months or longer, about 27 months or longer, about 30 months or longer, about 33 months or longer, about 36 months or longer, about 48 months or longer, about 60 months or longer, about 72 months or longer, about 84 months or longer, about 96 months or longer, about 108 months or longer, or about 120 months or longer.

[0496] In some embodiments, the T1 / 2 of the LNP, LNP suspension, freeze-dried LNP composition or LNP formulation of this disclosure is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, and the former is about 1 time or more, about 2 times or more, about 3 times or more, about 4 times or more, or about 5 times or more than the latter.

[0497] As used herein, “Tx” means the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of the LNP, LNP suspension, freeze-dried LNP composition, or LNP formulation to degrade to approximately X times the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, freeze-dried LNP composition, or LNP formulation. For example, “T80%” means the amount of time it takes for the nucleic acid integrity (e.g., mRNA integrity) of the LNP, LNP suspension, freeze-dried LNP composition, or LNP formulation to degrade to approximately 80% of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, freeze-dried LNP composition, or LNP formulation. In another example, "T1 / 2" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of the LNP, LNP suspension, freeze-dried LNP composition, or LNP preparation degrades to approximately half of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, freeze-dried LNP composition, or LNP preparation.

[0498] In some respects, when nucleic acids are present in lipid nanoparticles, they resist degradation by nucleases in aqueous solutions. The lipid nanoparticles contain nucleic acids, and methods for their preparation are disclosed in, for example, U.S. Patent Publications 2004 / 0142025, 2007 / 0042031 and PCT Publications WO 2013 / 016058 and WO 2013 / 086373, the entire disclosures of which are incorporated herein by reference for all purposes.

[0499] Some aspects described herein relate to compositions, methods, and uses involving more than one, such as two, three, four, five, six, or even more nucleic acid substances, such as RNA substances. In LNP formulations, various nucleic acid substances can be separately formulated as individual LNP formulations. In this case, each individual LNP formulation will contain one nucleic acid substance. Individual LNP formulations can exist as independent entities, such as in independent containers. Such formulations can be obtained by independently providing each nucleic acid substance (usually in the form of a nucleic acid-containing solution) along with a suitable cationic or cationic ionizable lipid or lipid-like material and a cationic polymer that allows LNP formation. The corresponding particles will contain only the specific nucleic acid substance provided when the particles are formed (individual particle formulations).

[0500] In some aspects, a pharmaceutical composition may contain more than one individual LNP formulation. The corresponding pharmaceutical composition is referred to as a mixed LNP formulation. A mixed LNP formulation according to the invention can be prepared by separately forming the individual LNP formulations as described above, followed by mixing the individual LNP formulations. Through the mixing step, a formulation containing a mixed population of LNPs containing nucleic acids can be obtained. The individual LNP populations may be contained together in a single container, representing a mixed population of individual LNP formulations.

[0501] Alternatively, different nucleic acid substances can be formulated together as a combined LNP formulation. Such formulations can be obtained by providing a combined formulation (usually a combined solution) of different RNA substances along with a suitable cationic or cationically ionizable lipid or lipid-like material and a cationic polymer that allows LNP formation. In contrast to mixed LNP formulations, combined LNP formulations typically contain LNPs containing more than one RNA substance. In combined LNP compositions, the different RNA substances are usually present together in a single particle.

[0502] The lipid component of an LNP may include, for example, cationic lipids, phospholipids (such as unsaturated lipids, e.g., DOPE or DSPC), PEG lipids, and structural lipids. The components of the lipid component may be provided in specific proportions.

[0503] In some embodiments, the LNP further comprises phospholipids, PEG lipids, structural lipids, or any combination thereof. Suitable phospholipids, PEG lipids, and structural lipids for the methods of this disclosure are further disclosed herein.

[0504] In some embodiments, the lipid component of the LNP comprises cationic lipids, phospholipids, PEG lipids, and structural lipids. In some embodiments, the lipid component of the lipid nanoparticles comprises about 30 mol% to about 60 mol% cationic lipids, about 0 mol% to about 30 mol% phospholipids, about 18.5 mol% to about 48.5 mol% structural lipids, and about 0 mol% to about 10 mol% PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the lipid nanoparticles comprises about 35 mol% to about 55 mol% cationic lipids, about 5 mol% to about 25 mol% phospholipids, about 30 mol% to about 40 mol% structural lipids, and about 0 mol% to about 10 mol% PEG lipids. In a particular embodiment, the lipid component comprises about 50 mol% of the cationic lipid, about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In another particular embodiment, the lipid component comprises about 40 mol% of the cationic lipid, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In other embodiments, the PEG lipids may be PEG-DMG and / or the structural lipids may be cholesterol.

[0505] The amount of therapeutic and / or preventative agents in the LNP depends on the size, composition, desired target and / or application, or on the nature of the lipid nanoparticles and the nature of the therapeutic and / or preventative agents. For example, the amount of RNA available in the LNP may depend on the size, sequence, and other properties of the RNA. The relative amounts of therapeutic and / or preventative agents (e.g., pharmaceutical substances) and other components (e.g., lipids) in the LNP may also differ. In some embodiments, the wt / wt ratio of the lipid component to the therapeutic and / or preventative agent in the LNP may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic and / or preventative agent can be from about 10:1 to about 40:1. In some embodiments, this wt / wt ratio is about 20:1. For example, the amount of the therapeutic and / or preventative agent in the LNP can be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0506] A. Cationic polymer materials

[0507] Due to their high chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Generally, cationic materials are used to electrostatically aggregate negatively charged nucleic acids into nanoparticles. These positively charged groups are typically composed of amines, which alter the protonation state in a pH range between 5.5 and 7.5, and are thought to cause ionic imbalance, leading to endosome disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have been used for nucleic acid delivery and are suitable as cationic materials in some aspects of this paper. Furthermore, some researchers have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have found widespread use in nucleic acid delivery due to their ease of synthesis and biodegradability. In some respects, such synthetic materials may be suitable as cationic materials in this paper.

[0508] As used herein, “polymer material” has its general meaning, such as a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some respects, these repeating units may be all identical; or, in some examples, more than one type of repeating unit may be present in the polymer material. In some examples, the polymer material is biologically derived, such as a biopolymer like a protein. In some cases, additional groups, such as targeting groups, may also be present in the polymer material, as described herein.

[0509] Those skilled in the art will understand that when a polymer (or polymer portion) contains more than one type of repeating unit, the polymer (or polymer portion) is referred to as a "polymer". In some aspects, the polymer (or polymer portion) utilized according to this disclosure may be a copolymer. The repeating units forming the copolymer may be arranged in any manner. For example, in some aspects, the repeating units may be arranged in a random order; alternatively or additionally, in some aspects, the repeating units may be arranged in an alternating order, or as a "block" copolymer, such as comprising one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block), etc. Block copolymers may have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0510] In some aspects, the polymeric materials used according to this disclosure are biocompatible. Biocompatible materials are those that generally do not cause significant cell death at moderate concentrations. In some aspects, the biocompatible materials are biodegradable, e.g., capable of chemically and / or biologically degrading within physiological environments (e.g., in vivo). In some aspects, the polymeric material may be or contains protamine or polyeneimide, particularly protamine.

[0511] Those skilled in the art will understand that the term "protamine" is generally used to refer to any of a variety of strongly basic proteins with relatively low molecular weight, rich in arginine and found to be particularly associated with DNA, replacing somatic histones in the sperm cells of various animals (such as fish). Specifically, the term "protamine" generally refers to proteins found in fish sperm that are strongly basic, water-soluble, do not coagulate upon heat, and primarily produce arginine upon hydrolysis. In purified forms, they are used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0512] In some respects, the term “protamine” as used herein means a protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and / or a multimeric form of such amino acid sequence or fragments thereof, as well as an artificial (synthetic) polypeptide specifically designed for a particular purpose and which cannot be isolated from a native or biological source.

[0513] In some respects, polyeneimides comprise polyacetylimide and / or polyacrylamide. In some respects, polyeneimides are polyethyleneimine (PEI). In some respects, polyeneimides are linear polyeneimides, such as linear polyethyleneimine (PEI).

[0514] The cationic materials (e.g., polymeric materials, including polycationic polymers) considered in this document comprise cationic materials capable of electrostatically binding nucleic acids. In some aspects, the cationic polymeric materials considered in this document comprise any cationic polymeric material capable of associating with nucleic acids, such as by forming complexes with nucleic acids or forming vesicles therein to encapsulate or embed nucleic acids.

[0515] In some respects, the particles described herein may comprise polymers other than cationic polymers, such as non-cationic polymer materials and / or anionic polymer materials. Generally, anionic and neutral polymer materials are referred to herein as non-cationic polymer materials.

[0516] B. Lipids and lipid-like materials

[0517] As used herein, the terms "lipid" and "lipid-like material" refer to molecules comprising one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. According to this disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist as uncharged or neutral zwitterionic forms at selected pH values.

[0518] The term "lipid" refers to a group of an organic compound that is insoluble in water but soluble in many organic solvents. Generally, lipids can be classified into eight categories: fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, sterol lipids, and sterol-containing metabolites such as cholesterol and prenol lipids. Examples of fatty acids include (but are not limited to) fatty acid esters and fatty acid amides. Examples of glycerolipids include (but are not limited to) glycosylglycerol and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include (but are not limited to) ceramides, sphingomyelins (e.g., sphingomyelin, phosphocholine), and glycosphingomyelins (e.g., cerebrosides, gangliosides). Examples of sterol lipids include (but are not limited to) cholesterol and its derivatives and tocopherols and their derivatives.

[0519] The terms "lipid-like materials," "lipid-like compounds," or "lipid-like molecules" refer to substances that are structurally and / or functionally related to lipids but may not be considered lipids in a narrow sense. For example, the term includes compounds capable of forming amphiphilic layers when present in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic portions. Generally, the term refers to molecules containing both hydrophilic and hydrophobic portions, having a different structural organization that may or may not resemble lipids.

[0520] In some aspects, the RNA solution and lipid preparation mixture or a combination thereof may contain cationic lipids, neutral lipids, cholesterol, and / or polymer (e.g., polyethylene glycol) conjugated lipids, forming lipid nanoparticles encapsulating RNA molecules. Therefore, in some aspects, LNPs may contain cationic lipids and one or more excipients, such as one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer conjugated lipids (e.g., PEG-lipids), or combinations thereof. In some aspects, LNPs encapsulate or encapsulate nucleic acid molecules.

[0521] i. Cationic lipids

[0522] Cationic or cationically ionizable lipids or lipid-like materials refer to lipids or lipid-like materials that are capable of carrying a positive charge and electrostatically binding to nucleic acids. As used herein, “cationic lipid” or “cationic lipid-like material” means a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety such as a sterol, an acyl chain, a diacyl or polyacyl chain, and a head group that typically carries a positive charge. Exemplary cationic lipids contain one or more positively charged amino groups. Cationic lipids may encapsulate negatively charged RNA.

[0523] In some respects, cationic lipids are ionizable, allowing them to exist in a positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. Without wishing to be bound by theory, this ionization behavior is thought to enhance efficacy by facilitating endosome escape and reducing toxicity compared to particles that remain in a cationic state at physiological pH. For the purposes of this disclosure, such “cationically ionizable” lipids or lipid-like materials are included in the terms “cationic lipid” or “cationically ionizable lipid material” unless otherwise specified.

[0524] In some aspects, cationic lipids may constitute about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids in the particles. In some aspects, cationic lipids may constitute at least, at most, exactly, or between any two of 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol% of the total lipids in the particles, or any range or value that is thus available.

[0525] Examples of cationic lipids include (but are not limited to): ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-bis-O-octadecyl-3-trimethylammonium propane (DOTMA), 3-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyl bis(octadecyl)ammonium bromide (DDAB); 1,2-dioleoyl-3-dimethylaminopropane (DODAP); 1,2-diacyloxy-3-dimethylaminopropane; 1,2-dialkoxy-3 -Dimethylaminopropane; dimethyl di(octadecyl)ammonium chloride (DODAC), 1,2-distearate-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazonium (DMRIE), 1,2-dilauryl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-dilauryl-3-trimethylaminopropane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-propylaminopropane trifluoroacetate (DOSPA), 1,2-di... Linolenic acid-N,N-dimethylaminopropane (DLinDMA), 1,2-di-linolenic acid-N,N-dimethylaminopropane (DLenDMA), 1,2-di-linolenic acid-N,N-dimethylaminopropane (DOGS), 3-dimethylamino-2-(cholesterol-5-en-3-β-oxybuten-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesterol-5-en-3-β-oxy)-3'-oxopentoxy]-3-dimethyl-1-(cis-9,12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N, N'-Dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinolenic acid-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinolenic acid-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinolenic acid-3-dimethylaminopropane (DLinCDAP), 2,2-dilinolenic acid-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinolenic acid-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinolenic acid-4-(2-dimethylaminoethyl)-[1,3]-Dioxolane (DLin-KC2-DMA), heptadec-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecoxy)-1-propylaminonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenoyl)-1-propylaminonium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecoxy)-1-propylaminonium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- Dimethyl-2,3-bis(tetradecoxy)-1-propylaminen bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecoxy)-1-propylaminen bromide (bAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propylaminen (DOBAQ), 2-({8-[(3b)-cholesterol-5-en-3-oxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9 [1,2-diene-1-oxy]propylamine (Octyl-CLinDMA), 1,2-dilauryl-3-dimethylaminopropane (DMDAP), 1,2-dipalmitoyl-3-dimethylaminopropane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxymethylamino)ethyl]-3,4-di[oleoyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (D OEPC), 2,3-bis(dodecoxy)-N-(2-hydroxyethyl)-N,N-dimethylpropylamine onium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecoxy)propylamine onium bromide (DMORIE), di((Z)-nonen-2-yl)8,8'-((((2-(dimethylamino)ethyl)thio)carbonyl)amino)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecoxy)propylamine (DLDMA), N,N-dimethyl-2,3-Di(tetradecoxy)propylamine (DMDMA), di((Z)-nonen-2-yl)-9-((4-(dimethylaminobutyryloxy)heptadecanoic acid ester (L319), N-dodecano-3-((2-dodecanoic acid)-{2-[(2-dodecanoic acid)-2-{(2-dodecanoic acid)-[2-(2-dodecanoic acid)-ethyl]-amino}-ethylamino)acrylamide (lipid 98N12-5), 1-[2-[bis(2-hydroxydodecanoic acid)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecanoic acid)amino]ethyl]piperazine-1-yl]ethyl]amino]dodec-2-ol (lipid 02-200); or heptadecano-9-yl 8-((2-hydroxyethyl) (6-Oxy-6-(Undecoxy)hexyl)amino)octacarbonate (SM-102). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cationic lipids may be excluded from the LNP of this disclosure.

[0526] In some respects, the ionizable cationic lipids disclosed herein include compounds of formula (I):

[0527]

[0528] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0529] R1 is C 5-30 Alkyl, C 5-20 alkenyl, -R YR”, -YR” or -R”M'R’;

[0530] R2 and R3 can be H and C independently. 1-14 Alkyl, C 2-14 alkenyl, -R YR", -YR", or -R OR", and / or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle;

[0531] R4 is C 3-6 Carbon ring, -(CH2) n Q、-(CH2) n CHQR, -CHQR, -CQ(R)2 or unsubstituted C 1-6 Alkyl group, where Q is a carbocyclic ring, heterocyclic ring, -OR-, or O(CH2). nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR or C(R)N(R)2C(O)OR, and / or each n can be independently 1, 2, 3, 4 or 5;

[0532] Each R5 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0533] Each R6 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0534] M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl group or heteroaryl group;

[0535] R7 is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0536] R8 is C 3-6 Carbon rings or heterocycles;

[0537] R9 represents H, CN, NO2, and C. 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 alkenyl, C 3-6 Carbon rings or heterocycles;

[0538] Each R is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0539] Each R' is C 1-18 Alkyl, C 2-18 alkenyl, -R YR", -YR", or H;

[0540] Each R” is C 3-14 Alkyl or C 3-14 alkenyl;

[0541] Each R Can be independently C 1-12 Alkyl or C 2-12 alkenyl;

[0542] Each Y can be independently represented by C. 3-6 Carbon rings;

[0543] Each X can be independently F, Cl, Br, or I; and

[0544] m can be 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0545] In some respects, a subset of compounds of formula (I) includes those where R4 is -(CH2). n Q、-(CH2) n When n is CHQR, -CHQR, or -CQ(R)2, then (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) when n is 1 or 2, Q is not a 5, 6, or 7-membered heterocyclic alkyl group.

[0546] In some respects, another subset of the compounds of formula (I) includes those where R1 is C 5-30 Alkyl, C 5-20 alkenyl, -R YR", -YR, or

[0547] -R”M'R';

[0548] R2 and R3 can be H and C independently. 1-14 Alkyl, C 2-14 alkenyl, -R YR", -YR", or -R OR", and / or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle;

[0549] R4 is C 3-6 Carbon ring, -(CH2) n Q、-(CH2) n CHQR, -CHQR,

[0550] -CQ(R)2 or unsubstituted C 1-6 Alkyl group, wherein Q is C 3-6A carbocyclic ring, a 5- to 14-membered heteroaryl group having one or more heteroatoms including N, O, or S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR or a 5 to 14-membered heterocyclic alkyl group having one or more heteroatoms including N, O and S, having a side oxygen group (=O), OH, amino, mono- or dialkyl amino or C 1-3 One or more substituents of the alkyl group are substituted and / or each n can be 1, 2, 3, 4 or 5 independently;

[0551] Each R5 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0552] Each R6 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0553] M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl group or heteroaryl group;

[0554] R7 is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0555] R8 is C 3-6 Carbon rings or heterocycles;

[0556] R9 represents H, CN, NO2, and C. 1-6Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 alkenyl, C 3-6 Carbon rings or heterocycles;

[0557] Each R can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0558] Each R' can be independently C 1-18 Alkyl, C 2-18 alkenyl, -R YR”

[0559] -YR" or H;

[0560] Each R can be independently C 3-14 Alkyl or C 3-14 alkenyl;

[0561] Each R Can be independently C 1-12 Alkyl or C 2-12 alkenyl;

[0562] Each Y can be independently represented by C. 3-6 Carbon rings;

[0563] Each X can be independently F, Cl, Br, or I; and

[0564] m is 5, 6, 7, 8, 9, 10, 11, 12 or 13 and / or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer.

[0565] In some respects, another subset of the compounds of formula (I) includes:

[0566] R1 is C 5-30 Alkyl, C 5-20 alkenyl, -R YR”, -YR” or -R”M'R’;

[0567] R2 and R3 can be H and C independently. 1-14 Alkyl, C 2-14 alkenyl, -R YR", -YR", or -R OR", and / or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle;

[0568] R4 is C 3-6 Carbon ring, -(CH2) n Q、-(CH2) n CHQR, -CHQR, -CQ(R)2 or unsubstituted C 1-6 Alkyl group, wherein Q is C3-6 Carbon rings, 5- to 14-membered heterocycles having one or more heteroatoms containing N, O, or S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR or -C(=NR9)N(R)2, and / or each n can be independently 1, 2, 3, 4 or 5; and / or when Q is a 5 to 14 member heterocyclic and (i)R4 is -(CH2). n Q where n is 1, 2, or (ii) R4 is -(CH2) n CHQR where n is 1, or (iii) when R4 is -CHQR and -CQ(R)2, then Q is a 5- to 14-membered heteroaryl or an 8- to 14-membered heterocycloalkyl.

[0569] Each R5 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0570] Each R6 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0571] M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl group or heteroaryl group;

[0572] R7 is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0573] R8 is C 3-6 Carbon rings or heterocycles;

[0574] R9 represents H, CN, NO2, and C. 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 alkenyl, C 3-6 Carbon rings or heterocycles;

[0575] Each R can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0576] Each R' can be independently C 1-18 Alkyl, C 2-18 alkenyl, -R YR", -YR", or H;

[0577] Each R can be independently C 3-14 Alkyl or C 3-14 alkenyl;

[0578] Each R Can be independently C 1-12 Alkyl or C 2-12 alkenyl;

[0579] Each Y can be independently represented by C. 3-6 Carbon rings;

[0580] Each X can be independently F, Cl, Br, or I; and

[0581] m is 5, 6, 7, 8, 9, 10, 11, 12 or 13 and / or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer.

[0582] In some respects, another subset of the compounds of formula (I) includes:

[0583] R1 is C 5-30 Alkyl, C 5-20 alkenyl, -R YR”, -YR” or -R”M'R’;

[0584] R2 and R3 can be H and C independently. 1-14 Alkyl, C 2-14 alkenyl, -R YR", -YR", or -R OR", and / or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle;

[0585] R4 is C 3-6 Carbon ring, -(CH2) n Q、-(CH2) n CHQR, -CHQR, -CQ(R)2 or unsubstituted C 1-6Alkyl group, wherein Q is C 3-6 A carbocyclic ring, a 5- to 14-membered heteroaryl group having one or more heteroatoms including N, O, or S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R )2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR or -C(=NR9)N(R)2, and / or each n can be independently 1, 2, 3, 4 or 5;

[0586] Each R5 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0587] Each R6 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0588] M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl group or heteroaryl group;

[0589] R7 is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0590] R8 is C 3-6 Carbon rings or heterocycles;

[0591] R9 represents H, CN, NO2, and C. 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 alkenyl, C 3-6 Carbon rings or heterocycles;

[0592] Each R can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0593] Each R' can be independently C 1-18 Alkyl, C 2-18 alkenyl, -R YR”

[0594] -YR" or H;

[0595] Each R can be independently C 3-14 Alkyl or C 3-14 alkenyl;

[0596] Each R Can be independently C 1-12 Alkyl or C 2-12 alkenyl;

[0597] Each Y can be independently represented by C. 3-6 Carbon rings;

[0598] Each X can be independently F, Cl, Br, or I; and

[0599] m is 5, 6, 7, 8, 9, 10, 11, 12 or 13 and / or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer.

[0600] In some respects, another subset of the compounds of formula (I) includes:

[0601] R1 is C 5-30 Alkyl, C 5-20 alkenyl, -R YR”, -YR” or -R”M'R’;

[0602] R2 and R3 can be H and C independently. 2-14 Alkyl, C 2-14 alkenyl, -R YR", -YR", or -R OR", and / or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle;

[0603] R4 is -(CH2) n Q or -(CH2) n CHQR, where Q is -N(R)2, and / or n is 3, 4 or 5;

[0604] Each R5 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0605] Each R6 can be independently C 1-3Alkyl, C 2-3 alkenyl or H;

[0606] M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl group or heteroaryl group;

[0607] R7 is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0608] Each R can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0609] Each R' can be independently C 1-18 Alkyl, C 2-18 alkenyl, -R YR”

[0610] -YR" or H;

[0611] Each R can be independently C 3-14 Alkyl or C 3-14 alkenyl;

[0612] Each R Can be independently C 1-12 Alkyl or C 1-12 alkenyl;

[0613] Each Y can be independently represented by C. 3-6 Carbon rings;

[0614] Each X can be independently F, Cl, Br, or I; and

[0615] m is 5, 6, 7, 8, 9, 10, 11, 12 or 13 and / or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer.

[0616] In some respects, another subset of the compounds of formula (I) includes:

[0617] R1 is C 5-30 Alkyl, C 5-20 alkenyl, -R YR”, -YR” or -R”M'R’;

[0618] R2 and R3 can be independently C 1-14 Alkyl, C 2-14 alkenyl, -R YR", -YR", or -R OR", and / or R2 and R3 together with the atoms to which they are attached form a heterocycle or a carbocycle;

[0619] R4 is -(CH2) n Q、-(CH2) n CHQR, -CHQR or -CQ(R)2, where Q is -N(R)2, and / or n is 1, 2, 3, 4 or 5;

[0620] Each R5 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0621] Each R6 can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0622] M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, aryl group or heteroaryl group;

[0623] R7 is C 1-3 Alkyl, C 2-3 alkenyl or H;

[0624] Each R can be independently C 1-3 Alkyl, C 2-3 alkenyl or H;

[0625] Each R' can be independently C 1-18 Alkyl, C 2-18 alkenyl, -R YR”

[0626] -YR" or H;

[0627] Each R can be independently C 3-14 Alkyl or C 3-14 alkenyl;

[0628] Each R Can be independently C 1-12 Alkyl or C 1-12 alkenyl;

[0629] Each Y can be independently represented by C. 3-6 Carbon rings;

[0630] Each X can be independently F, Cl, Br, or I; and

[0631] m is 5, 6, 7, 8, 9, 10, 11, 12 or 13 and / or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer.

[0632] In some respects, a subset of the compounds of formula (I) includes those of formula (IA):

[0633]

[0634] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein I is 1, 2, 3, 4, or 5; m is 5, 6, 7, 8, or 9; M1 is a bond or M'; and R4 is an unsubstituted C. 1-3 Alkyl or -(CH2) n Q, where Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl, or heterocyclic alkyl; M and M' can independently be -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, aryl, or heteroaryl groups; and R2 and R3 can independently be H, C, ... 1-14 Alkyl or C 2-14 Alkenyl group.

[0635] In some respects, a subset of the compounds of formula (I) includes those of formula (II):

[0636]

[0637] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein I is 1, 2, 3, 4, or 5; M1 is a bond or M'; and R4 is an unsubstituted C. 1-3 Alkyl or -(CH2) n Q, where n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl, or heterocyclic alkyl; M and M' can independently be -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, aryl, or heteroaryl groups; and R2 and R3 can independently be H, C, ... 1-14 Alkyl or C 2-14Alkenyl. In some respects, a subset of compounds of formula (I) includes those of formulas (IIa), (IIb), (IIc), or (IIe):

[0638]

[0639] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein R4 is as described herein.

[0640] In some respects, a subset of the compounds of formula (I) includes those of formula (IId):

[0641]

[0642] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein n is 2, 3, or 4; and m, R', R'', and R2 through R5 are as described herein. For example, each R2 and R3 may independently be C 5-14 Alkyl or C 5-14 Alkenyl group.

[0643] In some aspects, the ionizable cationic lipids of this disclosure comprise compounds having the following structures:

[0644] .

[0645] In some aspects, the ionizable cationic lipids of this disclosure comprise compounds having the following structures:

[0646] .

[0647] In some aspects, the ionizable cationic lipids of this disclosure comprise compounds having the following structures:

[0648]

[0649] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0650] L 1 or L 2 One of is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)=NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and L 1 or L 2The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)NRa-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;

[0651] G 1 and G 2 Each can be independently of the unsubstituted C1-C 12 Hydroxyl or C1-C 12 alkenyl group; G is C1-C 24 Hydroxyl group, C1-C 24 alkenyl, C3-C8 cycloalkylene, C3-C8 cycloalkenyl;

[0652] R a For H or C1-C 12 alkyl;

[0653] R1 and R2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl;

[0654] R 3 For H, OR 5 CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;

[0655] R 4 For C1-C 12 alkyl;

[0656] R 5 It is H or C1-C6 alkyl; and

[0657] x is 0, 1, or 2.

[0658] In some of the foregoing aspects, ionizable cationic lipids comprise compounds having one of the following structures:

[0659]

[0660] Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:

[0661] A is a 3- to 8-membered cycloalkyl or cycloalkylene ring;

[0662] R 6 Each time it appears, it can be independently H, OH, or Cl-C. 24Alkyl groups; and

[0663] n is an integer ranging from 1 to 15.

[0664] In some of the foregoing aspects, ionizable cationic lipids comprise ...

Claims

1. An RNA molecule comprising at least one open reading frame (ORF) and a 5' untranslated region (5' UTR), said ORF encoding a fimH antigen (FimH) polypeptide, wherein said 5' UTR comprises a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence shown in any one of SEQ ID NO: 95 to 101.

2. The RNA molecule of claim 1, wherein the 5' UTR comprises a nucleic acid sequence that is at least 92% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 95, 98, 99 and 101.

3. The RNA molecule of 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 NO: 95, 99 and 101.

4. The RNA molecule of any one of claims 1 to 3, wherein the 5' UTR comprises a nucleic acid sequence that is at least 98% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 99 and 101.

5. The RNA molecule of any one of claims 1 to 4, wherein the 5' UTR comprises a nucleic acid sequence that is at least 99% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 99 and 101.

6. The RNA molecule of any one of claims 1 to 5, wherein the 5' UTR comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 99 (5'UTR_BMD562); and SEQ ID NO: 101 (5'UTR_BMD576).

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

8. The RNA molecule of claim 7, wherein the 3' UTR comprises nucleotides having the sequence shown in SEQ ID NO: 103 (3'UTR_hHBB).

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

10. The RNA molecule of 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 of 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 NO: 1 to 64.

12. The RNA molecule of 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 NO: 1 to 64.

13. The RNA molecule of 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 an immunogenic fragment thereof.

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

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

16. The RNA molecule of claim 15, wherein the DAFgpi is a variant comprising a serine / glycine linker having a serine / glycine linker having the amino acid sequence GSSGSGSS (SEQ ID NO: 94) replacing 8 DAF amino acid residues near the ω site serine.

17. The RNA molecule of 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 shown in SEQ ID NO: 77, 79, 81, or 83.

18. The RNA molecule of 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 NO: 77, 79, 81 and 83.

19. The RNA molecule of any one of claims 1 to 18, wherein the open reading frame is transcribed from a nucleic acid, the 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 of claim 19, wherein the open reading frame is transcribed from a nucleic acid sequence comprising 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 of any one of claims 1 to 19, wherein the open reading frame comprises a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the sequences shown in SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO:

139.

22. The RNA molecule of claim 21, wherein the open reading frame comprises 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. The RNA molecule of any one of claims 1 to 22, wherein the RNA molecule further comprises a 5' cap portion or a 3' polyadenylate tail.

24. The RNA molecule according to any one of claims 1 to 23, wherein the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG or (m2 7,3 ' -O )Gppp(m 2 ' -O )ApG.

25. The RNA molecule of claim 24, wherein the polyadenylated tail comprises a sequence having SEQ ID NO:

92.

26. The RNA molecule of any one of claims 1 to 25, wherein the RNA molecule comprises nucleotides having the sequence shown in SEQ ID NO: 66 to 75, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO:

90.

27. The RNA molecule of claim 26, wherein the RNA molecule is 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 NO: 107 to 116 or SEQ ID NO: 120 to 124.

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

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

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

31. The RNA molecule of claim 30, wherein the modified nucleotide is pseudouridine, N1-methylpseuuridine, N1-ethylpseuuridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazo-pseuuridine, 2-thio-1-methylpseuuridine, 2-thio-5-aza-uridine, 2-thio-dihydropseuuridine, 2-thio-dihydrouridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-1-methylpseuuridine, 4-thio-pseuuridine, 5-aza-uridine, dihydropseuuridine, 5-methoxyuridine, or 2'-O-methyluridine.

32. The RNA molecule of claim 31, wherein the modified nucleotide is pseudouridine (Ψ) or N. 1 -Methylpseudouridine (m1Ψ).

33. The RNA molecule of claim 32, wherein each uridine in the RNA molecule is converted to pseudouridine (Ψ) or N. 1 -Methylpseudouridine (m1Ψ) substitution.

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

35. The RNA molecule of claim 34, wherein the RNA is a modRNA.

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

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

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

39. The composition of claim 37 or 38, wherein the PEGylated lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, 2-[(PEGylated)-2000]-N,N-bis(tetradecyl)acetamide, or a glycol lipid comprising: PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[((PEGylated monomethyl ether)2000)carbamoyl]-1,2-dimyristoxypropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG. Polyethylene glycol diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristate glyceryl ester (PEG-DMG), polyethylene glycol phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate) succinate (PEG-S-DMG), polyethylene glycol ceramide (PEG-cer), or PEG carbamate dialkoxypropyl ester, such as comethoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(o>-methoxy(polyethoxy)ethyl)carbamate.

40. The composition of claim 39, wherein the polyethylene glycol-modified lipid is 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159).

41. The composition of any one of claims 37 to 40, wherein the neutral lipid is distearate phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine. Amines such as 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-dipentanoyl-sn-glycerol-3-phosphoethanolamine (transDOPE).

42. The composition of claim 41, wherein the neutral lipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC).

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

44. The composition of any one of claims 36 to 43, wherein the composition is a vaccine.

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

83.

46. ​​The mutant FimH polypeptide of claim 45, wherein the mutant FimH polypeptide comprises amino acids having the sequence shown in SEQ ID NO:81 or SEQ ID NO:

83.

47. A polynucleotide encoding a mutant FimH polypeptide comprising at least 80% identity with any one of the amino acid sequences shown in SEQ ID NO: 77, 81 or 83.

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

139.

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

138.

50. A method for (i) inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli or (ii) inducing a subject to produce opsonization and / or neutralizing antibodies against extraintestinal pathogenic Escherichia coli, wherein the method comprises administering to the subject an effective amount of an RNA molecule, RNA-LNP, and / or vaccine as described in any one of claims 1 to 44.

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

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

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

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

55. Use of the RNA molecule, RNA-LNP and / or composition of any one of claims 1 to 44 in the manufacture of a medicament, wherein the medicament is used to (i) induce an immune response in a subject against extraintestinal pathogenic Escherichia coli or (ii) induce a subject to produce opsonization and / or neutralizing antibodies against extraintestinal pathogenic Escherichia coli.

56. The use as claimed in claim 55, wherein the infection, disease, or condition is a urinary tract infection.

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

58. The use as claimed in claim 55, wherein the subject is at risk of developing sepsis.

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

60. The method or use of any one of claims 50 to 59, wherein the subject is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older.

61. The method or use as claimed in any one of claims 50 to 59, wherein the subject is about 50 years of age or older.

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

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

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

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