CN118647397A - Polynucleotide compositions and uses thereof - Google Patents

Abstract

The present invention relates to RNA molecules encoding Escherichia coli fimbriae protein H antigen (FimH). The present disclosure also relates to compositions comprising RNA molecules formulated in lipid nanoparticles (RNA-LNPs). The present disclosure also relates to the use of RNA molecules, RNA-LNPs and compositions for preventing Escherichia coli infections (including urinary tract infections).

Description

Polynucleotide compositions and uses thereof

Related Applications

This application claims priority to U.S. Provisional Application No. 63/290,895, filed on December 17, 2021, and U.S. Provisional Application No. 63/384,607, filed on November 22, 2022. The entire contents of each of the foregoing applications are incorporated herein by reference in their entirety.

Field of the Invention

The present invention relates to compositions and methods for the preparation, manufacture and therapeutic use of RNA vaccines, which contain polynucleotide molecules encoding one or more Escherichia coli (E. coli) fimbriae antigens, such as fimbriae H antigen (FimH).

Sequence Listing Reference

This application is filed electronically via EFS-Web and includes an electronically filed sequence listing in .txt format. The .txt file contains a sequence listing named "PC72808-PRV2 Sequence Listing.xml" created on November 21, 2022 and having a size of 121KB. The sequence listing contained in the .xml file is part of the specification and is incorporated herein by reference in its entirety.

Background of the Invention

Urinary tract infections (UTIs) affect 1 in 5 women at least once in their lifetime, and UTIs cause substantial morbidity and mortality, placing a heavy burden on healthcare systems. While several different bacteria can cause UTIs, the most common cause (90-95% of cases) is the Gram-negative bacterium Escherichia coli (E. coli). Most E. coli UTIs are caused by uropathogenic E. coli (UPEC), which colonize the gastrointestinal tract and migrate from the fecal flora to the urogenital tract, where they adhere to the host's urothelial cells, establishing a reservoir for ascending infection of the urinary tract. Adhesion is facilitated by fimbrial adhesins, including type 1 fimbriae, which bind to mannosylated glycoproteins in the epithelial layer or are secreted into the urine. Type 1 pili are highly conserved among clinical UPEC isolates and are encoded by a group of genes called fim, which encode accessory proteins (FimC, FimD), multiple structural subunits (FimE, FimF, FimG), and an adhesin called FimH. FimH is essential for all features of UTI infection in a mouse model that mimics aspects of human bladder infection (Hannan et al. PLoS Pathog. 2010 Aug 12; 6(8):e1001042; doi:10.1371/journal.ppat.1001042; 10.1128/IAI.05339-11). Small molecule inhibitors targeting FimH by mimicking the mannosylated receptor further validated the role of FimH in UTI and showed promise as therapeutic agents in animal models (Cusumano CK, et al. Sci Transl Med. 2011; 3(109):109ra115. doi:10.1126/scitranslmed.3003021). In addition, FimH is positively selected in Escherichia coli human cystitis isolates (Chen SL, et al. Proc Natl Acad Sci U S A. 2009 Dec 29; 106(52): 22439-44. doi: 10.1073/pnas.0902179106) and the positively selected residues may affect virulence in a mouse cystitis model (Schwartz, D.J. et al. Proc Natl Acad Sci U S A 110, 15530-15537, doi: 10.1073/pnas.1315203110 (2013)).

FimH is composed of two domains, i.e., the lectin binding domain (FimH _LD ) and the pili protein domain responsible for binding to mannosylated glycoproteins. The pili protein domain is used to connect FimH to other structural subunits of pili (such as FimG) by a mechanism called donor chain exchange (Le Trong, I et al., J. Struct Biol. 2010 Dec; 172 (3): 380-8. doi: 10.1016/j.jsb.2010.06.002). The FimH pili protein domain forms an incomplete immunoglobulin fold, which produces a groove (groove) that provides a binding site for the N-terminal β chain of FimG, forming a strong intermolecular connection between FimH and FimG. Although FimH _LD can be expressed in a soluble, stable form, full-length FimH alone is unstable (Vetsch, M., et al. J. Mol. Biol. 322:827–840 (2002); Barnhart MM, et al., Proc Natl Acad Sci US A. (2000) Jul 5; 97(14):7709-14), unless formed in a complex with the chaperone FimC, or complemented with the donor strand peptide of FimG in peptide form or as a fusion protein (Barnhart MM, et al., Proc Natl Acad Sci US A. (2000) Jul 5; 97(14):7709-14; Sauer MM, et al. Nat Commun. (2016) Mar 7; 7:10738; Barnhart MM, et al. J Bacteriol. 2003 May; 185(9):2723-30). The design and expression of full-length FimH molecules by linking a FimG donor peptide to full-length FimH via a glycine-serine linker has been previously described (PCT International Publication No. WO2021/084429, published on May 6, 2021), and was designated FimH-DSG.

FimH _LD is considered a poor immunogen in terms of its ability to stimulate functional immunogenicity. Some studies have shown that although FimH _LD can induce binding antibody titers with and without adjuvants, functional neutralization titers can only be observed with adjuvants (PCT International Publication No. WO2021/084429, published on May 6, 2021). Studies have shown that locking FimH in an open conformation and reducing affinity for mannoside ligands can improve functional immunogenicity (Kisiela, DI et al., Proc Natl Acad Sci USA 110, 19089-19094 (2013)).

Therefore, there is a need for improved immunogenic compositions comprising FimH antigens having reduced affinity for mannoside ligands and improved biochemical properties that result in improved functional immunogenicity relative to wild-type FimH.

SUMMARY OF THE INVENTION

The present disclosure provides an unmet need for improved immunogenic compositions against E. coli infection, including as provided herein.

In one aspect, the present disclosure provides immunogenic compositions and methods for preventing, treating or ameliorating an infection, disease or condition in a subject, comprising administering an RNA molecule, such as an immunogenic RNA polynucleotide encoding an amino acid sequence of, for example, an immunogenic antigen, 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 an epitope of the FimH protein for inducing an immune response against FimH in a subject. RNA polynucleotides encoding immunogenic antigens are administered to provide (after appropriate target cells express the polynucleotide) antigens for inducing (e.g., stimulating, eliciting and/or amplifying) immune responses, such as antibodies and/or immune effector cells. In one aspect, the immune response induced according to the present disclosure is a B cell-mediated immune response, such as an antibody-mediated immune response and a T cell-mediated immune response. In one aspect, the immune response is an anti-FimH immune response.

Immunogenic compositions as described herein include RNA molecules, and the RNA molecules include RNA (as active ingredients) that can be translated into one or more proteins in the cells of the receptor. In addition to the wild type, codon optimized or mutant sequences encoding antigenic sequences, the RNA molecules may contain one or more structural elements, which are optimized for the maximum effectiveness of RNA in terms of stability and translation efficiency (5' cap, 5' UTR, subgenomic promoter, 3' UTR, poly A tail). In one aspect, the RNA molecules contain all these elements. RNA molecules as described herein can be compounded with lipids and/or proteins to generate RNA particles (e.g., lipid nanoparticles (LNP)) for administration. In one aspect, RNA molecules as described herein are compounded with lipids to generate RNA-lipid nanoparticles (e.g., RNA-LNP) for administration. In one aspect, RNA molecules as described herein are compounded with proteins for administration. In one aspect, RNA molecules as described herein are compounded with lipids and proteins for administration. If a combination of different RNA molecules is used, these RNA molecules can be compounded together or compounded with lipids and/or proteins to generate RNA particles for administration.

The present disclosure provides RNA molecules and RNA-LNPs comprising at least one open reading frame (ORF) encoding a FimH antigen. In some aspects, the FimH antigen is a FimH polypeptide. In some aspects, the FimH polypeptide is full-length, truncated, a fragment or variant thereof. In some aspects, the FimH polypeptide comprises at least one mutation.

The present disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF encoding a FimH polypeptide of Table 1. In some aspects, the FimH polypeptide comprises an amino acid sequence selected from SEQ ID NO: 67, 69, 71 or 73. In some aspects, the FimH polypeptide has, has at least, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity to any one of the amino acid sequences of Table 1, e.g., any one of SEQ ID NO: 67, 69, 71 or 73. In some aspects, the FimH polypeptide consists of any one of the amino acid sequences of Table 1, e.g., any one of SEQ ID NO: 67, 69, 71 or 73.

In another aspect, the present disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF encoding a FimH polypeptide, wherein the FimH polypeptide is FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), an immunogenic fragment thereof, or a combination of any two or more of the foregoing. In some aspects, the FimH polypeptide has, has at least, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity to any one of the amino acid sequences of SEQ ID NO: 59, 62 or 54.

The present disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF transcribed from at least one DNA nucleic acid of Table 2. In some aspects, the RNA molecule is transcribed from a nucleic acid sequence selected from SEQ ID NO: 66, 68, 70 or 72. In some aspects, the RNA molecule comprises an ORF transcribed from such a nucleic acid sequence, and the nucleic acid sequence has, has at least, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity with any nucleic acid sequence of Table 2, e.g., any one of SEQ ID NO: 66, 68, 70 or 72. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any nucleic acid sequence of Table 2, e.g., any one of SEQ ID NO: 66, 68, 70 or 72.

The present disclosure also provides RNA molecules and RNA-LNPs comprising at least one ORF comprising an RNA nucleic acid sequence of Table 3. In some aspects, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NOs: 82 to 85. In some aspects, the RNA molecule comprises a nucleic acid sequence having, having at least, or having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any nucleic acid sequence of Table 3, e.g., any one of SEQ ID NOs: 82 to 85. In some aspects, the RNA molecule comprises a nucleic acid sequence consisting of any nucleic acid sequence of Table 3, e.g., any one of SEQ ID NOs: 82 to 85. In some aspects, each uridine of any one of SEQ ID NOs: 82 to 85 is replaced by a 1-methyl-3'-pseudouridine group (Ψ).

The present disclosure also 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:75 or 77. 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:75 or 77. In some aspects, the 5'UTR comprises a sequence selected from any one of SEQ ID NO:75 or 77. In some aspects, the 5'UTR comprises a sequence consisting of any one of SEQ ID NO:75 or 77.

In some aspects, the RNA molecules and RNA-LNPs include a 3' untranslated region (3'-UTR). In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 76 or 78. In some aspects, the 3'UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity to any one of SEQ ID NO: 76 or 78. In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 76 or 78. In some aspects, the 3'UTR comprises a sequence consisting of any one of SEQ ID NO: 76 or 78.

The present disclosure also provides RNA molecules and RNA-LNPs including a 5' cap portion. The present disclosure also provides RNA molecules and RNA-LNPs including a 3' poly A tail. In some aspects, the poly A tail includes a sequence having SEQ ID NO:86.

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 poly A tail. In some aspects, the RNA molecule includes a 5' cap, a 3'UTR and a poly A tail.

In some aspects, the poly A tail length may contain +1/-1 A. In some aspects, the uridine is 1-methyl-3'-pseudouridine (Ψ).

The present disclosure also provides RNA molecules comprising at least one open reading frame that is codon optimized. The present disclosure also provides RNA molecules comprising stabilized RNA. The present disclosure further provides RNA molecules comprising RNA having at least one modified nucleotide. In some aspects, the modified nucleotide is pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxy-uridine or 2'-O-methyluridine. In some aspects, the modified nucleotide is 1-methyl-3'-pseudouridine base (Ψ).

The present disclosure further provides RNA molecules (which are messenger RNA (mRNA)), which can be nucleoside-modified RNA (modRNA). In some aspects, the RNA is mRNA. In other aspects, the RNA is modRNA.

The disclosure also provides immunogenic compositions comprising RNA molecules as described herein. In such immunogenic compositions, RNA molecules can be formulated, encapsulated, compounded, combined or adsorbed on lipid nanoparticles (LNP) (e.g., FimH RNA-LNP). In some aspects, lipid nanoparticles include at least one of cationic lipids, PEG-lipids, and at least one structural lipid (e.g., neutral lipids and steroids or steroid analogs).

In some aspects, the lipid nanoparticle comprises a cationic lipid. In some aspects, the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

In some aspects, lipid nanoparticles include polymer-conjugated lipids. In some aspects, lipid nanoparticles include PEG-lipids, also referred to as PEGylated lipids. In some aspects, PEG-lipids are PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, 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-ditetradecyl acetamide, ethylene glycol-lipids, including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000) carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG, PEGylated diacylglycerols (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3- Dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3 di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate. In some aspects, the PEG-lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159).

In some aspects, the lipid nanoparticles include at least one structural lipid, such as a neutral lipid. In some aspects, the neutral lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)- In some aspects, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

In some aspects, the lipid nanoparticle includes a second structural lipid, such as a steroid or steroid analog. In some aspects, the steroid or steroid analog is cholesterol.

In some aspects, the lipid nanoparticles have an average diameter of about 1 to about 500 nm.

In some aspects, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in a LNP having a lipid composition comprising a concentration of 0.8 The liquid RNA-LNP composition comprises a cationic lipid with a concentration of 0.15 to 0.3 mg/mL, a PEGylated lipid with a concentration of 0.05 to 0.15 mg/mL, a first structural lipid with a concentration of 0.1 to 0.25 mg/mL, and a second structural lipid with a concentration of 0.3 to 0.45 mg/mL, and the liquid RNA-LNP composition also comprises a buffer composition, wherein the buffer composition comprises a first buffer with a concentration of 0.15 to 0.3 mg/mL, a second buffer with a concentration of 1.25 to 1.4 mg/mL, and a stabilizer with a concentration of 95 to 110 mg/mL.

In some aspects, the liquid RNA-LNP immunogenic composition comprises an RNA polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in an LNP having a lipid composition comprising a concentration of 0. 8 to 0.95 mg/mL of ALC-0315, 0.05 to 0.15 mg/mL of ALC-0159, 0.1 to 0.25 mg/mL of DSPC, and 0.3 to 0.45 mg/mL of cholesterol, and the liquid RNA-LNP immunogenic composition further comprises a Tris buffer composition comprising tromethamine 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 sucrose at a concentration of 95 to 110 mg/mL.

In a specific aspect, the RNA-LNP immunogenic composition is a lyophilized RNA-LNP composition comprising an RNA polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in a LNP having a lipid composition comprising a concentration of 0. .8 to 0.95 mg/mL of cationic lipids, 0.05 to 0.15 mg/mL of PEGylated lipids, 0.1 to 0.25 mg/mL of first structural lipids, and 0.3 to 0.45 mg/mL of second structural lipids, and the RNA-LNP composition further comprises a first buffer at a concentration of 0.01 and 0.15 mg/mL, a second buffer at a concentration of 0.5 and 0.65 mg/mL, a stabilizer at a concentration of 35 to 50 mg/mL, and a salt at a concentration of 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of a carrier or diluent. The concentration in the lyophilized RNA-LNP composition is determined after reconstitution.

In a specific aspect, the lyophilized RNA-LNP composition comprises an RNA polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in an LNP having a lipid composition comprising a concentration of 0.8 to 0.95 mg /mL of ALC-0315, 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 the RNA-LNP composition also contains tromethamine at a concentration of 0.01 and 0.15 mg/mL, TrisHCl at a concentration of 0.5 and 0.65 mg/mL, sucrose at a concentration of 35 to 50 mg/mL, and NaCl at a concentration of 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of saline. The concentration in the lyophilized RNA-LNP composition is determined after reconstitution.

The present disclosure provides RNA molecules, RNA-LNPs, and immunogenic compositions that can administer FimH RNA encapsulated in LNPs to a subject at a dose of at least, at most, exactly, or between 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg.

The disclosure provides RNA molecules, RNA-LNP and immunogenic compositions that can be used in a single dose. The disclosure also provides RNA molecules, RNA-LNP and immunogenic compositions that can be used twice (for example, 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). The disclosure also provides RNA molecules, RNA-LNP and immunogenic compositions that can be used twice after day 0 and 2 months. The present disclosure also provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered twice, at day 0 and 6 months later. The present disclosure also provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more. In some aspects, it may be desirable to perform regular booster doses every 1-5 years to maintain the protective level of antibodies.

The present disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of RNA molecules, RNA-LNPs and/or immunogenic compositions described herein. The present disclosure also provides the use of RNA molecules, RNA-LNPs and/or immunogenic compositions described herein in the manufacture of a medicament for inducing an immune response in a subject.

The present disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of an RNA molecule and/or RNA-LNP or a composition as described herein, wherein the RNA molecule and/or RNA-LNP comprises at least one open reading frame encoding a FimH polypeptide. The present disclosure also provides the use of an RNA molecule and/or RNA-LNP comprising at least one open reading frame encoding a FimH polypeptide or a composition as described herein in the manufacture of a medicament for inducing an immune response in a subject.

The present disclosure provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of RNA molecules and/or RNA-LNPs or compositions described herein, wherein the RNA molecules and/or RNA-LNPs include at least one open reading frame encoding a polypeptide of a target gene. The present disclosure also provides the use of RNA molecules and/or RNA-LNPs or compositions described herein comprising at least one open reading frame encoding a polypeptide of a target gene in the manufacture of a medicament for inducing an immune response in a subject.

The present disclosure provides a method for preventing, treating or improving an infection, disease or condition of a subject, comprising administering to a subject an effective amount of RNA molecules, RNA-LNPs and/or immunogenic compositions as described herein. The present disclosure also provides the use of RNA molecules, RNA-LNPs and/or immunogenic compositions as described herein in the manufacture of a drug for preventing, treating or improving an infection, disease or condition of a subject. In some aspects, the infection or condition is associated with Escherichia coli FimH. In some aspects, the infection, disease or condition is urinary tract infection (UTI), urosepsis, cystitis or pyelonephritis.

The present disclosure provides a method for preventing, treating or improving an infection, disease or condition of a subject, comprising administering to the subject an effective amount of an RNA molecule and/or RNA-LNP or an immunogenic composition as described herein, wherein the RNA molecule and/or RNA-LNP comprises at least one open reading frame encoding a FimH polypeptide. The present disclosure also provides an RNA molecule and/or RNA-LNP comprising at least one open reading frame encoding a FimH polypeptide or an immunogenic composition as described herein for use in the manufacture of a medicament for preventing, treating or improving an infection, disease or condition of 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), urosepsis, cystitis or pyelonephritis.

The present disclosure also provides a method for preventing, treating or improving an infection, disease or condition of a subject, comprising administering to the subject an effective amount of RNA molecules and/or RNA-LNP or an immunogenic composition as described herein, wherein the RNA molecules and/or RNA-LNP include at least one open reading frame encoding a polypeptide of a target gene. The present disclosure also provides an RNA molecule and/or RNA-LNP or an immunogenic composition as described herein comprising at least one open reading frame encoding a polypeptide of a target gene for use in the manufacture of a drug for preventing, treating or improving an infection, disease or condition of a subject. In some aspects, infection, disease or condition is associated with a target gene.

In some aspects, the age of the experimenter is, at least or at most is less than about 1 year old, about 1 year old or larger, about 5 years old or larger, about 10 years old or larger, about 20 years old or larger, about 30 years old or larger, about 40 years old or larger, about 50 years old or larger, about 60 years old or larger, about 70 years old or larger or larger. In some aspects, the age of the experimenter is about 50 years old or larger. On the other hand, the age of the experimenter is 6 months to 1 year old, 1 year old to 2 years old, 1 year old to 3 years old, 1 year old to 4 years old, 1 year old to 5 years old, 6 months to 5 years old or 60 years old or larger. The whole birth cohort is all included in the relevant population of immunization. For example, this can be accomplished by the following, can be born to 6 months old, from 6 months old to 5 years old, in pregnant women (or women of childbearing age), by the passive transfer of antibody to protect their infants and in the experimenter greater than 50 years old, start immunization program at any time.

In some embodiments, the subject is a human. In some specific embodiments, the human is a child, such as an infant. In some other specific embodiments, the human is a female, particularly a pregnant woman. In some aspects, the subject is immunocompetent. In some aspects, the subject is immunocompromised.

The present disclosure provides methods or uses described herein, wherein the RNA molecules, RNA-LNPs and/or immunogenic compositions are administered as a vaccine.

The present disclosure provides the methods or uses described herein, wherein the RNA molecules, RNA-LNPs and/or immunogenic compositions are administered by intradermal or intramuscular injection.

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

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.

In another aspect of the E. coli vaccine, the RNA further comprises modified nucleotides.

In another aspect of the E. coli vaccine, at least one antigenic polypeptide is FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), an immunogenic fragment thereof, or a combination of any two or more of the foregoing.

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 FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62); or c) at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54).

In another aspect of the E. coli vaccine, the RNA encodes FimH fused to a C-terminal membrane targeting domain.

In another aspect of the E. coli vaccine, wherein the RNA encodes FimH fused to a C-terminal membrane targeting domain, and they are separated by a linker. In a preferred aspect of the E. coli vaccine, wherein the encoded linker has the amino acid sequence GGSSGGG (SEQ ID NO: 74).

In another aspect of the E. coli vaccine, the C-terminal membrane targeting domain is derived from a viral glycoprotein. In another aspect of the E. coli vaccine, the membrane targeting sequence is derived from HSV gD, SARS-CoV2 Spike protein, or human DAF protein GPI sequence, or a synthetic GPI sequence.

In another aspect of the E. coli vaccine, the FimH is secreted and lacks a C-terminal membrane targeting domain.

In another aspect of the E. coli vaccine, the open reading frame encoded by the RNA is codon optimized.

In another aspect of the E. coli vaccine, the vaccine further comprises a cationic lipid.

In another aspect of the E. coli vaccine, the vaccine comprises lipid nanoparticles comprising RNA molecules.

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

In another aspect of the E. coli vaccine, the lipid nanoparticle size is at least 40 nm. In another aspect of the E. coli vaccine, the lipid nanoparticle size is at most 180 nm.

In another aspect of the E. coli vaccine, at least 80% of the total RNA in the composition is encapsulated.

In another aspect of the E. coli vaccine, wherein the vaccine comprises ALC-0315 (4-hydroxybutyl) azanediyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate).

In another aspect of the E. coli vaccine, the vaccine comprises ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

In another aspect of the E. coli vaccine, wherein the vaccine comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

In another aspect of the E. coli vaccine, the RNA polynucleotide comprises a 5' cap, a 5' UTR, a 3' UTR and a poly A tail.

In another aspect of the E. coli vaccine, the 5'UTR comprises the sequence GAAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCACAGACΨCAGAGAGAACCCGCCACC (SEQ ID NO: 77).

In another aspect of the E. coli vaccine, the 5'UTR comprises the sequence GAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 75).

In another aspect of the E. coli vaccine, wherein the 3'UTR comprises the sequence CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCCΨΨΨCCCGΨCCΨGGGΨACCCCGAGΨCΨCCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨCCACCΨGCCCCACΨCACCACCΨCΨGCΨAGΨΨCCAGACA CCΨCCCAAGCACGCAGCAAΨGCAGCΨCAAAACGCΨΨAGCCΨAGCCACACCCCCACGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAACGAAAGΨΨΨAACΨAAGCΨAΨACΨAACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACACCCΨGGAGCΨAGC( SEQ ID NO:78).

In another aspect of the E. coli vaccine, the 3'UTR comprises the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC (SEQ ID NO: 76).

In another aspect of the E. coli vaccine, wherein Ψ is 1-methyl-3'-pseudouridine.

In another aspect of the E. coli vaccine, the length of the poly A tail is 80 nucleotides.

In another aspect of the E. coli vaccine, the FimH polypeptide comprises serine substitutions at positions N228 and N235.

Unless otherwise specified herein or clearly contradicted by context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to further illustrate the present disclosure and does not limit the scope of the claims. No language in the specification should be construed as indicating any unclaimed element essential to the implementation of the present disclosure.

Several documents are cited in this disclosure. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein shall be construed as an admission that the present disclosure is not entitled to be disclosed earlier than this.

It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

Other purposes, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate specific aspects of the present disclosure, they are given by way of illustration only, because from this detailed description, various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the FimH mRNA construct. Legend: SP, mouse IgG Kappa signal peptide; FimH _LD , FimH lectin domain; TMD, transmembrane domain; gpi, glycosylphosphatidylinositol anchor. FimH amino acid substitutions introduced to prevent N-glycosylation (N7S, N70S, N228S, and N235S) or stabilize conformation (G15A, G16A, V27A, or triple mutant "TM") are indicated; G, donor chain peptide stabilized addition to the C-terminus of the full-length FimH protein.

FIG2 depicts the FimH gene and protein sequences of the following constructs: FimH _LD TMCt60HSVgD (SEQ ID NO:66 and SEQ ID NO:67); FimH _LD TMCtΔ5Spike (SEQ ID NO:68 and SEQ ID NO:69); FimH _LD TMCtDAFgpI (SEQ ID NO:70 and SEQ ID NO:71); and secreted FimH _DSG -TM (N-deglycosylated) (SEQ ID NO:72 and SEQ ID NO:73). Legend: mouse IgK signal peptide is in italics; interdomain linkers are underlined; asterisks are stop codons.

Figure 3 depicts immunofluorescence microscopy imaging of FimH surface expression in HeLa cells. Representative confocal microscopy images of FimH expressed in HeLa cells 24 hours after transfection with 50ng of the indicated mRNA constructs. FimH was stained with a monoclonal antibody that recognizes an epitope that overlaps with the mannoside ligand binding site. In mock-transfected cells or cells transfected with mRNA encoding secreted FimH-DSG, only nuclear staining (with DAPI) and little or no FimH expression were observed. The percentages of cells showing FimH surface staining with mRNA encoding HSVgD chimeras, SARS-COV2ΔCtD5 chimeras, and DAF-GPI chimeras were 36% (8/20), 12% (3/24), and 82% (18/22), respectively.

Figures 4A and 4B depict FimH expression in transfected HeLa cells. Quantitative analysis of FimH expression in fixed/permeabilized HeLa cells (total FimH) (Figure 4A) compared to fixed cells (surface FimH) (Figure 4B). Image analysis software quantifies the signal in individual microplate wells."MFI" = Mean Fluorescence Intensity.

Figures 5A and 5B describe the expression of FimH mRNA in Expi293 cells detected by flow cytometry using the same monoclonal antibody used for immunofluorescence microscopy. FimH expression detected in permeabilized cells (total FimH; Figure 5A) and FimH expression detected in non-permeabilized Expi293 cells (surface FimH only; Figure 5B). The cell viability of all mRNA constructs at all transfection concentrations exceeded 90%. The baseline MFI of total staining or surface staining cells of the mock transfection control is shown as a dotted line (values are 938 and 222, respectively). "MFI" = mean fluorescence intensity.

Figure 6 depicts the titer of secreted FimH-DSG-TM in the supernatant of Expi293 cell transfection. The level of secreted his-tagged FimH antigen was detected in the cell culture supernatant by Octet biolayer interferometry.

FIG. 7 depicts the flow cytometry gating strategy in Example 2.

Figures 8A and 8B depict total and surface FimH expression in transfected human skeletal muscle cells (hSkMCs), showing quantitative analysis of FimH expression in fixed/permeabilized primary hSkMCs (Figure 8A, total FimH) compared to fixed cells (Figure 8B, surface FimH). The percentage of cells staining positive for FimH antigen expression was determined for each mRNA amount 24 hours after transfection of the indicated LNPs. The Opera confocal microscopy platform and Image analysis software quantifies the signal in individual microplate wells. The method is based on the HeLa cell transfection procedure described in Example 1.

Figures 9A and 9B depict FimH IgG dLIA titers, which show neutralizing antibody titers at PD1 and PD2 time points after mouse immunization. Figure 9A shows anti-FimH IgG at week 3 (PD1), and Figure 9B shows anti-FimH IgG at week 6 (PD2). The response to 1 μg or 10 μg FimH _DSG antigen mRNA LNP was significantly higher than the response to FimH _DSG protein antigen using LinA-2 adjuvant (****, p < 0.0001, ***, p < 0.001, **, p < 0.01). LLOQ, lower limit of quantification (0.61 μg/mL) was calculated based on the deviation of the standard curve. R, responder threshold is defined as a FimH IgG serum titer five times higher than the LLOQ (3.05 μg/mL).

Figure 10 depicts the FimH neutralizing antibody titers at PD1, PD2, and PD3 time points. The response to 1 μg of FimH _DSG antigen mRNA LNP at PD2 or PD3 was significantly higher than the response to FimH _DSG protein antigen adjuvanted with LiNA-2 (***, p < 0.0001). The initial dose of FimH _DSG protein antigen was 10 μg, followed by two 5 μg booster doses.

Figure 11 depicts representative flow cytometry dot plots illustrating the T cell gating strategy (top row) and antigen-specific stimulation of individual cytokines or surface markers associated with the Th1 or Th2 pathway ('FimH stim' versus 'DMSO'). The data in the figure show that FimH peptide stimulation results in the detection of cytokine-producing FimH-specific CD4+ and CD8+ T cells after immunization. 'FimH stim' indicates that the FimH (peptide) pool is stimulated.

Figure 12 depicts that modRNA constructs elicited stronger CD8+ T cell responses compared to protein subunits+LiNA-2, especially at higher concentrations.

13A to 13D depict that vaccination with membrane-associated modRNA constructs results in an increase in the frequency of FimH-specific Th1-biased CD4+ T cells compared to FimH _DSG (secreted) modRNA and protein subunit+LiNA2.

Figures 14A to 14C depict that all vaccinations generated FimH-specific CD4 T cells producing low Th2 and Th17 cytokines after three doses.

Sequence Identifier

SEQ ID NO: 1 shows the amino acid sequence of wild-type Escherichia coli FimH _LD (FimHLD_WT).

SEQ ID NO: 2 shows the amino acid sequence of mutant Escherichia coli FimHLD_G65A_V27A.

SEQ ID NO: 3 shows the amino acid sequence of mutant Escherichia coli FimHLD_F1I.

SEQ ID NO: 4 shows the amino acid sequence of mutant Escherichia coli FimHLD_F1L.

SEQ ID NO: 5 shows the amino acid sequence of mutant Escherichia coli FimHLD_F1V.

SEQ ID NO: 6 shows the amino acid sequence of mutant Escherichia coli FimHLD_F1M.

SEQ ID NO: 7 shows the amino acid sequence of mutant Escherichia coli FimHLD_F1Y.

SEQ ID NO: 8 shows the amino acid sequence of mutant Escherichia coli FimHLD_F1W.

SEQ ID NO: 9 shows the amino acid sequence of mutant Escherichia coli FimHLD_Q133K.

SEQ ID NO: 10 shows the amino acid sequence of mutant Escherichia coli FimHLD_G15A.

SEQ ID NO: 11 shows the amino acid sequence of mutant Escherichia coli FimHLD_G15P.

SEQ ID NO: 12 shows the amino acid sequence of mutant Escherichia coli FimHLD_G16A.

SEQ ID NO: 13 shows the amino acid sequence of mutant Escherichia coli FimHLD_G16P.

SEQ ID NO: 14 shows the amino acid sequence of mutant Escherichia coli FimHLD_G15A_G16A.

SEQ ID NO: 15 shows the amino acid sequence of mutant Escherichia coli FimHLD_R60P.

SEQ ID NO: 16 shows the amino acid sequence of mutant Escherichia coli FimHLD_G65A.

SEQ ID NO: 17 shows the amino acid sequence of mutant Escherichia coli FimHLD_P12C_A18C.

SEQ ID NO: 18 shows the amino acid sequence of mutant E. coli FimHLD_G14C_F144C.

SEQ ID NO: 19 shows the amino acid sequence of mutant Escherichia coli FimHLD_P26C_V35C.

SEQ ID NO: 20 shows the amino acid sequence of mutant Escherichia coli FimHLD_P26C_V154C.

SEQ ID NO: 21 shows the amino acid sequence of mutant Escherichia coli FimHLD_P26C_V156C.

SEQ ID NO: 22 shows the amino acid sequence of mutant Escherichia coli FimHLD_V27C_L34C.

SEQ ID NO: 23 shows the amino acid sequence of mutant Escherichia coli FimHLD_V28C_N33C.

SEQ ID NO: 24 shows the amino acid sequence of mutant E. coli FimHLD_V28C_P157C.

SEQ ID NO: 25 shows the amino acid sequence of mutant E. coli FimHLD_Q32C_Y108C.

SEQ ID NO: 26 shows the amino acid sequence of mutant Escherichia coli FimHLD_N33C_L109C.

SEQ ID NO: 27 shows the amino acid sequence of mutant E. coli FimHLD_N33C_P157C.

SEQ ID NO: 28 shows the amino acid sequence of mutant Escherichia coli FimHLD_V35C_L107C.

SEQ ID NO: 29 shows the amino acid sequence of mutant Escherichia coli FimHLD_V35C_L109C.

SEQ ID NO: 30 shows the amino acid sequence of mutant Escherichia coli FimHLD_S62C_T86C.

SEQ ID NO: 31 shows the amino acid sequence of mutant Escherichia coli FimHLD_S62C_L129C.

SEQ ID NO: 32 shows the amino acid sequence of mutant Escherichia coli FimHLD_Y64C_L68C.

SEQ ID NO: 33 shows the amino acid sequence of mutant Escherichia coli FimHLD_Y64C_A127C.

SEQ ID NO: 34 shows the amino acid sequence of mutant Escherichia coli FimHLD_L68C_F71C.

SEQ ID NO: 35 shows the amino acid sequence of mutant E. coli FimHLD_V112C_T158C.

SEQ ID NO: 36 shows the amino acid sequence of mutant E. coli FimHLD_S113C_G116C.

SEQ ID NO: 37 shows the amino acid sequence of mutant E. coli FimHLD_S113C_T158C.

SEQ ID NO: 38 shows the amino acid sequence of mutant Escherichia coli FimHLD_V118C_V156C.

SEQ ID NO: 39 shows the amino acid sequence of mutant E. coli FimHLD_A119C_V155C.

SEQ ID NO: 40 shows the amino acid sequence of mutant Escherichia coli FimHLD_L34N_V27A.

SEQ ID NO: 41 shows the amino acid sequence of mutant Escherichia coli FimHLD_L34S_V27A.

SEQ ID NO: 42 shows the amino acid sequence of mutant Escherichia coli FimHLD_L34T_V27A.

SEQ ID NO: 43 shows the amino acid sequence of mutant Escherichia coli FimHLD_A119N_V27A.

SEQ ID NO: 44 shows the amino acid sequence of mutant Escherichia coli FimHLD_A119S_V27A.

SEQ ID NO: 45 shows the amino acid sequence of mutant Escherichia coli FimHLD_A119T_V27A.

SEQ ID NO: 46 shows the amino acid sequence of mutant E. coli FimH-DSG_A115V.

SEQ ID NO: 47 shows the amino acid sequence of mutant Escherichia coli FimH-DSG_V163I.

SEQ ID NO: 48 shows the amino acid sequence of mutant Escherichia coli FimH-DSG_V185I.

SEQ ID NO: 49 shows the amino acid sequence of mutant E. coli FimH-DSG_DSG_V3I.

SEQ ID NO: 50 shows the amino acid sequence of mutant Escherichia coli FimHLD_G15A_V27A.

SEQ ID NO: 51 shows the amino acid sequence of mutant Escherichia coli FimHLD_G16A_V27A.

SEQ ID NO: 52 shows the amino acid sequence of mutant Escherichia coli FimHLD_G15P_V27A.

SEQ ID NO: 53 shows the amino acid sequence of mutant Escherichia coli FimHLD_G16P_V27A.

SEQ ID NO: 54 shows the amino acid sequence of mutant E. coli FimHLD_G15A_G16A_V27A.

SEQ ID NO: 55 shows the amino acid sequence of mutant Escherichia coli FimHLD_V27A_R60P.

SEQ ID NO: 56 shows the amino acid sequence of mutant Escherichia coli FimHLD_G65A_V27A.

SEQ ID NO: 57 shows the amino acid sequence of mutant E. coli FimHLD_V27A_Q133K.

SEQ ID NO: 58 shows the amino acid sequence of mutant Escherichia coli FimHLD_G15A_G16A_V27A_Q133K.

SEQ ID NO: 59 shows the amino acid sequence of the full-length FimH of wild-type E. coli, including the donor strand FimG peptide connected by a linker (FimH-DSG_WT).

SEQ ID NO: 60 shows the amino acid sequence of mutant Escherichia coli FimH-DSG_V27A.

SEQ ID NO: 61 shows the amino acid sequence of mutant E. coli FimH-DSG_G15A_V27A.

SEQ ID NO: 62 shows the amino acid sequence of mutant E. coli FimH DSG_G15A_G16A_V27A.

SEQ ID NO: 63 shows the amino acid sequence of mutant E. coli FimH DSG_V27A_Q133K.

SEQ ID NO: 64 shows the amino acid sequence of mutant E. coli FimH DSG_G15A_G16A_V27A_Q133K.

SEQ ID NO: 65 shows the amino acid sequence of the mouse Ig Kappa signal peptide sequence.

SEQ ID NO: 66 shows the nucleic acid sequence of the chimera FimH _LD TMCt60HSVgD.

SEQ ID NO: 67 shows the amino acid sequence of chimera FimH _LD TMCt60HSVgD.

SEQ ID NO: 68 shows the nucleic acid sequence of the chimera FimH _LD TMCtΔ5Spike.

SEQ ID NO: 69 shows the amino acid sequence of the chimera FimH _LD TMCtΔ5Spike.

SEQ ID NO: 70 shows the nucleic acid sequence of the chimera FimH _LD TMCtDAFgpi.

SEQ ID NO: 71 shows the amino acid sequence of chimera FimH _LD TMCtDAFgpi.

SEQ ID NO: 72 shows the nucleic acid sequence of secreted FimH-DSG-TM (N-deglycosylated).

SEQ ID NO: 73 shows the amino acid sequence of secreted FimH-DSG-TM (N-deglycosylated).

SEQ ID NO: 74 shows the amino acid sequence of the seven amino acid linker.

SEQ ID NO: 75 shows the nucleic acid sequence of 5'UTR.

SEQ ID NO: 76 shows the nucleic acid sequence of 3'UTR.

SEQ ID NO:77 shows the modified nucleic acid sequence of the 5′UTR shown in SEQ ID NO:75.

SEQ ID NO:78 shows the modified nucleic acid sequence of the 5′UTR shown in SEQ ID NO:76.

SEQ ID NO: 79 shows the amino acid sequence of the SARS-CoV2 spike protein [UniprotKB:P0DTC2].

SEQ ID NO: 80 shows the amino acid sequence of the C-terminal amino acids that constitute the ER retention motif of the SARS-CoV2 spike protein [UniprotKB:P0DTC2].

SEQ ID NO: 81 shows the amino acid sequence of the conserved charged region of the C-terminal cytoplasmic tail of the SARS-CoV2 spike protein [UniprotKB:P0DTC2].

SEQ ID NO: 82 shows the nucleic acid sequence of the chimera FimHLDTMCt60HSVgD.

SEQ ID NO: 83 shows the nucleic acid sequence of the chimera FimHLDTMCtΔ5Spike.

SEQ ID NO: 84 shows the nucleic acid sequence of the chimera FimHLDTMCtDAFgpi.

SEQ ID NO: 85 shows the nucleic acid sequence of secreted FimH-DSG-TM (N-deglycosylated).

SEQ ID NO: 86 shows the nucleic acid sequence of the 80A poly A tail.

DETAILED DESCRIPTION OF THE INVENTION

By referring to the following detailed description of the embodiments of the present invention and the examples included herein, the present invention can be more easily understood. It should be understood that the present invention is not limited to specific preparation methods, which can certainly vary. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments and are not intended to be limiting.

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

E1. A ribonucleic acid polynucleotide (RNA) molecule comprising at least one open reading frame (ORF) encoding a FimH antigen polypeptide.

E2. The RNA molecule of clause E1, wherein the FimH antigen polypeptide is full-length, truncated, a fragment or a variant thereof.

E3. The RNA molecule of any one of clauses E1-E2, wherein the FimH antigenic polypeptide comprises at least one mutation.

E4. The RNA molecule of any one of clauses E1-E3, wherein the FimH antigen polypeptide comprises the amino acids of Table 13, including but not limited to any one of SEQ ID NOs: 1 to 64.

E5. The RNA molecule of any one of clauses E1-E4, wherein the FimH antigenic polypeptide comprises FimH-DSG (SEQ ID NO: 59), FimH-DSG triple mutant (G15A, G16A, V27A) (SEQ ID NO: 62), FimHLD triple mutant (G15A, G16A, V27A) (SEQ ID NO: 54), or an immunogenic fragment thereof.

E6. The RNA molecule of any one of clauses E1 to E5, wherein the FimH polypeptide is at least 90%, 95, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 64.

E7. The RNA molecule of any one of clauses E1-E6, wherein the RNA is fused to a C-terminal membrane targeting domain.

E8. The RNA molecule of clause E7, wherein the RNA molecule and the C-terminal membrane targeting domain are separated by a linker.

E9. The RNA molecule of clause E8, wherein the linker has the amino acid sequence GGSSGGG (SEQ ID NO: 74).

E10. The RNA molecule of any one of clauses E7-E9, wherein the C-terminal membrane targeting domain is derived from a viral glycoprotein.

E11. The RNA molecule of clause E10, wherein the viral glycoprotein is selected from the group consisting of HSV gD, SARS-CoV2 spike protein and human DAFgpi.

E12. The RNA of clause E9, wherein the C-terminal membrane targeting domain is an E. coli G peptide.

E13. The RNA molecule of any one of clauses E1-E12, wherein the open reading frame is codon-optimized.

E14. The RNA molecule of clause E11, wherein the FimH antigen polypeptide comprises the amino acids of Table 1, including but not limited to any one of SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84.

E15. The RNA molecule of clause E14, wherein the FimH antigen polypeptide comprises the amino acid having SEQ ID NO:84.

E16. The RNA molecule of clause E12, wherein the FimH antigen polypeptide comprises the amino acid having SEQ ID NO:85.

E17. The RNA molecule of any one of E1-E16, wherein the open reading frame is transcribed from the nucleic acid sequence of Table 2, including but not limited to any one of SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70 or SEQ ID NO:72.

E18. The RNA molecule of any one of E1-E17, wherein the open reading frame comprises the nucleic acid sequence of Table 3, including but not limited to any one of SEQ ID NO: 82 to 85.

E19. The RNA of clause E18, wherein each uridine in any one of SEQ ID NOs: 82 to 85 is replaced by a 1-methyl-3'-pseudouridine group (Ψ).

E20. The RNA molecule of any one of clauses E1-E19, further comprising a 5' untranslated region (5'UTR).

E21. The RNA molecule of clause E21, wherein the 5'UTR comprises nucleotides having SEQ ID NO:75.

E22. The RNA molecule of any one of clauses E1-E21, further comprising a 3' untranslated region (3'UTR).

E23. The RNA molecule of clause E22, wherein the 3'UTR comprises nucleotides having SEQ ID NO:76.

E24. The RNA molecule of any one of clauses E1 to E23, wherein the RNA molecule comprises a 5' cap moiety.

E25. The RNA molecule of clause E24, wherein the 5' cap portion is m7G(5')ppp(5')(2'OMeA)pG.

E26. The RNA molecule of any one of clauses E1-E25, further comprising a 3' poly A tail.

E27. The RNA of clause E26, wherein the poly-A tail comprises a sequence having SEQ ID NO:86.

E28. The RNA molecule of any one of clauses E1-E27, wherein the RNA molecule comprises a 5'UTR and a 3'UTR.

E29. The RNA molecule of any one of clauses E1-E28, wherein the RNA molecule comprises a 5' cap, a 5' UTR and a 3' UTR.

E30. The RNA molecule of any one of clauses E1-E29, wherein the RNA molecule comprises a 5' cap, a 5' UTR, a 3' UTR and a poly A tail.

E31. The RNA molecule of any one of clauses E1-E30, wherein the RNA molecule comprises stabilized RNA.

E32. The RNA molecule of any one of clauses E1 to E31, wherein the RNA comprises at least one modified nucleotide.

E33. The RNA molecule of clause E32, wherein the modified nucleotide is pseudouridine, 1-methyl-3'-pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, or 5-methoxyuridine or 2'-O-methyluridine.

E34. The RNA molecule of clause E33, wherein the modified nucleotide is 1-methyl-3'-pseudouridine (Ψ).

E35. The RNA molecule of any one of clauses E1-E25, wherein the RNA is mRNA.

E36. A composition comprising the RNA molecule of any one of clauses E1-E35, wherein the RNA molecule is formulated in lipid nanoparticles (RNA-LNP).

E37. The composition of clause E36, wherein the lipid nanoparticles comprise at least one of a cationic lipid, a PEG-lipid, a neutral lipid, and a steroid or a steroid analog.

E38. The composition of clause E36 or E37, wherein the lipid nanoparticles comprise cationic lipids.

E39. The composition of clause E38, wherein the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

E40. The composition of any one of E36-E39, wherein the lipid nanoparticles comprise PEG-lipid.

E41. The composition of clause E40, wherein the PEG-lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide, ethylene glycol-lipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000)carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG, PEGylated diacylglycerol In some embodiments, the present invention can be used to prepare PEG-DAG such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-bis(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

E42. The composition of clause E40 or E41, wherein the PEG-lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecanoylacetamide (ALC-0159).

E43. The composition of any one of clauses E36-E42, wherein the lipid nanoparticles comprise neutral lipids.

E44. The composition of clause E43, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (transDOPE).

E45. The composition of clause E43 or E44, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

E46. The composition of any one of clauses E36-E45, wherein the lipid nanoparticles comprise a steroid or a steroid analog.

E47. The composition of clause 46, wherein the steroid or steroid analog is cholesterol.

E48. The composition of any one of clauses E36-E47, wherein the lipid nanoparticles have an average diameter of about 1 to about 500 nm.

E49. The composition of any one of clauses E36-E48, wherein the composition is a vaccine.

E50. The composition of any one of clauses E36-E49, wherein the lipid nanoparticles are at least 40 nm in size.

E51. The composition of any one of clauses E36-E49, wherein the lipid nanoparticles are at most 180 nm in size.

E52. A method for (i) inducing an immune response against extraintestinal pathogenic Escherichia coli in a subject, or (ii) inducing the production of opsonophagocytic and/or neutralizing antibodies specific to extraintestinal pathogenic Escherichia coli in a subject, wherein the method comprises administering to the subject an effective amount of the RNA molecule, RNA-LNP and/or vaccine of any one of clauses E1-E51.

E53. The method of clause E52, wherein the subject is at risk of developing a urinary tract infection.

E54. The method of clause E52, wherein the subject is at risk of developing bacteremia.

E55. The method of clause E52, wherein the subject is at risk of developing urosepsis.

E56. The method of clause E52, wherein the subject is at risk of developing cystitis.

E57. Use of the RNA molecule, RNA-LNP and/or composition of any one of clauses E1-E56 in the manufacture of a medicament for (i) inducing an immune response against extraintestinal pathogenic Escherichia coli in a subject, or (ii) inducing the production of opsonophagocytic and/or neutralizing antibodies specific to extraintestinal pathogenic Escherichia coli in a subject.

E58. The use of clause E57, wherein the infection, disease or condition is a urinary tract infection.

E59. The use of clause E57, wherein the subject is at risk of developing bacteremia.

E60. The use of clause E57, wherein the subject is at risk of developing sepsis.

E61. The use of clause E57, wherein the subject is at risk of developing cystitis.

E62. The method or use of any of clauses E52-E61, 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.

E63. The method or use of any one of clauses E52-E61, wherein the subject is about 50 years old or older.

E64. The method or use of any one of clauses E52-E61, wherein the subject is a pregnant woman.

E65. The method or use of any one of clauses E52-E64, wherein the RNA molecule or composition is administered as a vaccine.

E66. The method or use of any one of clauses E52-E65, wherein the RNA molecule or composition is administered by intradermal injection or intramuscular injection.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patent publications, UniProtKB accession numbers, are hereby incorporated by reference as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

1. Example of definition

Unless otherwise defined herein, scientific and technical terms related to the present invention have the meanings that are commonly understood by one of ordinary skill in the art.

Throughout this application, the term "about" is used according to its plain and ordinary meaning in the arts of cell and molecular biology to indicate a deviation of ±10% from the value with which it is associated.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

The word "a" or "an" when used in combination with the term "comprising" may mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

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

The phrase "substantially all" is defined as "at least 95%"; if substantially all members of a group have a certain property, then at least 95% of the members of the group have that property. In some aspects, substantially all means that 95%, 96%, 97%, 98%, 99% or 100% of any one, at least any one, or any two between them of the group have that property.

Compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed throughout the specification. In this specification, unless the context requires otherwise, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are all inclusive or open-ended, and will be understood to imply the inclusion of a stated step or ingredient or group of steps or ingredients, but not the exclusion of any other step or ingredient or group of steps or ingredients. It is contemplated that aspects described herein in the context of the term "comprising" may also be implemented in the context of the term "consist of" or "consist essentially of." Compositions and methods that "consist essentially of any disclosed ingredients or steps" limit the scope of the claim to specific materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The phrase "consisting of" (and any form of "consisting of," such as "consist of" and "consist of") means including, but not limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" means that the listed ingredients are required or mandatory, and no other ingredients may be present.

In this specification, references to "an aspect", "aspect", "particular aspects", "related aspects", "an aspect", "additional aspects" or "another aspect" or combinations thereof indicate that a particular feature, structure or characteristic described in relation to that aspect is included in at least one aspect of the present disclosure. Therefore, the appearance of the aforementioned phrases in multiple places in this specification do not necessarily all refer to the same aspect. Furthermore, particular features, structures or characteristics may be combined in any suitable manner in one or more aspects.

The terms "inhibit," "reduce," or "lower," or any variation of these terms, include any measurable reduction (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction) or complete inhibition to achieve a desired outcome. The terms "improve," "promote," or "increase," or any variation of these terms, include any measurable increase (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% increase) to achieve a desired outcome or production of a protein or molecule.

As used herein, the terms "reference," "standard," or "control" describe a value relative to which a comparison is made. For example, a target agent, subject, population, sample, or value is compared to a reference, standard, or control agent, subject, population, sample, or value. A reference, standard, or control can be tested and/or determined substantially simultaneously, and/or simultaneously with the target test or determination of a target agent, subject, population, sample, or value, and/or can be determined or characterized under conditions or circumstances comparable to the target agent, subject, population, sample, or value being evaluated.

The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA technology) or chemical precursors or other chemicals (when synthesized by chemical synthesis) from which it is derived. In addition, an isolated compound refers to a compound that can be administered to a subject as an isolated compound; in other words, if the compound is adhered to a column or embedded in an agarose gel, the compound cannot be simply regarded as "isolated". In addition, an "isolated nucleic acid fragment" or "isolated peptide" refers to a nucleic acid or protein fragment that does not exist naturally in the form of a fragment and/or is not usually in a functional state and/or is altered or removed from the natural state by human intervention. For example, DNA naturally present in living animals is not "isolated", but synthetic DNA, or DNA partially or completely separated from coexisting materials in its natural state is "isolated". An isolated nucleic acid may be present in a substantially purified form, or may be present in a non-natural environment, for example, a cell to which the nucleic acid has been delivered.

As used herein, "nucleic acid" is a molecule comprising a nucleic acid component, and refers to a DNA or RNA molecule. It can be used interchangeably with the term "polynucleotide". Nucleic acid molecules are polymers comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. Nucleic acids can also include modified nucleic acid molecules, such as DNA or RNA molecules with base modifications, sugar modifications, or backbone modifications. Nucleic acids can exist in various forms, such as: isolated segments and recombinant vectors or recombinant polynucleotides encoding polypeptides (such as one or two chains of an antigen or antibody, or fragments, derivatives, mutant proteins, or variants thereof) incorporated into sequences, polynucleotides sufficient to be used as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting polynucleotide expression, mRNA, modRNA, and complementary sequences as described above herein. Nucleic acids can encode epitopes to which antibodies can bind.

The term "epitope" refers to a part specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some aspects, an epitope comprises a plurality of chemical atoms or groups on an antigen. In some aspects, when an antigen adopts a relevant three-dimensional conformation, such chemical atoms or groups are exposed to the surface. In some aspects, when an antigen adopts this conformation, these chemical atoms or groups are physically close to each other in space. In some aspects, when an antigen adopts an alternative conformation (e.g., linearization), at least some of such chemical atoms or groups are physically separated from each other.

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

The term "polynucleotide" refers to a nucleic acid molecule that can be recombinant or separated from a total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, etc. In some aspects, a polynucleotide includes a regulatory sequence that is substantially separated from its naturally occurring gene or protein coding sequence. A polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), its analogs or combinations thereof. Additional coding or non-coding sequences may be present but need not be present in a polynucleotide.

In some aspects, there are polynucleotide variants with substantial identity to the sequences disclosed herein; compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters), comprising any one, at least any one, at most any one, or any two of those polynucleotide variants of sequence identity equal to 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher. In some aspects, the isolated polynucleotide will include a nucleotide sequence encoding a polypeptide that has at least 90% identity to the amino acid sequence described herein over the entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide. In some aspects, the isolated polynucleotide will include a nucleotide sequence encoding a polypeptide that has at least 95% identity to the amino acid sequence described herein over the entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide.

Nucleic acid segments, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., so that their overall length can vary widely. Nucleic acids can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides, any one, at least any one, at most any one, or between any two, and/or can contain one or more additional sequences, such as regulatory sequences, and/or be part of a larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of almost any length can be used, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.

In this regard, the term "gene" is used to refer to nucleic acids encoding proteins, polypeptides or peptides (including any sequences required for correct transcription, post-translational modification or localization). It will be understood by those skilled in the art that the term encompasses genomic sequences, expression cassettes, cDNA sequences and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and mutants. A nucleic acid encoding all or part of a polypeptide may contain a continuous (contiguous) nucleic acid sequence encoding all or part of the polypeptide. It is also envisioned that a specific polypeptide may be encoded by a nucleic acid containing variations that have slightly different nucleic acid sequences, but still encode the same or substantially similar polypeptides.

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

In general, the term "engineered" refers to aspects that have been artificially manipulated. For example, a polynucleotide is considered "engineered" when two or more sequences that are not sequentially linked together in nature are directly linked to each other in an engineered polynucleotide by artificial manipulation, and/or when specific residues in a polynucleotide are non-naturally occurring and/or are linked to entities or parts that are not linked in nature by artificial manipulation.

As used herein, the term "DNA" refers to a nucleic acid molecule comprising nucleotides (e.g., deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate monomers), which are composed of a sugar portion (deoxyribose), a base portion, and a phosphate portion and are polymerized by a characteristic backbone structure. The backbone structure is usually formed by a phosphodiester bond between the sugar portion of the first nucleotide (e.g., deoxyribose) and the phosphate portion of the second adjacent monomer. The specific order of monomers, such as the order of bases connected to the sugar/phosphate backbone, is called a DNA sequence. DNA may be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand are usually hybridized with the nucleotides of the second strand, for example, by 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-ribofuranosyl group. Without limitation, DNA may include double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

As used herein, the term "RNA" refers to a nucleic acid molecule comprising nucleotides (e.g., adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers) that are interconnected along a so-called backbone. The backbone is formed by a phosphodiester bond between a first sugar (e.g., ribose) and the phosphate portion of a second adjacent monomer. RNA can be obtained by transcription of a DNA sequence, such as transcription in a cell. In eukaryotic cells, transcription is typically performed in the nucleus or mitochondria. In vivo, DNA transcription can produce immature RNA that is processed into messenger RNA (mRNA). For example, in eukaryotic organisms, the processing of immature RNA includes a variety of post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and export 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 a peptide or protein. Mature mRNA may include a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a poly A tail sequence. RNA may contain all or most of the ribonucleotide residues. As used herein, the term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. In one aspect, RNA can be messenger RNA (mRNA), which relates to an RNA transcript encoding a peptide or protein. As known to those skilled in the art, mRNA generally contains a 5' untranslated region (5'UTR), a polypeptide coding region, and a 3' untranslated region (3'UTR). Without limitation, RNA can include double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, and modified RNA (modRNA).

"Isolated RNA" is defined as an RNA molecule that may be recombinant or that has been separated from total genomic nucleic acid. An isolated RNA molecule or protein may exist in a substantially purified form, or may exist in a non-native environment, such as a host cell.

"Modified RNA" or "modRNA" refers to an RNA molecule having at least one addition, deletion, substitution and/or change of one or more nucleotides compared to a naturally occurring RNA. Such changes may refer to the addition of non-nucleotide substances to internal RNA nucleotides, or to the 5' and/or 3' ends of the RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as a change in a nucleotide base. For example, the modified nucleotide can replace one or more uridine and/or cytidine nucleotides. For example, these replacements may occur in each uridine and/or cytidine in the RNA sequence, or may only occur in selected uridine and/or cytidine nucleotides. Such changes in standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide in the RNA sequence can be replaced by N1-methyl pseudouridine. Other such changed nucleotides are known to those skilled in the art. Such changed RNA molecules are considered to be analogs of naturally occurring RNA. In some aspects, RNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some aspects, the RNA can be a replicon RNA (replicon), particularly a self-replicating RNA, or a self-amplifying RNA (saRNA).

As contemplated herein, without any limitation, RNA can be used as a therapeutic modality to treat and/or prevent a variety of conditions in mammals (including humans). Methods described herein include administering RNA described herein to mammals, such as humans. For example, in one aspect, this method using RNA includes antigen encoding RNA vaccines to induce powerful neutralizing antibodies and accompanying/accompanied T cell responses to achieve protective immunity. In some aspects, a minimum dose of vaccine is administered to induce powerful neutralizing antibodies and accompanying/accompanied T cell responses to achieve protective immunity. In one aspect, the RNA administered is in vitro transcribed RNA. For example, this RNA can be used to encode at least one antigen intended to produce an immune response in the mammal. Pathogenic antigens are peptides or protein antigens from pathogens associated with infectious diseases. In a specific aspect, pathogenic antigens are peptides or protein antigens derived from Escherichia coli FimH. Conditions and/or diseases that can be treated with RNA disclosed herein include, but are not limited to, those conditions and/or diseases caused and/or affected by bacterial infection. These bacteria include, but are not limited to, Escherichia coli.

As used herein, "prevent" or "prevention," when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder, and/or condition and/or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention is considered to be accomplished when the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

As can be understood from the context, the "risk" of a disease, illness and/or the patient's condition refers to the possibility that a specific individual will have the disease, illness and/or the patient's condition. In some respects, risk is expressed as a percentage. In some respects, risk is, at least or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 until 100%. In some respects, risk is expressed as the risk relative to the risk associated with a reference sample or a reference sample group. In some respects, a reference sample or a reference sample group has a known disease, illness, the patient's condition and/or event risk. In some respects, a reference sample or a reference sample group is from an individual comparable to a specific individual. In some respects, risk may reflect one or more genetic attributes, for example, it may make an individual prone to (or not occur) a specific disease, illness and/or the patient's condition. In some respects, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyles or environmental events or attributes. Susceptible: Individuals who are "susceptible" to a disease, illness, and/or condition refer to individuals who have a higher risk of the disease, illness, and/or condition occurring than members of the general public. In some aspects, individuals who are susceptible to a disease, illness, and/or condition may not have been diagnosed with the disease, illness, and/or condition. In some aspects, individuals who are susceptible to a disease, illness, and/or condition may show symptoms of the disease, illness, and/or condition. In some aspects, individuals who are susceptible to a disease, illness, and/or condition may not show symptoms of the disease, illness, and/or condition. In some aspects, individuals who are susceptible to a disease, illness, and/or condition will have the disease, illness, and/or condition. In some aspects, individuals who are susceptible to a disease, illness, and/or condition will not have the disease, illness, and/or condition.

The terms "protein", "polypeptide" or "peptide" are used synonymously herein and refer to a polymer of amino acid monomers, such as a molecule comprising at least two amino acid residues. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments of the foregoing and other equivalents, variants and analogs. Polypeptides can be single molecules or multimolecular complexes, such as dimers, trimers or tetramers. Proteins contain one or more peptides or polypeptides and can fold into a three-dimensional form, which may be necessary for the protein to exert its biological function.

As used herein, the term "wild type" or "WT" or "native" refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, a wild type version of a protein or polypeptide is employed, however, in other aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The above terms can be used interchangeably.

A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered relative to a wild-type protein or polypeptide. In some aspects, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is particularly contemplated that a modified/variant protein or polypeptide may be altered in one activity or function but retains wild-type activity or function in other aspects, such as immunogenicity. When a protein is specifically mentioned herein, it is generally referred to as a natural (wild-type) or recombinant (modified) protein. The protein can be isolated directly from an organism in which it naturally occurs, produced by recombinant DNA/exogenous expression methods, produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In specific aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antigens or fragments thereof). The term "recombinant" can be used in combination with the name of a polypeptide or a specific polypeptide, and generally refers to a polypeptide that has been produced by a nucleic acid molecule manipulated in vitro or a replication product of such a molecule.

The term "fragment" refers to a portion of an amino acid sequence with respect to an amino acid sequence (peptide or protein), for example, a sequence representing an amino acid sequence shortened at the N-terminus and/or C-terminus. For example, a fragment shortened at the C-terminus (N-terminal fragment) can be obtained by translating a truncated open reading frame lacking the 3' end of the open reading frame. For example, a fragment shortened at the N-terminus (C-terminal fragment) can be obtained by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains a start codon for initiating translation. A fragment of an amino acid sequence comprises, 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 the present disclosure, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least, at most, exactly, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or between any two of 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% sequence identity to the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived.

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

As used herein, in the context of molecules (e.g., nucleic acids, proteins, or small molecules), the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but is structurally different from the reference molecule, e.g., in the presence or absence or level of one or more chemical moieties compared to the reference entity. In some aspects, a variant is also functionally different from its reference molecule. In general, whether a particular molecule is properly considered a "variant" of a reference molecule depends on the degree of structural identity between it and the reference molecule. It will be appreciated by those skilled in the art that any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that has one or more such characteristic structural elements, but is different from a reference molecule in at least one aspect. In some aspects, a variant polypeptide or nucleic acid may be different from a reference polypeptide or nucleic acid due to one or more differences in amino acid or nucleotide sequence and/or one or more differences in a chemical moiety (e.g., carbohydrate, lipid, phosphate group) as a covalent component of a polypeptide or nucleic acid (e.g., attached to a polypeptide or nucleic acid backbone). In some aspects, the overall sequence identity exhibited by the variant polypeptide or nucleic acid and the reference polypeptide or nucleic acid is at least, at most, just 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99%, or between any two of 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 element 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 shares 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, if the polypeptide or nucleic acid of interest has the same amino acid or nucleotide sequence as a reference polypeptide or nucleic acid, but has a small amount of sequence changes at a specific position, the polypeptide or nucleic acid of interest is considered to be a "variant" of the reference polypeptide or nucleic acid. Preferably, the variant polypeptide or nucleic acid sequence has at least one modification, such as 1 to about 20 modifications, compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 10 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 5 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 4 modifications compared to the reference polypeptide or nucleic acid sequence. Typically, 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 the residues in the variant are substituted, inserted or deleted compared to the reference. Typically, a variant polypeptide or nucleic acid comprises a minimal amount (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (e.g., residues involved in a specific biological activity) relative to a reference. In some aspects, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to a reference. In some aspects, a variant polypeptide or nucleic acid comprises less 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 typically less than about 5, about 4, about 3, or about 2 additions or deletions compared to a reference. In some aspects, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some aspects, comprises no additions or deletions.

In some aspects, the reference polypeptide or nucleic acid is a "wild type" or "WT" or "native" sequence found in nature, including allelic variations. A wild type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For the purposes of the present disclosure, a "variant" of an amino acid sequence (peptide, protein or polypeptide) comprises an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and/or an amino acid substitution variant. A "variant" of a nucleotide sequence comprises a nucleotide insertion variant, a nucleotide addition variant, a nucleotide deletion variant and/or a nucleotide substitution variant. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those variants that occur in nature. The term "variant" particularly includes fragments of an amino acid or nucleic acid sequence.

Can introduce change by nucleic acid mutation, thereby cause the change of the aminoacid sequence of its encoded polypeptide (for example, antigen or antibody or antibody derivative).Can use any technology known in the art to introduce sudden change.In one aspect, use for example site-directed mutagenesis scheme to change one or more specific amino acid residues.On the other hand, use for example random mutagenesis scheme to change one or more randomly selected residues.In some respects, no matter how to prepare, can express mutant polypeptide and screen desired property.

Mutations can be introduced into nucleic acids without significantly changing the biological activity of the polypeptides encoded therein. For example, nucleotide substitutions can be made, resulting in amino acid substitutions of non-essential amino acid residues. Alternatively, one or more mutations can be introduced into nucleic acids, which selectively change the biological activity of the polypeptides encoded therein. For example, mutations may quantitatively or qualitatively change biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating activity. Examples of qualitative changes include changing the antigenic specificity of an antibody.

"Sequence similarity" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

The terms "% identical", "% identity" or similar terms are intended to refer specifically to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment, for segments or "comparison windows" to identify local regions of corresponding sequences. Can carry out optimal comparison manually for comparison, or by Smith and Waterman, 1981, Ads App.Math.2,482 local homology algorithm, by Neddleman and Wunsch, 1970, J.Mol.Biol.48,443 local homology algorithm, by Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA88,2444 similarity search algorithm, or by computer program (Wisconsin Genetics Software Package, GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Genetics Computer Group) using said algorithm.In some respects, use BLASTN or BLASTP algorithm to determine the percentage identity of two sequences, as available on the website of United States National Center forBiotechnology Information (NCBI).

The percent identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing that number by the number of positions being compared (eg, the number of positions in the reference sequence) and multiplying the result by 100.

In some respects, similarity or degree of identity is provided for at least, at most, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the entire length of the reference sequence or between about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of any two of the entire length of the reference sequence. For example, if the reference nucleic acid sequence is composed of 200 nucleotides, the degree of identity is provided for at least, at most, about 100, about 120, about 140, about 160, about 180 or about 200 nucleotides or between about 100, about 120, about 140, about 160, about 180 or about 200 nucleotides (in some respects continuous nucleotides) between any two. In some respects, similarity or degree of identity is provided for the entire length of the reference sequence.

Homologous amino acid sequences can exhibit at least, at most, exactly 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% or between any two of 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity of the amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 95% identity of the amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 98% identity of the amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 99% identity of the amino acid residues.

A fragment or variant of an amino acid sequence (peptide or protein) may be a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequences from which it is derived, for example, it is functionally equivalent. For an antigen or antigenic sequence, a specific function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence, which comprises an amino acid sequence in which one or more amino acids are changed compared to the amino acid sequence of a parent molecule or sequence, and which is still able to achieve one or more functions of the parent molecule or sequence, such as inducing an immune response. In one aspect, the modification in the amino acid sequence of a parent molecule or sequence does not significantly affect or change the properties of the molecule or sequence. The terms "mutant" of wild-type E. coli FimH protein, "mutant" of E. coli FimH protein, "E. coli FimH protein mutant" or "modified E. coli FimH protein" refer to a polypeptide that displays introduced mutations relative to the wild-type FimH protein and is immunogenic against the wild-type FimH protein.

An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof. For example, one of ordinary skill in the art will appreciate that antigens suitable for use herein can be altered so that they differ in sequence from the naturally occurring or native sequences from which they are derived while retaining the desired activity of the native sequence.

In the present disclosure, vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. A vector can be used to integrate a nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors. A vector can be an RNA vector or a DNA vector. In some aspects, a vector is a DNA molecule. In some aspects, a vector is a plasmid vector. In some aspects, a vector is a viral vector. Typically, an expression vector will contain the desired coding sequence and other appropriate sequences necessary for expressing an operably connected coding sequence in a specific host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are typically used to transform and amplify a certain desired fragment (typically a DNA fragment), and may lack the functional sequences required for expressing the desired fragment.

As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be an immunogenic composition. In some aspects, the active agent is present in a unit dosage amount suitable for use in a treatment regimen, and when applied to a relevant population, it exhibits a statistically significant probability of achieving a predetermined therapeutic effect. In some aspects, the pharmaceutical composition may be specifically formulated for parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection, such as as a sterile solution or suspension or a sustained release formulation.

As used herein, the term "vaccination" refers to the administration of an immunogenic composition intended to produce an immune response, such as an immune response to a disease-associated (e.g., disease-causing) pathogen (e.g., bacteria). In some aspects, vaccinations can be administered before, during, and/or after exposure to disease-associated pathogens, while in some aspects, vaccinations can be administered shortly before, during, and/or after exposure to pathogens. In some aspects, vaccinations include multiple administrations of vaccine compositions at appropriate time intervals. In some aspects, vaccinations produce an immune response to infectious pathogens. In some aspects, vaccinations produce an immune response to tumors; in some of these aspects, vaccinations are "personalized" because they are partially or entirely directed to epitopes (e.g., which may be or include one or more new epitopes) that are determined to be present in a specific individual tumor.

An immune response refers to a humoral response, a cellular response, or both a humoral and a cellular response of an organism. An immune response can be measured by a variety of assays, including but not limited to assays that measure the presence or quantity of antibodies that specifically recognize proteins or cell surface proteins, assays that measure T cell activation or proliferation, and/or assays that measure the activity of one or more cytokines or the regulation of expression.

As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some aspects, two or more regimens may be administered simultaneously; in some aspects, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen are administered); in some aspects, such agents are administered with overlapping dosing regimens. In some aspects, the "administration" of a combination therapy may involve administering one or more agents or treatments to a subject receiving other agents or treatments in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or not even necessarily simultaneously), although in some aspects, two or more agents or their active portions may be administered together in a combination composition, or may even be administered together in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Those skilled in the art will appreciate that the term "dosage regimen" can be used to refer to a group of unit doses (usually more than one) administered to a subject individually, usually separated by time periods. 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 multiple doses, each of which is separated from other doses in time. In some aspects, each dose is separated from each other by a time period of the same length; in some aspects, a dosing regimen comprises multiple doses and at least two different time periods separating each dose. In some aspects, all doses within a dosing regimen are the same unit dose amount. In some aspects, different doses within a dosing regimen have different amounts. In some aspects, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses of a second dose amount different from the first dose amount. In some aspects, a dosing regimen comprises a first dose of a first dose amount, followed by one or more additional doses of a second dose amount identical to the first dose amount. In some aspects, a dosing regimen is associated with a desired or beneficial result when administered in a relevant population (e.g., a therapeutic dosing regimen).

II. Escherichia coli fimbriae antigen H (FimH)

As used herein, the term "FimH antigenic polypeptide" includes any FimH polypeptide or an immunogenic mutant thereof, including but not limited to the FimH polypeptide shown in SEQ ID No: 1-64, 67, 69, 71 or 73.

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

FimH antigen polypeptides are described in PCT International Publication No. WO2022/137078, which is incorporated herein by reference in its entirety.

Embodiments of the present disclosure provide RNA (eg, mRNA) vaccines comprising a polynucleotide encoding an E. coli FimH antigen. The E. coli FimH RNA vaccines provided herein can be used to induce a balanced immune response, including cellular immunity and humoral immunity.

Some embodiments provide an E. coli vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a FimH protein and a pharmaceutically acceptable carrier or excipient, formulated in cationic lipid nanoparticles. In some embodiments, the FimH protein is selected from FimH-DSG, FimH-DSG triple mutant (G15A, G16A, V27A) or FimH _LD triple mutant (G15A, G16A, V27A).

As used herein, the term "TM" when used in conjunction with an antigen shall refer to a triple mutant, particularly a triple mutant of a FimH _LD or FimH-DSG polypeptide having mutations at amino acid positions G15A, G16A and V27A. Thus, the terms "FimH-DSG triple mutant (G15A, G16A, V27A)" and "FimH-DSG TM" are interchangeable. In addition, "FimH _LD triple mutant (G15A, G16A, V27A)" and "FimH _LD TM" are interchangeable.

As used herein, the abbreviation "Ct" refers to the C-terminal domain of a polypeptide or polynucleotide.

Some embodiments provide a method for preventing or treating E. coli infection, comprising administering any vaccine described herein to a subject. In some embodiments, the antigen-specific immune response comprises a T cell response. In some embodiments, the antigen-specific immune response comprises a B cell response. In some embodiments, the antigen-specific immune response comprises a T cell response and a B cell response. In some embodiments, the method for producing an antigen-specific immune response involves a single administration of the vaccine. In some embodiments, the vaccine is administered to a subject by intradermal injection, intramuscular injection, subcutaneous injection, intranasal inoculation, or oral administration.

In some embodiments, the RNA (eg, mRNA) polynucleotide or a portion thereof can encode one or more polypeptides of E. coli FimH or fragments thereof as an antigen.

III. RNA molecules

In some aspects, RNA molecules as described herein are coding RNA molecules. Coding RNA includes functional RNA molecules that can be translated into peptides or polypeptides. In some aspects, coding RNA molecules include at least one open reading frame (ORF) encoding at least one peptide or polypeptide. Open reading frame includes codon sequences that can be translated into peptides or proteins. Coding RNA molecules can include one (monocistron), two (bicistrons) or more (polycistronic) OFRs, which can be codon sequences that can be translated into target polypeptides or proteins.

There are a variety of mRNA vaccine platforms in the prior art. The basic structure of in vitro transcribed (IVT) mRNA is very similar to "mature" eukaryotic mRNA and consists of: (i) a protein-coding open reading frame (ORF) flanked by (ii) 5' and 3' untranslated regions (UTRs), and terminated by (iii) a 7-methylguanosine 5' cap structure and (iv) a 3' poly (A) tail. Non-coding structural features play an important role in mRNA pharmacology and can be individually optimized to modulate mRNA stability, translation efficiency, and immunogenicity.

By incorporating modified nucleosides, mRNA transcripts known as "nucleoside-modified mRNA" or "modRNA" can be generated with reduced immunostimulatory activity, thus achieving an improved safety profile. In addition, modified nucleosides allow the design of mRNA vaccines with greatly enhanced stability and translation capacity, because they can avoid direct antimicrobial pathways induced by IFN-type, which are programmed to degrade and inhibit invading mRNA. For example, replacing uridine with pseudouridine in in vitro transcribed (IVT) mRNA reduces the activity of 2'-5'-oligoadenylate synthetase, an enzyme that regulates the cleavage of mRNA by RNase L. In addition, a decrease in the activity of protein kinase R, an enzyme involved in the inhibition of the mRNA translation process, was measured.

In addition to the incorporation of modified nucleotides, other approaches have been shown to improve the translational capacity and stability of mRNA. One example is the development of "sequence engineered mRNA". In this context, mRNA expression can be significantly increased by sequence optimization of the ORF and UTR of the mRNA, for example by enriching the GC content, or by selecting the UTR of a natural long-lived mRNA molecule.

In addition, some modifications are made to the terminal structure of mRNA. The anti-reverse cap (ARCA) modification can ensure the correct cap orientation at the 5' end, which produces an almost complete mRNA portion that can effectively bind to ribosomes. Other cap modifications, such as phosphorothioate cap analogs, can further improve the affinity for eukaryotic translation initiation factor 4E and increase resistance to RNA decapping complexes.

Conversely, by modifying its structure, the potency of mRNA in triggering innate immune responses can be further improved, but at the expense of translational capacity. Stabilizing mRNA by using a phosphorothioate backbone, or by precipitation with the cationic protein protamine, can reduce antigen expression but achieve greater immunostimulatory capacity.

In one aspect, the present invention relates to an immunogenic composition comprising an mRNA molecule encoding one or more polypeptides of Escherichia coli FimH or a fragment thereof as an antigen. In some embodiments, the mRNA molecule comprises a nucleoside-modified mRNA. The RNA molecule can encode one or more target polypeptides, such as an antigen 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 can also encode more than one target polypeptide, such as an antigen, such as a bicistronic or tricistronic RNA molecule encoding different or identical antigens.

The sequence of RNA molecule can be codon optimized or go optimized to express in the host (for example human cell) of expectation.In some respects, target gene (for example, antigen) as described herein is encoded by encoding sequence, and described encoding sequence is codon optimized and/or its guanosine/cytidine (G/C) content increases compared with wild-type encoding sequence.In some respects, compared with the corresponding sequence region of wild-type encoding sequence, one or more sequence regions of encoding sequence are codon optimized and/or G/C content increases.In some respects, codon optimized and/or increase the sequence of the amino acid sequence that G/C content can not change coding.

The term "codon optimization" is understood by those skilled in the art to refer to the codons of the coding region of a nucleic acid molecule to reflect the typical codon selection of the host organism, but without changing the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, in some aspects, the coding region is codon optimized to achieve optimal expression in a subject to be treated with the RNA polynucleotides described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of tRNA molecules in the cell. Therefore, the RNA sequence can be modified so that the codons of the frequently occurring tRNA molecules are inserted in place of "rare codons".

In some respects, the G/C content of the coding region of RNA (for example, target gene sequence) is compared with the G/C content of the corresponding coding sequence of the wild-type RNA encoding the target gene and increases, wherein in some respects, the amino acid sequence of this RNA encoding is compared with the amino acid sequence of wild-type RNA encoding and is not modified.This modification of RNA sequence is based on such a fact: the sequence in any RNA region to be translated is important for the effective translation of this mRNA.The sequence that G (guanosine)/C (cytidine) content increases is more stable than the sequence that A (adenosine)/U (uridine) content increases.In view of the fact that several codons encode a kind of and identical amino acid (so-called genetic codon degeneracy), it is possible to determine that the codon (so-called alternative codon selection) that is most favorable to stability.According to the amino acid of RNA encoding, compared with its wild-type sequence, the modification of RNA sequence has multiple possibilities.Specifically, the codon containing A and/or U nucleoside 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 A and/or U nucleoside of lower content. Thus, in some aspects, the G/C content of the RNA coding region described herein is increased by at least, at most, exactly 10%, 20%, 30%, 40%, 50%, 55% or even more, or between any two of 10%, 20%, 30%, 40%, 50%, 55% or even more, compared to the G/C content of the wild-type RNA coding region.

In some aspects, the RNA molecule comprises from about 20 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 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 00, 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).

In some aspects, the RNA molecule has 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, 420 0, 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, 22 000, 24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 4 or between any two of 4000, 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.

In some aspects, the RNA molecule comprises at least 100 nucleotides. For example, in some aspects, the length of the 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; between 7,000 and 25,000 nucleotides. In some aspects, the RNA molecule has 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, ,2050,2100,2150,2200,2250,2300,2350,2400,2450,2500,2550,2600,2650,2700,2750,2800,2850,2900,2950,3000,3050,3100,3150,3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 40 50, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 525 0, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050 , 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 81 00, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 930 0, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10 100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 111 00, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 1 1800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12700, 12750, 12 800, 12850, 12900, 12950, 13000, 13050, 13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450 , 1400, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000 nucleotides.

The mRNA useful in the present disclosure generally includes a first region (e.g., a coding region) encoding a linked nucleoside of a polypeptide of interest, a first flanking region (e.g., a 5'-UTR) located at the 5'-end of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3'-end of the first region, at least one 5' cap region and a 3' stabilizing region. In some embodiments, the mRNA of the present invention also includes a poly A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, the mRNA of the present invention may contain one or more intronic nucleotide sequences that can be excised from the polynucleotide. In some embodiments, the mRNA of the present invention may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence and/or a polyadenylation signal. Any region of a nucleic acid may include one or more alternative components (e.g., alternative nucleosides). For example, the 3'-stabilizing region can contain alternative nucleosides, such as L-nucleosides, inverted thymidines, or 2'-0-methyl nucleosides, and/or the coding region, 5'-UTR, 3'-UTR, or cap region can include alternative nucleosides, such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine), and/or 5-substituted cytidines (e.g., 5-methyl-cytidine).

In some embodiments, the RNA disclosed herein comprises the following components in the 5' to 3' direction: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region (5'UTR) comprising a cap-proximal sequence, a sequence encoding a payload (e.g., E. coli FimH protein); a 3' untranslated region (3'UTR); and a poly A sequence.

In some embodiments, LNP includes one or more RNA, and one or more RNA, lipid and its amount can be selected to provide a specific N: P ratio. The N in the composition: P ratio refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In general, a lower N: P ratio is preferred. One or more RNA, lipid and its amount can 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 certain embodiments, the N: P ratio can 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 about 5.0:1, about 5.5:1, about 6.0:1, about 6.5:1, or about 7.0:1.

A. Modified Nucleobases

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

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

The mRNA of the present invention may include one or more alternative components, as described herein, which confer useful properties, including increased stability and/or lack of significant induction of the innate immune response of the cells into which the polynucleotides are introduced. For example, compared with the corresponding unmodified mRNA, the modRNA may show reduced degradation in the cells into which the modRNA is introduced. These alternative species can enhance the efficiency of protein production, the intracellular retention of the polynucleotides and/or the viability of the contacted cells, and have reduced immunogenicity.

The mRNA of the present invention can include one or more modified (for example, changed or alternative) core bases, nucleosides, nucleotides or their combinations. Useful mRNA in LNP can include any useful modification or change, for example, modification or change of the key between core base, sugar or nucleoside (for example, to connecting phosphate/to phosphodiester bond/to phosphodiester backbone). In certain embodiments, each in the key between core base, sugar and nucleoside has change (for example, one or more changes). Changes according to the present disclosure can be changes of ribonucleic acid (RNA), for example, 2'-OH of ribofuranosyl ring is replaced with 2'-H, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or its hybrid.

The mRNA of the present invention may or may not be uniformly changed along the entire length of the molecule. For example, in the mRNA, or in a given predetermined sequence region thereof, one or more or all types of nucleotides (e.g., purine or pyrimidine, or one or more or all of A, G, U, C) may or may not be uniformly changed. In some cases, all nucleotides X in the mRNA (or in a given sequence region thereof) are changed, wherein X can be any one of the nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

There may be different sugar changes and/or internucleoside bonds (e.g., backbone structures) at multiple positions of a polynucleotide. It will be appreciated by those of ordinary skill in the art that nucleotide analogs or other changes may be located at any position of a polynucleotide such that the function of the polynucleotide is not substantially reduced. The change may also be a 5' or 3'-terminal change. In some embodiments, a polynucleotide includes a change at the 3'-terminal. A polynucleotide may contain from about 1% to about 100% alternative nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intervening 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 %, 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 will be appreciated that any remaining percentages are explained by the presence of canonical nucleotides (e.g., A, G, U, or C).

Polynucleotide can contain minimum zero and maximum 100% alternative nucleotide, or any percentage therebetween, for example at least 5% alternative nucleotide, at least 10% alternative nucleotide, at least 25% alternative nucleotide, at least 50% alternative nucleotide, at least 80% alternative nucleotide or at least 90% alternative nucleotide.For example, polynucleotide may contain alternative pyrimidine, for example 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% uracil is replaced by alternative uracil (for example, 5-substituted uracil) in polynucleotide.Alternative uracil can be replaced by the compound with single unique structure or can be replaced by a plurality of compounds with different structures (for example, 2,3,4 or more unique structures). In some cases, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosines in the polynucleotide are replaced by alternative cytosines (e.g., 5-substituted cytosines). Alternative cytosines can be replaced by compounds with a single unique structure or can be replaced by multiple compounds with different structures (e.g., 2, 3, 4 or more unique structures).

In some cases, the nucleic acid does not substantially induce an innate immune response in the cell into which the polynucleotide (e.g., mRNA) is introduced. Characteristics of the induced innate immune response include 1) increased expression of proinflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.), and/or 3) termination or reduction of protein translation.

In some embodiments, mRNA comprises one or more alternative nucleosides or nucleotides.Alternative nucleosides and nucleotides can include alternative core bases.The core base of nucleic acid is an organic base, such as purine or pyrimidine or its derivatives.Core base can be a standard base (e.g., adenine, guanine, uracil, thymine and cytosine).These core bases can be changed or completely replaced to provide a polynucleotide molecule with enhanced properties, for example, enhanced stability (such as resistance to nuclease).Non-standard or modified bases can include, for example, one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy and/or thio substitution; one or more condensed rings or open rings; oxidation; and/or reduction.

In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides with alternative uracils include pseudouracil (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (S ² U), 4-thiouracil (S ⁴ U), 4-thiopseudouracil (S 4 ψ), 2-thiopseudouracil (S 2 ψ), 5-hydroxyuracil (ho ⁵ U), 5-aminoallyluracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil), 3-methyluracil (m ³ U), 5-methoxyuracil (mo ⁵ U), uracil 5-hydroxyacetic acid (cmo ⁵ U), uracil 5-hydroxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyluracil (cm ⁵ U), 1-carboxymethyl pseudouridine, 5-carboxyhydroxymethyluracil (chm ⁵ U), 5-carboxyhydroxymethyluracil methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyluracil (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thiouracil (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-carboxymethylaminomethyluracil (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thiouracil (cmnmVu), 5-propargyl uracil, 1-propargyl-pseudouracil, 5-taurylmethyl-uracil (xm ⁵ U), 1-taurylmethyl-pseudouridine, 5-taurylmethyl-2-thiouracil (xm ⁵ s ² U), 1-taurylmethyl-4-thio-pseudouridine, 5-methyl-uracil (m ⁵ U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (mV), 5-methyl-2-thiouracil (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (ms4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m\|/), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl) uracil (acpU), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acpψ), 5-(isopentenylaminomethyl) uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2'-0-dimethyl-uridine (m5Um), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mem Um), 5-carbamoylmethyl-2'-0-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm5Um), 3,2'-0-dimethyl-uridine (mUm) and 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxyethenyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyl-uracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propyleneamino)]uracil. "Pseudouridine" is an example of a modified nucleoside and is an isomer of uridine in which the uracil is linked to the pentose ring by a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides with alternative cytosines include 5-azacytosine, 6-azacytosine, pseudoisocytosine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methylcytosine (m4C), 5-methylcytosine (m5C), 5-halocytosine (e.g., 5-iodo-cytosine), 5-hydroxymethylcytosine (hm5C), 1-methylpseudoisocytosine, pyrrolocytosine, pyrrolopseudoisocytosine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thiopseudoisocytosine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebular ine), 5-aza-zebularine, 5-methyl-1-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine (k2C), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acetyl-2'-0-methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formyl-2'-0-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxycytosine, 5-(3-azidopropyl)cytosine and 5-(2-azidoethyl)cytosine.

In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides with an alternative adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7- Deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (m1A), 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-glycylaminoformyl-adenine (g6A), N6-threonylaminoformyl-adenine (t6A), N6-methyl-N6-threonylaminoformyl-adenine (m6t6A), 2-methylthio-N6-threonylaminoformyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A) , N6-hydroxynorvalylaminoformyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylaminoformyl-adenine (ms2hn6A), N6-acetyladenine (ac6A), 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, N6,2'-0-dimethyladenosine (m6Am), N6,N6,2'-0-trimethyladenosine (m62Am), 1,2'-0-dimethyladenosine (ml Am), 2-amino-N6-methylpurine, 1-thioadenine, 8-azidoadenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyladenine, N6-formyladenine and N6-hydroxymethyladenine.

In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides with alternative guanines include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxy wyosine (o2yW), hydroxy wyosine (OHyW), low-modified hydroxy wyosine (OHyW*), 7-deaza-guanine, braided glycoside (Q), epoxy braided glycoside (oQ), galactosyl galQ, mannosyl galQ, 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl), archaeosin (G+), 7-deaza-8-aza-guanine, 6-thioguanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl -6-thio-guanine, N2-methyl-2'-0-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-0-methyl-guanosine (m22Gm), 1-methyl-2'-0-methyl-guanosine (mlGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'-0-methyl-inosine (Im), 1,2'-0-dimethyl-inosine (mllm), 1-thio-guanine and O-6-methyl-guanine.

The alternative nucleobase of nucleotide can be purine, pyrimidine, purine or pyrimidine analog independently.For example, nucleobase can be the alternative of adenine, cytosine, guanine, uracil or hypoxanthine.In another embodiment, nucleobase can also include for example naturally occurring and synthetic base derivatives, including pyrazolo [3,4-d] pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propargyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen (for example 8-bromine), 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; or 1,3,5-triazine. When the abbreviations A, G, C, T or U are used to describe a nucleotide, each letter refers to a representative base and/or its derivatives, for example, A includes adenine or an adenine analog, such as 7-deazaadenine.

In some aspects, the RNA molecule comprises a nucleic acid sequence in which at least one uridine is replaced by a pseudouridine. In some aspects, the RNA molecule comprises a nucleic acid sequence 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%, 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%, In some aspects, the RNA molecule comprises a nucleotide sequence in which all uridines are replaced by pseudouridines.

B.5'CAP

mRNA may contain a 5'-cap structure. The 5'-cap structure of the polynucleotide is involved in nuclear export and increases the stability of the polynucleotide, and binds to the mRNA cap binding protein (CBP), which is responsible for the stability of the polynucleotide in the cell, and causes translation competence by binding of CBP to poly A binding protein to form a mature circular mRNA species. The cap further helps remove 5'-proximal introns during mRNA splicing.

Endogenous polynucleotide molecules may be 5'-capped, creating a 5'-ppp-5'-triphosphate linkage between the terminal guanylate cap residue of the polynucleotide and the sense nucleotide transcribed at the 5'-terminus. The 5'-guanylate cap may then be methylated, creating an N7-methylguanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides at the 5' end of the polynucleotide may also be optionally 2'-O-methylated. 5'-decapping by hydrolysis and cleavage of the guanylate cap structure can target polynucleotide molecules, such as mRNA molecules, for degradation.

Alterations of the polynucleotide may generate a non-hydrolyzable cap structure that prevents decapping, thereby increasing the half-life of the polynucleotide. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, alternative nucleotides may be used during the capping reaction. For example, Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with α-thioguanosine nucleotides to generate a phosphorothioate linkage in the 5'-ppp-5' cap according to the manufacturer's instructions.

Additional alternative guanosine nucleotides can be used, such as α-methylphosphonic acid and selenophosphate nucleotides. Additional changes include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminal and/or 5'-preterminal nucleotides of the polynucleotide on the 2'-hydroxyl of the sugar (as described above). A variety of different 5'-cap structures can be used to generate the 5'-cap of the mRNA molecule.

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

The cap can be a dinucleotide cap analog. As a non-limiting example, a dinucleotide cap analog can be modified with a boranophosphate group or a phophoroselenoate group at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Pat. No. 8,519,110, the cap structure of which is incorporated herein by reference.

Alternatively, the cap analog can be a N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and/or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5)ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5)ppp(5')G cap analogs (see, e.g., Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for a variety of cap analogs and methods for synthesizing cap analogs; their cap structures are incorporated herein by reference). In other cases, the cap analog useful in the polynucleotides of the present disclosure is a 4-chloro/bromophenoxyethyl analog.

Although cap analogs allow simultaneous capping of polynucleotides during in vitro transcription reactions, up to 20% of transcripts remain uncapped. This phenomenon, along with the structural differences between cap analogs and the endogenous 5'-cap structure of polynucleotides produced by the endogenous cellular transcription machinery, may lead to reduced translational capacity and cellular stability.

Enzymes can also be used to cap alternative polynucleotides after transcription to produce more realistic 5'-cap structures. As used herein, the phrase "more realistic" refers to features that closely reflect or mimic endogenous or wild-type features in structure or function. That is, "more realistic" features better represent endogenous, wild-type, natural or physiological cell functions and/or structures compared to synthetic features or analogs of the prior art, or are superior to corresponding endogenous, wild-type, natural or physiological features in one or more aspects. Non-limiting examples of more realistic 5'-cap structures useful in the polynucleotides of the present disclosure are those structures that have enhanced cap-binding protein binding, increased half-life, reduced sensitivity to 5'-endonucleases and/or reduced 5'-decapping compared to synthetic 5'-cap structures (or wild-type, natural or physiological 5'-cap structures) known in the art. For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can establish a canonical 5'-5'-triphosphate connection between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, wherein the cap guanosine contains N7-methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl group. This structure is referred to as a Capl structure. Compared to other 5' cap analog structures known in the art, the cap can produce higher translational capacity, cell stability, and reduced activation of cellular proinflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N, pN2p(Cap0), 7mG(5')ppp(5')NlmpNp(Cap 1), 7mG(5')-ppp(5')NlmpN2mp(Cap 2) and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up(Cap 4).

Because the alternative polynucleotide can be capped after transcription, and because the process is more efficient, nearly 100% of the mRNA can be capped, as opposed to about 80% when the cap analog is attached to the polynucleotide during an in vitro transcription reaction.

The 5'-terminal cap may include an endogenous cap or a cap analog. The 5'-terminal cap may include a guanosine analog. Useful guanosine analogs include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine. In some cases, the polynucleotide contains a modified 5'-cap. The modification on the 5'-cap can increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and improve the translation efficiency of the polynucleotide. The modified 5'-cap may include, but is not limited to, one or more of the following modifications: modification at the 2'- and/or 3'-position of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (which produces a carbocyclic ring) with a methylene moiety (CH2), modification at the triphosphate bridging portion of the cap structure, or modification at the nucleobase (G) portion.

C. Untranslated region (UTR)

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

In some aspects, UTR is derived from naturally abundant mRNA in a specific tissue (e.g., lymphoid tissue) in which mRNA expression is targeted. In some aspects, UTR increases protein synthesis. Not subject to the constraints of mechanism or theory, UTR can increase protein synthesis by increasing the time (information stability) of mRNA staying in translation polyribosomes and/or the rate (information translation efficiency) at which ribosomes start information translation. Therefore, UTR sequences may extend protein synthesis in a tissue-specific manner.

In some aspects, 5'UTR and 3'UTR sequences are calculated. In some aspects, 5'UTR and 3'UTR derive from naturally abundant mRNA in tissue. The tissue can be, for example, liver, stem cell or lymphoid tissue. Lymphoid tissue can include, for example, any of lymphocytes (for example, 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 aspects, 5'UTR and 3'UTR derive from alphavirus. In some aspects, 5'UTR and 3'UTR are from wild-type alphavirus.

In some aspects, the RNA disclosed herein comprises a 5'UTR. If present, the 5'UTR is located at the 5' end and starts from the transcription start site upstream of the start codon of the protein coding region. The 5'UTR is located downstream of the 5' cap (if present), for example, directly adjacent to the 5' cap. The 5'UTR may contain a variety of regulatory elements, such as a 5' cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in controlling translation initiation.

In some aspects, the 5'UTR disclosed herein comprises a cap-proximal sequence, e.g., as disclosed herein. In some aspects, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In some aspects, the cap-proximal sequence comprises nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

5'-UTR can be provided as the flanking region of mRNA.5'-UTR can be homologous or heterologous to the coding region found in the polynucleotide.Multiple 5'-UTRs can be included in the flanking region, and these 5'-UTRs can be the same sequence or different sequences.Any part of the flanking region (including the non-flanking region) can be codon optimized, and before and/or after codon optimization, any part may independently contain one or more different structural or chemical changes.

In order to change one or more properties of mRNA, a 5'UTR heterologous to the mRNA coding region can be engineered. The mRNA can then be applied to a cell, tissue, or organism, and the results, such as protein levels, localization, and/or half-life, can be measured to assess the beneficial effects that the heterologous 5'UTR may have on the mRNA. Variants of the 5'UTR can be used, in which one or more nucleotides, including A, T, C, or G, are added or removed at the end. The 5'UTR can also be codon optimized or altered in any manner described herein.

In some aspects, the RNA molecule includes a 5' untranslated region (5'-UTR). In some aspects, the 5'UTR comprises a sequence selected from any one of SEQ ID NO: 75 or 77. 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 to any one of SEQ ID NO: 75 or 77. In some aspects, the 5'UTR comprises a sequence selected from any one of SEQ ID NO: 75 or 77. In some aspects, the 5'UTR comprises a sequence consisting of any one of SEQ ID NO: 75 or 77.

In some aspects, the RNA disclosed herein comprises a 3'UTR. If present, the 3'UTR is located downstream of the protein coding sequence open reading frame, for example, downstream of the termination codon of the protein coding region. The 3'UTR is usually a part of the mRNA, which is located between the protein coding sequence and the poly A tail of the mRNA. Therefore, in some aspects, the 3'UTR is located upstream of the poly A sequence (if present), for example, directly adjacent to the poly A sequence. The 3'UTR may be involved in regulatory processes, including transcript cleavage, stability and polyadenylation, translation and mRNA localization.

3'UTR can also include elements that are not encoded in the template of the transcribed RNA, but are added after transcription during the maturation process, such as a poly A tail. The 3'UTR of mRNA will not be translated into an amino acid sequence. In some aspects, the RNA disclosed herein comprises a 3'UTR that comprises an F element and/or an I element. In some aspects, the 3'UTR or its proximal sequence comprises a restriction site. In some aspects, the restriction site is a BamHI site. In some aspects, the restriction site is a Xhol site.

In some aspects, the RNA molecules and RNA-LNPs include a 3' untranslated region (3'-UTR). In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 76 or 78. In some aspects, the 3'UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity to any one of SEQ ID NO: 76 or 78. In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 76 or 78. In some aspects, the 3'UTR comprises a sequence consisting of any one of SEQ ID NO: 76 or 78.

mRNA may include stem loops, such as but not limited to histone stem loops. The stem loop can be a nucleotide sequence of about 25 or about 26 nucleotides in length. The histone stem loop can be located at the 3' side of the coding region (e.g., at the 3'-end of the coding region). As a non-limiting example, the stem loop can be located at the 3' end of the polynucleotide described herein. In some cases, the mRNA includes more than one stem loop (e.g., two stem loops). The stem loop can be located at the second terminal region of the polynucleotide. As a non-limiting example, the stem loop can be located in the untranslated region (e.g., 3'-UTR) in the second terminal region. In some cases, the mRNA including the histone stem loop can be stabilized by adding a 3'-stable region (e.g., a 3'-stable region including at least one chain terminating nucleoside). Without wishing to be bound by theory, adding at least one chain terminating nucleoside can slow down the degradation of the polynucleotide, thereby increasing the half-life of the polynucleotide. In other cases, the mRNA including the histone stem loop can be stabilized by changing the 3' region of the polynucleotide, and the change can prevent and/or inhibit the addition of oligio (U). In some cases, the mRNA including the histone stem loop can be stabilized by adding oligonucleotides terminated with 3'-deoxynucleosides, 2', 3'-dideoxynucleosides, 3'-O-methyl nucleosides, 3-O-ethyl nucleosides, 3'-arabinoside and other alternative nucleosides known in the art and/or described herein. In some cases, the mRNA of the present disclosure may include a histone stem loop, a poly A region and/or a 5'-cap structure. The histone stem loop may be located before and/or after the poly A region. The polynucleotides including the histone stem loop and the poly A region sequence may include the chain termination nucleosides described herein. In other cases, the polynucleotides of the present disclosure may include a histone stem loop and a 5'-cap structure. The 5'-cap structure may include, but is not limited to, those structures described herein and/or known in the art. In some cases, the conservative stem loop region may include the miR sequence described herein. As a non-limiting example, the stem loop region may include the seed sequence of the miR sequence described herein. In another non-limiting example, the stem loop region may include the miR-122 seed sequence.

The mRNA may include at least one histone stem-loop and a poly A region or polyadenylation signal. In some cases, the polynucleotide encoding the histone stem-loop and the poly A region or polyadenylation signal may encode a pathogen antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and the poly A region or polyadenylation signal may encode a therapeutic protein. In some cases, the polynucleotide encoding the histone stem-loop and the poly A region or polyadenylation signal may encode a tumor antigen or a fragment thereof. In other cases, the polynucleotide encoding the histone stem-loop and the poly A region or polyadenylation signal may encode an allergenic antigen or an autoimmune self-antigen.

D. Open reading frame (ORF)

5' and 3' UTR can be operably connected to an open reading frame (ORF), which can be a codon sequence that can be 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. ORF can start with a start codon, for example, a combination of three subsequent nucleotides at its 5' end, usually encoding amino acid methionine (ATG or AUG), and a subsequent region, which usually shows a length of a multiple of 3 nucleotides. An open reading frame can 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 respects, an open reading frame can terminate with one, two, three, four or more stop codons, which is known in the art. An open reading frame can be isolated, or it can also be integrated into a longer nucleic acid sequence, for example in a vector or mRNA. An open reading frame can also be referred to as a "(protein) coding region" or "coding sequence".

As described herein, an RNA molecule can include one (monocistronic), two (bicistronic), or more (polycistronic) open reading frames.

The present disclosure provides RNA molecules comprising at least one open reading frame encoding an E. coli FimH polypeptide as described herein. In some aspects, the RNA molecule comprises at least one open reading frame encoding an E. coli FimH protein as described herein.

E. Target gene

The RNA molecules described herein may include a target gene. The target gene encodes a target polypeptide. Non-limiting examples of target polypeptides include, for example, biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secretory polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting moieties, those polypeptides encoded by the human genome for which therapeutic indications have not yet been identified but which have utility in the field of research and discovery, or combinations thereof. Those skilled in the art may use public and private databases (e.g. ) to easily identify the sequence of a specific gene of interest.

In some aspects, the RNA molecule includes the coding region of the target gene. In some aspects, the target gene is or comprises an antigenic polypeptide or its immunogenic variant or immunogenic fragment. In some aspects, the antigenic polypeptide comprises an epitope from an antigen. In some aspects, the antigenic polypeptide comprises a plurality of different epitopes from an antigen. In some aspects, the antigenic polypeptide comprising a plurality of different epitopes from an antigen is polyepitopic. In some aspects, the antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasite antigenic polypeptide, an antigenic polypeptide from an infectious pathogen, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide.

The term "antigen" may refer to a substance that can be recognized by the immune system (e.g., an adaptive immune system) and that can elicit an antigen-specific immune response, such as by forming antibodies and/or antigen-specific T cells as part of an adaptive immune response. An antigen may be or may include a peptide or protein that can be presented to a T cell by MHC. An antigen may be a provided nucleic acid molecule, such as a translation product of an RNA molecule comprising at least one coding sequence as described herein. In addition, fragments, variants, and derivatives of an antigen (e.g., a peptide or protein) comprising at least one epitope are also understood to be antigens.

In some aspects, RNA encoding a target gene (e.g., an antigen) is expressed in cells of a subject treated to provide a target gene (e.g., an antigen). In some aspects, RNA is transiently expressed in the cells of a subject. In some aspects, the expression of a target gene (e.g., an antigen) is on the cell surface. In some aspects, the target gene (e.g., an antigen) is expressed and presented in the context of MHC. In some aspects, the expression of a target gene (e.g., an antigen) enters the extracellular space, such as the antigen is secreted.

In some aspects, RNA molecules include the coding region of a target gene (e.g., an antigen). In some aspects, RNA molecules include the coding region of a target gene (e.g., an antigen derived from a pathogen associated with an infectious disease). In some aspects, RNA molecules include the coding region of a target gene (e.g., an antigen derived from an Escherichia coli fimbriae antigen (FimH)).

In some aspects, the RNA polynucleotides described herein or the compositions or pharmaceutical preparations comprising them comprise nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides comprise sequences having at least 80% identity to the nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides comprise sequences encoding polypeptides having at least 80% identity to the polypeptide sequences disclosed herein. In some aspects, the RNA polynucleotides described herein or the compositions or pharmaceutical preparations comprising them are transcribed by DNA templates. In some aspects, the DNA templates for transcribing the RNA polynucleotides described herein comprise sequences complementary to the RNA polynucleotides. In some aspects, the target genes described herein are encoded by the RNA polynucleotides described herein comprising the nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides encode polypeptides having at least 80% identity to the polypeptide sequences disclosed herein. In some aspects, the polypeptides described herein are encoded by RNA polynucleotides, which are transcribed by DNA templates comprising sequences complementary to the RNA polynucleotides.

In some aspects, the RNA molecule encodes a FimH protein comprising the sequence of any one of SEQ ID NO: 67, 69, 71 or 73, or a fragment or variant thereof.

In some aspects, the RNA molecule encodes an E. coli FimH protein synthesized from a nucleic acid sequence comprising any one of SEQ ID NOs: 66, 68, 70, 72, or 82-85, or a fragment or variant thereof.

F. Poly A tail

In some aspects, the RNA molecules disclosed herein comprise a polyadenylic acid (poly A) sequence, e.g., as described herein. In some aspects, the poly A sequence is located downstream of the 3'UTR, e.g., adjacent to the 3'UTR. A "poly A tail" or "poly A sequence" refers to a continuous stretch of adenine residues that can be attached to the 3' end of an RNA molecule. The poly A sequence is known to those skilled in the art and can be located after the 3'UTR in the RNA molecules described herein. The poly A tail can increase the half-life of the RNA molecule.

mRNA can include a poly A sequence and/or a polyadenylation signal. The poly A sequence can be composed of all or most of adenine nucleotides or their analogs or derivatives. The poly A sequence can be a tail located near the 3' untranslated region of the nucleic acid. During RNA processing, long chains of adenylic acid (poly A region) are usually added to messenger RNA (mRNA) molecules to increase the stability of the molecule. After transcription, the 3' end of the transcript is immediately cleaved to release the 3'-hydroxyl. Then, poly A polymerase adds adenine nucleotide chains to RNA. This process is called polyadenylation, adding a poly A region with a length of 100 to 250 residues. The unique length of the poly A region can provide certain advantages for the alternative polynucleotides of the present disclosure. Typically, the length of the poly A region of the present disclosure is at least 30 nucleotides. In another embodiment, the length of the poly A 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, on the alternative polynucleotide molecules described herein, the length of the poly A region can be 80 nucleotides, 120 nucleotides, 160 nucleotides. In other cases, on alternative polynucleotide molecules described herein, the length of the poly A district can be 20, 30, 40, 80, 100, 120, 140 or 160 nucleotides. In some cases, the poly A district is designed relative to the length of the overall alternative polynucleotide. The design can be based on the length of the coding region of the alternative polynucleotide, the specific features of the alternative polynucleotide (e.g., mRNA) or the length of the final product expressed from the alternative polynucleotide. When relative to any feature of the alternative polynucleotide (e.g., except for the mRNA portion including the poly A district), the length of the poly A district can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% longer than the additional feature. The poly A district can also be designed as a part of the alternative polynucleotide to which it belongs. In this context, the poly A region may 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 length of the poly A region.

In some cases, expression can be enhanced by the combination of engineered binding sites and/or mRNA with poly A binding proteins. The engineered binding sites can be sensor sequences that can serve as binding sites for ligands of the local microenvironment of the mRNA. As a non-limiting example, the mRNA can include at least one engineered binding site to change the binding affinity of poly A binding protein (PABP) and its analogs. The incorporation of at least one engineered binding site can increase the binding affinity of PABP and its analogs.

In addition, a variety of different mRNAs can be connected to PABP (poly A binding protein) through the 3' end using alternative nucleotides at the 3' end of the poly A region. Transfection experiments can be performed in relevant cell lines, and the production of protein can be detected by ELISA at 12 hours, 24 hours, 48 hours, 72 hours and the 7th day after transfection. As a non-limiting example, transfection experiments can be used to evaluate the effect of adding at least one engineered binding site on the binding affinity of PABP or its analogs. In some cases, the poly A region can be used to regulate translation initiation. Although it is not desired to be bound by theory, the poly A region recruits PABP, which can interact with the translation initiation complex and may therefore be essential for protein synthesis. In some cases, the poly A region can also be used in the present disclosure to protect from 3'-5'-nuclease digestion. In some cases, mRNA may include a poly A-G quadruplex. The G quadruplex is a cyclic hydrogen bond array of four guanosine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G quadruplex is incorporated into the end of the poly A region. The stability of the obtained mRNA, protein production, and other parameters including the half-life at multiple time points can be tested. It has been found that poly A-G quadruplexes result in protein production that is at least 75% of the protein production seen using a poly A region of 120 nucleotides alone. In some cases, the mRNA may include a poly A region and may be stabilized by adding a 3'-stabilizing region. The mRNA with the poly A region may also include a 5'-cap structure. In other cases, the mRNA may include a poly A-G quadruplex. The mRNA with the poly A-G quadruplex may also include a 5'-cap structure. In some cases, the 3'-stabilizing region that can be used to stabilize the mRNA includes a poly A region or a poly A-G quadruplex. In other cases, the 3'-stabilizing region that can be used with the present disclosure includes chain terminating nucleosides, 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'-deoxynucleosides or O-methyl nucleosides. In other cases, mRNAs including poly A regions or poly A-G quadruplets can be stabilized by altering the 3' region of the polynucleotide, which alterations can prevent and/or inhibit the addition of oligio (U). In still other cases, mRNAs that include poly A regions or poly A-G quadruplexes can be stabilized by the addition of oligonucleotides that terminate with 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3-O-methyl nucleosides, 3'-O-ethyl nucleosides, 3'-arabinoside, and other alternative nucleosides known in the art and/or described herein.

In one aspect, the RNA disclosed herein comprises a poly A tail comprising a sequence that is at least, at most, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO: 86, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the poly A tail comprises the sequence of SEQ ID NO: 86.

IV. RNA transcription

In some aspects, RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of the present disclosure, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, RNA can be translated into a peptide or protein.

According to the present disclosure, "transcription" includes "in vitro transcription" or "IVT", which refers to the process in which transcription occurs in vitro in a non-cell system to produce a synthetic RNA product, which can be used in a variety of applications, including, for example, the production of proteins or polypeptides. Cloning vectors can be used to produce transcripts. These cloning vectors are generally referred to as transcription vectors, and according to the present invention, they are covered by the term "vector". According to a specific aspect, the RNA used is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter controlling the transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. Preferably, the in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

The synthesized IVT RNA products can be translated in vitro or introduced directly into cells, where they can be translated. In terms of RNA, the term "expression" or "translation" refers to the process by which the mRNA chain in the ribosome of the cell guides the assembly of an amino acid sequence to produce a peptide or protein. Such synthetic RNA products include, for example, but are not limited to, mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, etc. The IVT reaction typically utilizes a DNA template (e.g., a linear DNA template) described and/or utilized herein, ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates) and an appropriate RNA polymerase.

In some aspects, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some aspects, the RNA disclosed herein is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter controlling transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In some aspects, starting materials for IVT can include linearized DNA template, nucleotides, RNase inhibitors, pyrophosphatase, and/or T7 RNA polymerase. In some aspects, the IVT process is performed in a bioreactor. The bioreactor can include a mixer. In some aspects, nucleotides can be added to the bioreactor throughout the IVT process.

In some aspects, after the IVT process, one or more post-IVT reagents are added to the IVT mixture containing RNA in the bioreactor. Exemplary post-IVT reagents can include DNAse I configured to digest the linearized DNA template and proteinase K configured to digest DNAse I and T7 RNA polymerase. In some aspects, the post-IVT reagents are incubated with the mixture in the bioreactor after IVT. In some aspects, the bioreactor can contain at least, at most, exactly, or between any two of 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. The IVT mixture can have an RNA concentration of at least, at most, just, or between any two of 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg/mL or more RNA.

In some aspects, the IVT mixture can include residual spermidine, residual DNA, residual protein, peptides, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+, Na+, Ca2+), RNA fragments, residual nucleotides, free phosphate, or any combination thereof.

In some aspects, at least a portion of the IVT mixture is filtered. The IVT mixture can be filtered by ultrafiltration and/or diafiltration to remove at least some impurities from the IVT mixture and/or to change the buffer solution of at least a portion of the IVT mixture to produce a concentrated RNA solution as a retentate.

In some aspects, "ultrafiltration" and "diafiltration" both refer to membrane filtration processes. Ultrafiltration typically uses a membrane with a pore size of at least, at most, just or between any two of the following: 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 aspects, ultrafiltration membranes are typically classified according to molecular weight cut-off (MWCO) rather than pore size. For example, the MWCO can be at least, at most, exactly, or between any two of the following: 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, kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa, 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. It will be appreciated by those skilled in the art that, depending on the application, the filter membrane can be made of different suitable materials, including, for example, polymers, cellulose, ceramics, etc. In some aspects, membrane filtration may be more desirable for large-capacity purification processes.

In some aspects, ultrafiltration and diafiltration of the IVT mixture used to purify RNA can include (1) direct flow filtration (DFF), also known as "dead-end" filtration, which applies a feed flow perpendicular to the membrane surface and attempts to pass 100% of the fluid through the membrane, and/or (2) tangential flow filtration (TFF), also known as cross-flow filtration, in which the feed flow passes parallel to the membrane surface, as a portion passes through the membrane (permeate), while the remainder (retentate) is retained and/or recycled back to the feed tank.

In some aspects, filtration of the IVT mixture is performed by TFF, which includes an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some aspects, the first diafiltration step is performed in the presence of ammonium sulfate. The first diafiltration step can be configured to remove a majority of impurities from the IVT mixture. In some aspects, the second diafiltration step is performed in the absence of ammonium sulfate. The second diafiltration step can be configured to transfer the RNA to a DS buffer formulation.

In the TFF process, a filter membrane with an appropriate MWCO can be selected for ultrafiltration. The MWCO of the TFF membrane determines which solutes can pass through the membrane into the filtrate and which solutes will be retained in the retentate. The MWCO of the TFF membrane can be selected so that substantially all the solutes of interest (e.g., the desired synthetic RNA material) are retained in the retentate, while undesirable components (e.g., excess ribonucleotides, small nucleic acid fragments (e.g., digested or hydrolyzed DNA templates), peptide fragments (e.g., digested proteins and/or other impurities) enter the filtrate. In some aspects, the retentate containing the desired synthetic RNA material can be recycled to the feed reservoir for re-filtration in additional cycles. In some aspects, the TFF membrane can have a MWCO that is equal to at least, at most, just, 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, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, In some aspects, the TFF membrane can have a MWCO of about 250-350kDa. In some aspects, the TFF membrane (e.g., a cellulose-based membrane) can have a MWCO of about 30-300kDa; in some aspects, about 50-300kDa, about 100-300kDa, or about 200-300kDa.

Diafiltration can be performed discontinuously or continuously. For example, in continuous diafiltration, the diafiltration solution can be added to the sample feed reservoir at the same rate as the filtrate is produced. In this way, the volume in the sample reservoir remains constant, but the small molecules (e.g., salts, solvents, etc.) that can freely permeate through the membrane are removed. Taking solvent removal as an example, each additional diafiltration volume (DV) will further reduce the solvent concentration. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting volume. The process is then repeated until the desired concentration of small molecules (e.g., salts, solvents, etc.) remaining in the reservoir is reached. Each additional diafiltration volume (DV) will further reduce the concentration of small molecules (e.g., solvents). Continuous diafiltration generally requires a minimum volume for a given reduction in the molecules to be filtered. On the other hand, discontinuous diafiltration allows rapid changes in the state of the retentate, such as pH, salt content, etc. In some aspects, a first diafiltration step is performed with a diafiltration volume equal to at least, at most, exactly, or between any two of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some aspects, a second diafiltration step is performed with a diafiltration volume equal to at least, at most, exactly, or between any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some aspects, a first diafiltration step is performed with 5 diafiltration volumes and a second diafiltration step is performed with 10 diafiltration volumes.

In some aspects, for ultrafiltration and/or diafiltration, the IVT mixture is filtered at a rate equal to at least, at most, exactly, or between any two of 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L/m2 of filtration area/hour, or faster. The concentrated RNA solution can contain at least, at most, exactly, or between any two of 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL of single-stranded RNA.

In some aspects, the bioburden of the concentrated RNA solution obtained by filtering the RNA product solution can also be reduced. One or more filters can be used to filter and reduce the bioburden. One or more filters can include a pore size of at least, at most, just 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.

As an example, reducing the bioburden can include discharging a retentate tank containing a retentate obtained by ultrafiltration and/or diafiltration to obtain a retentate. Reducing the bioburden can include flushing a filtration system for ultrafiltration and/or diafiltration with a wash buffer solution to obtain a wash tank solution containing residual RNA remaining in the filtration system. The retentate can be filtered to obtain a filtered retentate. The wash tank solution can be filtered using a first 0.2 μm filter to obtain a filtered wash tank solution. The retentate can be filtered using a first 0.2 μm filter or another 0.2 μm filter.

The filtered wash pool solution and the filtered retentate 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.

V. RNA Packaging

The RNA in the RNA product solution can be encapsulated, and the RNA solution can also include at least one encapsulating agent. In one aspect, the encapsulating agent includes lipids, lipid nanoparticles (LNP), lipid complexes (lipoplexes), polymer particles, polymer complexes (polyplexes) and overall (monolithic) delivery systems and combinations thereof.

Lipid nanoparticle can comprise lipid component and one or more additional components, for example therapeutic component and/or prevention component.LNP can be designed for one or more specific applications or targets.Can be based on specific applications or targets, and/or based on the effectiveness, toxicity, expense, ease of use, availability or other characteristics of one or more compositions to select the composition of LNP.Similarly, can be according to for example, the effectiveness and toxicity of specific composition combination, for specific application or target select specific formulation (formulation) of LNP.The effectiveness and tolerability of LNP preparation may be subject to the influence of formulation stability.

Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and/or prophylactic agents (e.g., RNA) to specific cells, tissues, organs, systems, or groups thereof in a mammal.

The physicochemical properties of lipid nanoparticles can be changed to increase the selectivity to specific body targets. For example, the particle size can be adjusted according to the fenestration sizes of different organs. The therapeutic agent and/or preventive agent included in the LNP can also be selected according to the desired delivery target or multiple targets. For example, a therapeutic agent and/or preventive agent can be selected for a specific indication, a patient's condition, a disease or a disorder and/or be delivered to a specific cell, tissue, organ or system or its group (such as local or specific delivery). In certain embodiments, LNP can include an mRNA encoding a polypeptide of interest, which can be translated to produce a polypeptide of interest in the cell. Such compositions can be designed to be delivered specifically to a specific organ. In some embodiments, the composition can be designed to be delivered specifically to a mammal's liver. In some embodiments, the composition can be designed to be delivered specifically to a lymph node. In some embodiments, the composition can be designed to be delivered specifically to a mammal's spleen.

In one aspect, the encapsulating agent is a lipid, and what is produced is the RNA encapsulated by lipid nanoparticles (LNP). Without being bound by any theory, it is believed that cationic or cationic ionizable lipids or lipid-like materials and/or cationic polymers are combined with nucleic acids to form aggregates, and this aggregation produces colloidally stable particles. Lipid can be a naturally occurring lipid or a synthetic lipid. However, lipid is generally a biological substance. Biological lipids are well known in the art, and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lyso-lipids, glycosphingolipids, glycolipids, sulfatides (sulphatide), lipids and polymerizable lipids and combinations thereof of fatty acids with ether and ester connections. Lipid is a material that is insoluble in water and can be extracted with an organic solvent. Compounds other than those specifically described herein are understood by those skilled in the art as lipids, and these compounds are included in the compositions and methods of the present disclosure. Lipid components and non-lipid components can be connected to each other in a covalent or non-covalent manner.

In some respects, LNP can be designed to protect RNA molecules (for example, mRNA) from the effect of extracellular RNases and/or can be designed to RNA systemically delivered to target cells. In some respects, when RNA molecules are intravenously administered to experimenters in need, such LNP may be particularly used to deliver RNA molecules (for example, mRNA, modRNA). In some respects, when RNA molecules are intramuscularly administered to experimenters in need, such LNP may be particularly used to deliver RNA molecules (for example, mRNA).

In one aspect, the RNA concentration in the RNA solution is <1 mg/mL. On the other hand, the concentration of RNA is at least about 0.05 mg/mL. On the other hand, the concentration of RNA is at least about 0.5 mg/mL. On the other hand, the concentration of RNA is at least about 1 mg/mL. On the other hand, the concentration of RNA is about 0.05 mg/mL to about 0.5 mg/mL. On the other hand, the concentration of RNA is at least 10 mg/mL. On the other hand, the concentration of RNA is at least 50 mg/mL. In some aspects, the concentration of RNA is at least, at most, just or between 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 any two of higher.

The disclosure provides RNA solution and lipid preparation mixture or its composition, it comprises at least one RNA encoding for example antigen (for example Escherichia coli FimH protein), described RNA is compounded with one or more lipids, is encapsulated in one or more lipids and/or is prepared together with one or more lipids, and forms lipid nanoparticle (LNP), liposome, lipid complex and/or nanoliposome.In some aspects, said composition comprises lipid nanoparticle.

Lipid nanoparticle or LNP refers to the particle of any form produced when cationic lipid and optional one or more other lipid combinations (for example in aqueous environment and/or in the presence of RNA).In some respects, lipid nanoparticle is included in preparation, and the preparation can be used for active agent or therapeutic agent, for example nucleic acid (for example, mRNA, modRNA) is delivered to the target position of purpose (for example, cell, tissue, organ, tumor etc.).In some respects, lipid nanoparticle of the present disclosure comprises nucleic acid.This type of lipid nanoparticle generally comprises cationic lipid and one or more excipients, for example one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids or its combination.In some respects, active agent or therapeutic agent, for example nucleic acid (for example, mRNA, modRNA), can be encapsulated in the lipid part of lipid nanoparticle or in the aqueous space wrapped by some or all lipid parts of lipid nanoparticle, thereby protecting it from other undesirable influences (for example unfavorable immune response) caused by enzymatic degradation or the mechanism of host organism or cell.Nucleic acid (for example, mRNA, modRNA) or its part can also be associated with lipid nanoparticle and compound. The lipid nanoparticle may comprise any lipid capable of forming a particle to which the nucleic acid is attached or within which one or more nucleic acids are encapsulated.

In some aspects, provided RNA molecules (e.g., mRNA, modRNA) can be formulated with LNPs. In some aspects, the lipid nanoparticles can have an average diameter of about 1 to 500 nm. In some aspects, the lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, just, or between any two of 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, and are substantially non-toxic. The term "mean diameter" refers to the average hydrodynamic diameter of the particles measured by dynamic laser scattering (DLS), using the so-called cumulant algorithm for data analysis, which provides the results as the so-called Z-average with a length dimension and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "mean diameter", "diameter", "size" or "mean size" of the particles is used synonymously with the Z-average.

The LNPs described herein may 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 less. For example, the LNPs can exhibit a polydispersity index of 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 aspects, the polydispersity index is calculated from dynamic light scattering measurements by so-called cumulant analysis, as mentioned in the definition of "mean diameter." Under certain prerequisites, it can be considered a measure of the size distribution of the nanoparticle population.

Lipid nanoparticles 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 LNP. Zeta potential can be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Dynamic light scattering can also be used to measure particle size. Instruments such as ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure the various characteristics of LNP, such as particle size, polydispersity index, and zeta potential.

The average size of the LNPs may be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). For example, the average size may be from about 40 nm to about 150 nm, for example, 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 LNP can be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In certain embodiments, the average size of LNP can be from about 70nm to about 100nm. In specific embodiments, the average size can be about 80nm. In other embodiments, the average size can be about 100nm.

LNP may be relatively uniform. Polydispersity index can be used to indicate the homogeneity of LNP, such as the particle size distribution of lipid nanoparticles. Small (e.g., less than 0.3) polydispersity index generally represents a narrow particle size distribution. LNP can have a polydispersity index of 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 LNP can be about 0.10 to about 0.20.

The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low charge (positive or negative) are generally desirable because the species with higher charge may interact undesirably with cells, tissues, and other components in the body. In some embodiments, the zeta potential of the LNP can 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.

In some aspects, when nucleic acid (for example, RNA molecule) is present in the LNP provided, it can resist the degradation of nuclease in aqueous solution.In some aspects, LNP is the lipid nanoparticle of targeting liver.In some aspects, LNP is the cationic lipid nanoparticle comprising one or more cationic lipids (for example, a kind of cationic lipid as described herein).In some aspects, cationic LNP can comprise at least one cationic lipid, at least one polymer-conjugated lipid and at least one auxiliary lipid (for example, at least one neutral lipid).

In certain aspects, the RNA solution and lipid formulation mixture or a composition thereof can have, have at least, or have at least, at most, just, or between any two of the following: 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 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about %, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 3%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% of a particular lipid, lipid type, or non-lipid component (e.g., lipid-like material and/or cationic polymer or adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or known to those of skill in the art).

LNP as described herein can be prepared using a variety of methods, which may involve obtaining a colloid from at least one cationic or cationic ionizable lipid or lipid-like material and/or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain nucleic acid particles. As used herein, the term "colloid" refers to a class of uniform mixtures in which dispersed particles are not precipitated. The insoluble particles in the mixture are microscopic, with a particle size between 1 and 1000 nanometers. The mixture can be referred to as a colloid or colloidal suspension. Sometimes, the term "colloid" refers only to the particles in the mixture rather than the entire suspension.

In order to prepare a colloid comprising at least one cationic or cationic ionizable lipid or lipid-like material and/or at least one cationic polymer, conventional methods for preparing liposome vesicles and appropriately adjusted can be applied herein. The most commonly used methods for preparing liposome vesicles have the following basic stages: (i) lipid dissolution in an organic solvent, (ii) drying the resulting solution, and (iii) hydration of the dried lipids (using a variety of aqueous media). In the film hydration method, the lipids are first dissolved in a suitable organic solvent and then dried to form a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposome dispersion. In addition, an additional size reduction step may also be included.

Reverse phase evaporation is an alternative method for thin film hydration of liposome vesicles, which involves the formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of the mixture is required to homogenize the system. Removal of the organic phase under reduced pressure yields a milky gel, which is subsequently converted into a liposome suspension.

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, such as 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 (e.g., cationic lipids and additional 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.

The term "extrusion" or "extrusion" refers to the creation of particles with a fixed cross-sectional profile. Specifically, it refers to the reduction of particle size, thereby forcing the particles to pass through a filter with a defined pore size.

According to the present invention, other methods having organic solvent-free properties can also be used to prepare colloids.

In some respects, can under the condition of triggering lipid component solubility sudden change (it drives lipid to self-assemble in the form of LNP), by quickly mixing RNA solution as herein described (for example, RNA product solution) and lipid preparation as herein described (comprising for example, at least one cationic lipid in organic solvent and optional one or more other lipid components) to produce the RNA of LNP encapsulation.In some respects, suitable buffer comprises tris (tris), histidine, citrate, acetate, phosphate or succinate.The pH value of liquid preparation is relevant with the pKa of encapsulating agent (for example cationic lipid).The pH value of acidified buffer can be at least half pH value (pH scale) less than the pKa value of encapsulating agent (for example cationic lipid), and the pH value of final buffer can be at least half pH value (pH scale) greater than the pKa value of encapsulating agent (for example cationic lipid).In some respects, select the property of cationic lipid so that by being combined with the oppositely charged backbone of nucleic acid (for example, RNA), the primary formation of particle occurs. In this way, particles are formed around the nucleic acid, which can, for example, in some aspects result in an encapsulation efficiency that is much higher than that achieved in the absence of interaction between the nucleic acid and the at least one lipid component.

The encapsulation efficiency of therapeutic and/or prophylactic agent describes the amount of therapeutic and/or prophylactic agent encapsulated or otherwise associated with LNP after preparation relative to the initial amount provided. It is expected that the encapsulation efficiency is high (e.g., close to 100%). For example, the encapsulation efficiency can be measured by comparing the amount of therapeutic and/or prophylactic agent in the solution containing lipid nanoparticles before and after breaking the lipid nanoparticles with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agent (e.g., RNA) in the solution. For lipid nanoparticles as described herein, the encapsulation efficiency of therapeutic and/or prophylactic agent can be at least 50%, such as 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 can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

The LNP may optionally include one or more coatings. For example, the LNP may be formulated into a capsule, a film, or a tablet having a coating. The capsule, film, or tablet comprising the compositions described herein may have any useful size, tensile strength, hardness, or density.

The preparation comprising amphiphilic polymers and lipid nanoparticles can be formulated as a pharmaceutical composition in whole or in part. The pharmaceutical composition can include one or more amphiphilic polymers and one or more lipid nanoparticles. For example, the pharmaceutical composition can include one or more amphiphilic polymers and one or more lipid nanoparticles including one or more different therapeutic agents and/or preventive agents. The pharmaceutical composition can also include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the preparation and manufacture of pharmaceutical compositions and medicaments can be found in, for example, Remington's The Science and Practice of Pharmacy, 21st edition, A.R.Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, unless any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the LNP in the preparation of the present disclosure or one or more amphiphilic polymers. If its combination with the components or amphiphilic polymers may cause any undesirable biological effects or other harmful effects, the excipient or auxiliary ingredient may be incompatible with the amphiphilic polymers of the components of the LNP or the preparation.

In some embodiments, one or more excipients or auxiliary components may account for more than 50% of the total mass or volume of the pharmaceutical composition including LNP. For example, one or more excipients or auxiliary components may account for 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration (United States Food and Drug Administration). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (United States Pharmacopoeia (USP)), the European Pharmacopoeia (European Pharmacopoeia (EP)), the British Pharmacopoeia (British Pharmacopoeia) and/or the International Pharmacopoeia (International Pharmacopoeia). According to the present disclosure, the relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients and/or any additional ingredients in the pharmaceutical composition will vary according to the identity, size and/or condition of the subject being treated, and also according to the route by which the composition is to be administered. For example, a pharmaceutical composition may include 0.1% to 100% (wt wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition may include 0.1% to 15% (wt/vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10% or 12.5% w/v).

In certain embodiments, the lipid nanoparticles and/or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and/or transportation (e.g., stored at a temperature of 4°C or less, such as a temperature between about -150°C and about 0°C or a temperature 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, a pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., by lyophilization) that is stored at, for example, about -20°C. In some embodiments, the present disclosure also relates to a method for increasing the stability of lipid nanoparticles by adding an effective amount of an amphiphilic polymer and storing the lipid nanoparticles and/or pharmaceutical compositions thereof at a temperature of 4°C or less (e.g., a temperature between about -150°C and about 0°C or a temperature between about -80°C and about -20°C, such as 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).

The chemical properties of the LNPs, LNP suspensions, lyophilized LNP compositions or LNP formulations of the present disclosure can be characterized by a variety of methods. In some embodiments, electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reversed phase liquid chromatography) can be used to check mRNA integrity.

In some embodiments, the LNP integrity of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure is about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more.

In some embodiments, the LNP integrity of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present 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, about 100% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, about 200% or more, about 210% or more, about 220% or more, about 230% or more, about 240% or more, about 250% or more, about 260% or more, about 270% or more, about 280% or more, about 290% or more, about 300% or more, about 310% or more, about 320% or more, about 330% or more, about 340% or more, about 350% or more, about 360% or more, about 370% or more, about 380% or more, about 390% or more, about 400% or more, about 410% or more, about 420% or more, about 430% or more, about 440% or more, about 450% or more, about 460% or more, about 470% or more, about 480% or more, about 490% or more, about 500% or more, about 500% or more, about 510% or more, about 520% or more, about 5 times or more, about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, about 50 times or more, about 100 times or more, about 200 times or more, about 300 times or more, about 400 times or more, about 500 times or more, about 1000 times or more, about 2000 times or more, about 3000 times or more, about 4000 times or more, about 5000 times or more, or about 10000 times or more.

In some embodiments, the Txo% of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure 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, about 120 months or longer.

In some embodiments, the Txo% of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present 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, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more longer than the Txo% of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations produced by comparable methods.

In some embodiments, the T1/2 of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure 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, about 120 months or longer.

In some embodiments, the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present 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, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more longer than the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method.

As used herein, "Tx" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation is degraded to about X of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For example, "T80%" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation is degraded to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For another example, "T1/2" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation is degraded to about 1/2 of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.

In some aspects, when nucleic acids are present in lipid nanoparticles, they can resist degradation by nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods for preparing the same are disclosed in, for example, U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Publication Nos. WO 2013/016058 and WO 2013/086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

Some aspects described herein relate to compositions, methods and uses comprising more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species (e.g., RNA species). In LNP formulations, each nucleic acid species may be formulated separately as a separate LNP formulation. In this case, each separate LNP formulation will contain a nucleic acid species. A single LNP formulation can exist as a separate entity, e.g., in a separate container. Such formulations can be obtained by providing each nucleic acid species (usually each nucleic acid species is in the form of a nucleic acid-containing solution) and suitable cations or cation-ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs, respectively. Each particle will specifically contain a specific nucleic acid species (separate particle formulation) provided when the particle is formed.

In some aspects, a composition (e.g., a pharmaceutical composition) comprises more than one separate LNP formulation. Each pharmaceutical composition is referred to as a mixed LNP formulation. A mixed LNP formulation according to the present invention can be obtained by forming separate LNP formulations as described above, followed by a step of mixing separate LNP formulations. By a mixing step, a formulation comprising a mixed LNP population of nucleic acid-containing can be obtained. Separate LNP populations can be placed together in a container, which comprises a mixed population of separate LNP formulations.

Alternatively, different nucleic acid species can be formulated together as a combined LNP formulation. This formulation can be obtained by providing a combined formulation (usually a combined solution) of different RNA species and a suitable cation or cation ionizable lipid or lipid-like material and a cationic polymer that allows the formation of LNP. Contrary to mixed LNP formulations, combined LNP formulations typically include LNPs containing more than one RNA species. In the combined LNP compositions, different RNA species are typically present together in a single particle.

The lipid component of LNP can include, for example, cationic lipids, phospholipids (eg, unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The ingredients of the lipid component can be provided in specific fractions.

In some embodiments, the LNP further comprises a phospholipid, a PEG lipid, a structured lipid, or any combination thereof. Suitable phospholipids, PEG lipids, and structured lipids for use in the disclosed methods are further disclosed herein.

In some embodiments, the lipid components of LNP include cationic lipids, phospholipids, PEG lipids and structural lipids. In certain embodiments, the lipid components of lipid nanoparticles include cationic lipids of about 30mol% to about 60mol%, phospholipids of about 0mol% to about 30mol%, structural lipids of about 18.5mol% to about 48.5mol%, and PEG lipids of about 0mol% to about 10mol%, with the premise that total mol% is no more than 100%. In some embodiments, the lipid components of lipid nanoparticles include cationic lipid compounds of about 35mol% to about 55mol%, phospholipids of about 5mol% to about 25mol%, structural lipids of about 30mol% to about 40mol%, and PEG lipids of about 0mol% to about 10mol%. In a specific embodiment, lipid components include the described cationic lipids of about 50mol%, the phospholipids of about 10mol%, the structural lipids of about 38.5mol%, and the PEG lipids of about 1.5mol%. In another specific embodiment, the lipid component includes about 40mol% of the cationic lipid, about 20mol% of the phospholipid, about 38.5mol% of the structural lipid, and about 1.5mol% of the PEG lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and/or the structural lipid can be cholesterol.

In some embodiments, the amount of therapeutic and/or preventive agent in LNP may depend on the size, composition, desired target and/or application or other properties and the property of therapeutic and/or preventive agent of lipid nanoparticle.For example, the amount of RNA useful in LNP may depend on the size, sequence and other characteristics of RNA.The relative amount of therapeutic and/or preventive agent (i.e. drug substance) and other components (e.g. lipid) in LNP may also change.In some embodiments, the wt/wt ratio of lipid component and therapeutic and/or preventive agent in LNP can be 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 prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt/wt ratio is about 20: 1. For example, the amount of the therapeutic and/or prophylactic agent in the LNP can be measured using absorption spectroscopy (e.g., UV-visible spectroscopy).

A. Cationic polymer materials

Considering its high chemical flexibility, polymeric materials are usually used for delivery based on nanoparticles. Usually, cationic materials are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups are usually composed of amines, which change their protonation state within the pH range between 5.5 and 7.5, which is considered to cause ion imbalance, thereby causing endosome rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine and naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable for use as cationic materials useful in some aspects of this article. In addition, some researchers have synthesized polymeric materials specifically for nucleic acid delivery. Especially poly (P-amino esters), due to its easy synthesis and biodegradability, has been widely used in nucleic acid delivery. In some respects, this type of synthetic material may be suitable for use as cationic materials herein.

As used herein, "polymeric material" is given its usual meaning, for example, a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some aspects, these repeating units can all be the same; or, in some cases, more than one type of repeating unit can be present in the polymeric material. In some cases, the polymeric material is of biological origin, for example, a biopolymer, such as a protein. In some cases, additional moieties, such as the targeting moieties described herein, may also be present in the polymeric material.

It is known to those skilled in the art that when there is more than one type of repeating unit in a polymer (or polymer portion), the polymer (or polymer portion) is referred to as a "copolymer". In some aspects, the polymer (or polymer portion) used in accordance with the present 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, for example, comprising one or more regions, each region comprising a first repeating unit (e.g., a first block), and one or more regions, each region comprising a second repeating unit (e.g., a second block), etc. Block copolymers may have two (diblock copolymers), three (triblock copolymers), or a plurality of different blocks.

In some aspects, the polymeric material used according to the disclosure is biocompatible. Biocompatible materials generally refer to those materials that do not cause a large amount of cell death under moderate concentrations. In some aspects, biocompatible materials are biodegradable, for example can be chemically and/or biologically degraded in a physiological environment (for example in vivo). In some aspects, polymeric material can be or comprise protamine or polyalkyleneimine (polyalkyleneimine), particularly protamine.

Those skilled in the art will appreciate that the term "protamine" is generally used to refer to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found to be particularly associated with DNA, replacing somatic histones in sperm cells of a variety of animals (e.g., fish). Specifically, the term "protamine" is generally used to refer to strongly basic proteins found in fish sperm that are soluble in water, do not coagulate due to heat, and produce primarily arginine when hydrolyzed. In purified form, they are used in long-acting insulin preparations and to neutralize the anticoagulant effect of heparin.

In some aspects, as used herein, the term "protamine" refers to a protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and/or polymeric forms of the amino acid sequence or its fragments, as well as artificial (synthetic) polypeptides that are specifically designed for a specific purpose and cannot be isolated from a natural or biological source.

In some aspects, the polyalkyleneimine comprises polyethyleneimine and/or polypropyleneimine. In some aspects, the polyalkyleneimine is polyethyleneimine (PEI). In some aspects, the polyalkyleneimine is a linear polyalkyleneimine, such as linear polyethyleneimine (PEI).

Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those materials capable of electrostatically binding nucleic acids. In some aspects, cationic polymer materials contemplated for use herein include any cationic polymer material with which nucleic acids can associate, such as by forming a complex with the nucleic acid or forming a vesicle that is enclosed or encapsulated in the vesicle.

In some aspects, the particles described herein can include polymers other than cationic polymers, such as non-cationic polymer materials and/or anionic polymer materials. Anionic and neutral polymer materials are collectively referred to herein as non-cationic polymer materials.

B. Lipids and lipid-like materials

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 the present disclosure, lipids and lipid-like materials can be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH value.

The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. In general, lipids can be divided 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 esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramide phospho-sphingolipids (e.g., sphingomyelin, phosphorylcholine) and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives.

The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" refers to substances that are structurally and/or functionally related to lipids but cannot be considered as lipids in the strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they exist in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment, and includes surfactants or synthetic compounds with hydrophilic and hydrophobic parts. In general, the term refers to molecules that contain hydrophilic and hydrophobic parts with different structural organizations, which may or may not be similar to lipids.

In some respects, RNA solution and lipid formulation mixture or its compositions can comprise the lipid that cationic lipid, neutral lipid, cholesterol and/or polymer (such as polyethylene glycol) are put together, and it forms the lipid nanoparticle that comprises RNA molecule.Therefore, in some respects, LNP can comprise cationic lipid and one or more excipients, such as one or more neutral lipids, charged lipid, steroid or steroid analog (such as cholesterol), polymer put together lipid (such as PEG-lipid), or its combination.In some respects, LNP comprises or encapsulates nucleic acid molecule.

i. Cationic lipids

Cation or cation ionizable lipid or lipid-like material refers to a lipid or lipid-like material that can be positively charged and can electrostatically bind nucleic acid. As used herein, "cationic lipid" or "cationic lipid-like material" refers to a lipid or lipid-like material with a net positive charge. Cationic lipid or lipid-like material is combined with negatively charged nucleic acid by electrostatic interaction. In general, cationic lipid has a lipophilic part, such as sterol, acyl chain, diacyl or more acyl chain, and the head group of lipid is usually positively charged. Exemplary cationic lipids include one or more amine groups with a positive charge. Cationic lipid can encapsulate negatively charged RNA.

In some aspects, cationic lipids are ionizable, so that they can exist in positively charged or neutral form according to pH value. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. Without wishing to be bound by theory, compared with particles that maintain cationic state at physiological pH, this ionizable behavior is considered to enhance effectiveness by helping endosome escape and reducing toxicity. For purposes of this disclosure, unless there is a contradiction in the situation, this type of "cationic ionizable" lipid or lipid-like material is included in the term "cationic lipid" or "cationic lipid-like material".

In some aspects, the cationic lipid can account for about 10mol% to about 100mol%, about 20mol% to about 100mol%, about 30mol% to about 100mol%, about 40mol% to about 100mol%, or about 50mol% to about 100mol% of the total lipid present in the particle. In some aspects, the cationic lipid can be at least, at most, just, or between any two of 10mol%, 20mol%, 30mol%, 40mol%, 50mol%, 60mol%, 70mol%, 80mol%, 90mol% or 100mol%, or any range or value that can be derived therein.

Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl) azanediyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), dimethyldioctadecyl ammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; dioctadecyl dimethyl ammonium chloride (DODAC ), 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-bis(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylammonium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DL enDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxopentyloxy)-3-dimethyl-1-(cis, cis-9',12'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N '-Dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptathriacont ... Methyl-2,3-bis(tetradecyloxy)-1-propanium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propanium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanium bromide (bAE-DMRIE), N-(4-carboxybenzyl)- N,N-dimethyl-2,3-bis(oleyloxy)propan-1-ammonium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3- Ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-ammonium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl) dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy) heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipid 02-200); or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).

In some aspects, the lipid nanoparticle comprises one or more cationic lipids. In one aspect, the lipid nanoparticle comprises (4-hydroxybutyl) azanediyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), which has the formula:

Cationic lipids are disclosed in, for example, US 10,166,298, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Representative cationic lipids include:

In some aspects, RNA-LNP comprises cationic lipid, RNA molecule as described herein and one or more neutral lipid, steroid, pegylated lipid or its combination.If more than one cationic lipid is incorporated in LNP, then these percentages are applicable to the cationic lipid of combination.In one aspect, cationic lipid is present in LNP with following amount: for example, respectively at least, at most, just or between about 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 or 60 mole percent in any two between.

In some aspects of the disclosure, the LNP comprises a combination or mixture of any of the lipids described above.

ii. Polymer-conjugated lipids

In some aspects, LNP comprises polymer-conjugated lipids. The term "polymer-conjugated lipids" refers to molecules comprising a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide, etc.

In some aspects, LNP comprises additional stable lipids, which are polyethylene glycol lipids (PEGylated lipids). The polymer-conjugated lipid (e.g., PEG-lipid) refers to a molecule comprising a lipid portion and a polymer portion. The example of a polymer-conjugated lipid is a PEG-lipid. PEG-lipid refers to a molecule comprising a lipid portion and a polyethylene glycol portion. PEG-lipids include, but are not limited to, 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. Representative polyethylene glycol lipids include PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG. In one aspect, polyethylene glycol-lipid is N-[(methoxypolyethylene glycol) 2000) aminoformyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). In one aspect, the polyethylene glycol-lipid is PEG-2000-DMG. In one aspect, the polyethylene glycol-lipid is PEG-c-DOMG. In other aspects, the LNP comprises a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a polyethylene glycol succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a polyethylene glycol dialkoxypropyl carbamate, such as co-methoxy(polyethoxy)ethyl-N-(2,3-bis(tetradecyloxy)propyl)carbamate or 2,3-bis(tetradecyloxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate. PEG-lipids are disclosed, for example, in US Pat. 9,737,619, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

In some aspects, the lipid nanoparticle comprises a polymer-conjugated lipid. In one aspect, the lipid nanoparticle comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide (ALC-0159), which has the formula:

On the other hand, the polymer can be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. For example, the polymer can include polycaprolactone (PCL), ethylene vinyl acetate polymer (EVA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), poly-L-lactic acid-glycolic acid copolymer (PLLGA), poly-D,L-lactide (PDLA), poly-L-lactide (PLLA), poly-D,L-lactide-caprolactone copolymer, poly-D,L-lactide-caprolactone-glycolide copolymer, poly-D,L-lactide-PEO-D,L-lactide copolymer, poly-D,L-lactide ester-PPO-D,L-lactide copolymer, polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyethylene ethers, polyethylene esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinyl pyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic acid polymers such as poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(methacrylic acid), acrylate), poly(butyl acrylate), poly(octadecyl acrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and copolymers and mixtures thereof, polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, polyorthoesters, polybutyric acid, polyvaleric acid, poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

In various aspects, the molar ratio of the cationic lipid to the PEGylated lipid or polymeric lipid ranges from about 100: 1 to about 20: 1, such as about 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1, or any range or value derivable therein.

In certain aspects, the PEG-lipid or polymer lipid is present in the LNP in an amount of about 1 to about 10 mole percent (mol %) (e.g., at least, at most, exactly, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %) relative to the total lipid content of the nanoparticle.

iii. Additional lipids

In some aspects, LNP comprises one or more extra lipids or lipid-like materials, which stabilize the formation of particles in the particle formation process. Suitable stable or structural lipids include non-cationic lipids, such as neutral lipids and anionic lipids. Without being bound by any theory, by also adding other hydrophobic parts (such as cholesterol and lipid) to optimize the preparation of LNP except ionizable/cationic lipids or lipid-like materials, the effectiveness of particle stability and nucleic acid delivery can be enhanced.

As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. In some aspects, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

Representative neutral lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. Exemplary phospholipids include, for example, phosphatidylcholine, for example, diacylphosphatidylcholine, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidonoylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DPPC), dioctano ... phosphatidylcholine (DBPC), ditricosylphosphatidylcholine (DTPC), didemethylneuroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholestylesuccinyl-sn-glycero-3-phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycero-3-phosphocholine (C16 and phosphatidylethanolamines, such as diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), diphytanoylphosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one aspect, the neutral lipid is 1,2-distearoyl-sn-glycero-3 phosphocholine (DSPC), which has the formula:

In some aspects, the LNP comprises a neutral lipid, and the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, or SM.

In various aspects, the LNP further comprises a steroid or steroid analog. A "steroid" is a compound comprising the following carbon skeleton:

In certain aspects, the steroid or steroid analog is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In one aspect, cholesterol has the formula:

Without being bound by any theory, the amount of at least one cationic lipid can affect important nucleic acid particle properties, such as charge, particle size, stability, tissue selectivity and biological activity of the nucleic acid, compared to the amount of at least one additional lipid. Therefore, in some aspects, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1, or from about 10:0 to about 1:9, from about 4:1 to about 1:2, or from about 3:1 to about 1:1.

In some aspects, non-cationic lipids, such as neutral lipids (e.g., one or more phospholipids and/or cholesterol), can comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 0 mol% to about 70 mol%, about 0 mol% to about 60 mol%, or about 0 mol% to about 50 mol% of the total lipids present in the particle. In some aspects, non-cationic lipids, such as neutral lipids (e.g., one or more phospholipids and/or cholesterol), can be at least, at most, just 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol% or 90 mol% of the total lipids present in the particle, or between any two of 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol% or 90 mol% of the total lipids present in the particle.

C. Other Materials

Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, dasyphylline, bromhexine, carbocysteine, eprazone, mesna, ambroxol, sobrexol, domiol, letosteine, sitoronin, tiopronin, gelsolin, thymosin β4, dornase α, netexin, and erdosteine), and DNases (e.g., rhDNase). The surface-altering agent may be located within the nanoparticle and/or on the surface of the LNP (e.g., by coating, adsorption, covalent bonding, or other processes).

LNP can also comprise one or more functionalized lipids.For example, lipid can be functionalized with alkyne groups, and when exposed to azide under appropriate reaction conditions, cycloaddition reaction can take place.Specifically, lipid bilayer can be functionalized by one or more groups that help to promote membrane penetration, cell recognition or imaging in this way.The surface of LNP can also be put together with one or more useful antibodies.Functional groups and conjugates that can be used for target cell delivery, imaging and membrane penetration are well known in the art.

In addition to these components, lipid nanoparticles may also include any substance that can be used in pharmaceutical compositions. For example, lipid nanoparticles may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, surfactants, buffers, preservatives and other types.

Surfactants and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], glycerol monooleate, sorbitan monooleate[ 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [ 30]), polyvinyl pyrrolidone, diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F68, 188. Cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and/or a combination thereof.

The example of preservative may include but is not limited to antioxidant, chelating agent, free radical scavenger, antibacterial preservative, antifungal preservative, alcohol preservative, acidic preservative and/or other preservative. The example of antioxidant includes but is not limited to alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite and/or sodium sulfite. The example of chelating agent includes ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, ethylenediaminetetraacetic acid, fumaric acid, malic acid, phosphoric acid, sodium ethylenediaminetetraacetate, tartaric acid and/or trisodium ethylenediaminetetraacetate. The example of antimicrobial preservative includes but is not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerine, hexetidine, imidazolidinyl urea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol and/or thimerosal. The example of antifungal preservative includes but is not limited to butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and/or sorbic acid. The example of alcohol preservative includes but is not limited to ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate and/or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT Methylparaben, 115. II, NEOLONE ^™ , KATHON ^™ and/or Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

Examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium valerate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, sulfamate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and/or combinations thereof.

In some embodiments, the preparation comprising LNP may also include salts, such as chloride salts. In some embodiments, the preparation comprising LNP may also include sugars, such as disaccharides. In some embodiments, the preparation also includes sugars but does not include salts, such as chloride salts. In some embodiments, LNP may also include one or more small hydrophobic molecules, such as vitamins (such as vitamin A or vitamin E) or sterols. Carbohydrates may include monosaccharides (such as glucose) and polysaccharides (such as glycogen and derivatives thereof and analogs).

The characteristics of LNP may depend on its components. For example, LNPs including cholesterol as structural lipids may have different characteristics from LNPs including different structural lipids. As used herein, the term "structural lipid" refers to sterols and lipids containing sterol moieties. As defined herein, "sterols" are the steroid subgroups consisting of steroids. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.

In some embodiments, the characteristic of LNP may depend on the absolute amount or relative amount of its components. For example, LNP comprising a higher mole fraction of phospholipids may have different characteristics from LNP comprising a lower mole fraction of phospholipids. Characteristics may also vary because of the preparation method and conditions of lipid nanoparticles. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

Phospholipid part can be selected from the non-restrictive group such as being made up of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin.Fatty acid part can be selected from the non-restrictive group such as being made up of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.Specific phospholipid can promote the fusion with film.In some embodiments, cationic phospholipid can interact with one or more negatively charged phospholipids of film (for example, cell or intracellular membrane).Phospholipid and membrane fusion can allow one or more compositions (for example, therapeutic agent) containing lipid composition (for example, LNP) to pass through film, thereby allow for example one or more compositions to be delivered to target tissue. Non-natural phospholipid species are also contemplated, including natural species that have been modified and substituted (including branching, oxidation, cyclization, and alkynes). In some embodiments, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, copper-catalyzed cycloaddition reactions can occur when alkyne groups are exposed to azides. Such reactions can be used to functionalize the lipid bilayer of nanoparticle compositions to promote membrane penetration or cell recognition, or to conjugate nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes). Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phospholipids, such as sphingomyelin. In some embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC.

VI. Characterization and Analysis of RNA Molecules

A variety of methods can be used to analyze and characterize RNA molecules described herein. The analysis can be performed before or after capping. Alternatively, the analysis can be performed before or after affinity purification based on poly A capture. On the other hand, the analysis can be performed before or after additional purification steps (e.g., anion exchange chromatography, etc.). For example, the RNA template quality can be determined using an electrophoresis system based on a Bioanalyzer chip. In other aspects, analytical reversed-phase HPLC is used to analyze RNA template purity, respectively. For example, total nuclease digestion can be used, and then the MS/MS quantification of dinucleotide cap species relative to uncapped GTP species is used to analyze the capping efficiency. For example, in vitro efficacy can be analyzed by transfecting RNA molecules into human cell lines. Methods such as ELISA or flow cytometry can be used to quantify the protein expression of the target polypeptide. Immunogenicity can be analyzed by, for example, transfecting RNA molecules into cell lines (e.g., PBMC) indicating innate immune stimulation. Cytokine induction can be analyzed by quantitative analysis of cytokines (e.g., interferon-α) using methods such as ELISA. Biodistribution can be analyzed by, for example, bioluminescence measurement.

In some aspects, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or pharmaceutical formulation comprising the RNA polynucleotide is administered, increased expression of a target gene (e.g., an antigen) is observed relative to an appropriate reference; the duration of expression of the target gene (e.g., an antigen) is increased (e.g., expression is prolonged); the expression of the target gene (e.g., an antigen) is increased and the duration of expression is increased (e.g., expression is prolonged); the interaction of the RNA polynucleotide with IFIT1 is reduced; and the translation of the RNA polynucleotide is increased.

In some aspects, the reference comprises an organism administered with an otherwise similar RNA polynucleotide that does not have a m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2 cap. In some aspects, the reference comprises an organism administered with an otherwise similar RNA polynucleotide that does not have a cap-proximal sequence disclosed herein. In some aspects, the reference comprises an organism administered with an otherwise similar RNA polynucleotide that has a self-hybridizing sequence.

In some aspects, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours or at least 120 hours after using a composition or pharmaceutical preparation comprising RNA polynucleotides, the expression of elevation is measured. In some aspects, at least 24 hours after using a composition or pharmaceutical preparation comprising RNA polynucleotides, the expression of elevation is measured. In some aspects, at least 48 hours after using a composition or pharmaceutical preparation comprising RNA polynucleotides, the expression of elevation is measured. In some aspects, at least 72 hours after using a composition or pharmaceutical preparation comprising RNA polynucleotides, the expression of elevation is measured. In some aspects, at least 96 hours after using a composition or pharmaceutical preparation comprising RNA polynucleotides, the expression of elevation is measured. In some aspects, at least 120 hours after using a composition or pharmaceutical preparation comprising RNA polynucleotides, the expression of elevation is measured.

In some aspects, the expression of raising is measured in about 24-120 hours after the composition or pharmaceutical preparation comprising RNA polynucleotide is used.In some aspects, the expression of raising is measured in about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours or about 110-120 hours after the composition or pharmaceutical preparation comprising RNA polynucleotide is used.

In some aspects, the expression of the target gene (e.g., antigen) increases by at least 2 times to at least 10 times. In some aspects, the expression of the target gene (e.g., antigen) increases by at least 2 times. In some aspects, the expression of the target gene (e.g., antigen) increases by at least 3 times. In some aspects, the expression of the target gene (e.g., antigen) increases by at least 4 times. In some aspects, the expression of the target gene (e.g., antigen) increases by at least 6 times. In some aspects, the expression of the target gene (e.g., antigen) increases by at least 8 times. In some aspects, the expression of the target gene (e.g., antigen) increases by at least 10 times.

In some aspects, the expression of a gene of interest (e.g., antigen) increases by about 2 times to about 50 times. In some aspects, the expression of a gene of interest (e.g., antigen) increases by about 2 times to about 45 times, about 2 times to about 40 times, about 2 times to about 30 times, about 2 times to about 25 times, about 2 times to about 20 times, about 2 times to about 15 times, about 2 times to about 10 times, about 2 times to about 8 times, about 2 times to about 5 times, about 5 times to about 50 times, about 10 times to about 50 times, about 15 times to about 50 times, about 20 times to about 50 times, about 25 times to about 50 times, about 30 times to about 50 times, about 40 times to about 50 times, or about 45 times to about 50 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least, at most, just, or between any two of 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, or 50 times, or any range or value derivable therein.

In some aspects, after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of target gene (e.g., antigen) increases (e.g., expression duration increases) for at least, at most, just or between any two of 24 hours, 48 hours, 72 hours, 96 hours or 120 hours. In some aspects, the expression of target gene (e.g., antigen) increases after application for at least 24 hours. In some aspects, the expression of target gene (e.g., antigen) increases after application for at least 48 hours. In some aspects, the expression of target gene (e.g., antigen) increases after application for at least 72 hours. In some aspects, the expression of target gene (e.g., antigen) increases after application for at least 96 hours. In some aspects, the expression of target gene (e.g., antigen) increases after application for at least 120 hours.

In some respects, the expression of target gene (for example, antigen) rises and continues about 24-120 hours after using the compositions or pharmaceutical preparation comprising RNA polynucleotide.In some respects, after using the compositions or pharmaceutical preparation comprising RNA polynucleotide, expression rises and continues about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours or about 110-120 hours. In some aspects, the increased expression of the gene of interest (e.g., antigen) persists for at least, at most, exactly, or between any two of 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or any range or value derivable therein.

VII. Immune Response

As discussed herein, the present disclosure relates to causing or inducing an immune response in a subject against a FimH protein (e.g., a wild-type or variant FimH protein). In one aspect, the immune response can protect or treat a subject suffering from, suspected of suffering from, or at risk of developing an infection or related disease, particularly those diseases associated with E. coli FimH. One use of the immunogenic compositions of the present disclosure is to prevent E. coli infection by inoculating or vaccinating a subject.

In some aspects of the present disclosure, RNA molecules encoding E. coli FimH protein, RNA-LNPs and compositions thereof confer protective immunity to a subject. Protective immunity refers to the ability of the human body to produce a specific immune response that can protect the subject from a specific disease or condition involving a pathogen to which the immune response is directed. An immunologically effective amount can confer protective immunity to a subject.

As used herein, the phrase "immune response" or its equivalent "immunological response" refers to the occurrence of a humoral response (antibody-mediated), a cellular response (mediated by antigen-specific T cells or their secretory products), or a humoral and cellular response to an antigen. This response can be an active response or a passive response. The cellular immune response is caused by the presentation of polypeptide epitopes associated with class I or class II MHC molecules to activate antigen-specific CD4(+) T helper cells and/or CD8(+) cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred by a subject in response to the presence of an antigen (e.g., the E. coli FimH protein encoded by the RNA molecules of the present disclosure) by producing antibodies.

As used herein, "passive immunization" includes, but is not limited to, administering activated immune effectors, including cellular mediators or protein mediators (e.g., monoclonal and/or polyclonal antibodies) of immune responses. Monoclonal or polyclonal antibody compositions can be used for passive immunization to treat, prevent, or mitigate the severity of a disease caused by infection with an organism carrying an antigen recognized by the antibody. Antibody compositions can include antibodies that are bound to a variety of antigens, which in turn may be associated with a variety of organisms. The antibody component can be a polyclonal antiserum. In some aspects, one or more antibodies are affinity purified from an animal or a second subject that has been attacked by an antigen. Alternatively, an antibody mixture can be used, which is a mixture of monoclonal antibodies and/or polyclonal antibodies directed against an antigen present in the same, related, or different microorganisms or organisms (e.g., bacteria, including but not limited to Escherichia coli).

Passive immunity can be conferred to a patient or subject by administering immunoglobulins (Ig) and/or other immune factors obtained from donors or other non-patient sources with known immunoreactivity to the patient. In other aspects, the immunogenic composition of the present disclosure can be administered to a subject, who is then used as a source or donor of globulins produced in response to an attack by an immunogenic composition ("hyperimmune globulin") containing antibodies against E. coli or other organisms. The subject so treated will donate plasma, from which hyperimmune globulins are then obtained by conventional plasma fractionation methods and administered to another subject to confer resistance to or treat E. coli infection.

For the purpose of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to sites on antigens that B cells and/or T cells respond to or recognize. B cell epitopes can be formed by continuous amino acids or non-continuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes usually include at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. For example, see Epitope Mapping Protocols (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to the target antigen. T cells recognize a continuous epitope of about 9 amino acids for CD8 cells or a continuous epitope of about 13-15 amino acids for CD4 cells. T cells recognizing the epitope can be identified by in vitro assays measuring antigen-dependent proliferation, such as by ^3H -thymidine incorporation by T cells elicited in response to the epitope (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion assays.

The presence of a cell-mediated immune response can be determined by proliferation assays (CD4(+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogenic composition can be distinguished by isolating IgG and T cells, respectively, from immunized syngeneic animals and measuring the protective or therapeutic effect in a second subject.

As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an immune response in an animal or recipient, including IgG, IgD, IgE, IgA, IgM, and related proteins. Under normal physiological conditions, antibodies are present in plasma and other body fluids and in the membranes of certain cells and are produced by lymphocytes of a type designated as B cells or their functional equivalents.

As used herein, the terms "immunogenic agent" or "immunogen" or "antigen" are used interchangeably to describe a molecule that is capable of inducing an immune response against itself when administered to a recipient alone, in combination with an adjuvant, or on a presentation vehicle.

VIII. Composition

In some aspects, the RNA molecules and/or RNA-LNP disclosed herein can be administered in the form of a pharmaceutical composition or a medicine, and can be administered in the form of any suitable pharmaceutical composition. In some aspects, the pharmaceutical composition is used for therapeutic or preventive treatment. In one aspect, the disclosure relates to a composition for administration to a host. In some aspects, the host is a human. In other aspects, the host is a non-human.

In some aspects, RNA molecules and/or RNA-LNP disclosed herein can be used in the form of a pharmaceutical composition, which can be formulated into a solid, semisolid, liquid, lyophilized, frozen or gaseous preparation. In some aspects, RNA molecules and/or RNA-LNP disclosed herein can be used in the form of a pharmaceutical composition, which can include a pharmaceutically acceptable carrier and can optionally include one or more adjuvants, stabilizers, salts, buffers, preservatives and optional other therapeutic agents. In some aspects, pharmaceutical compositions disclosed herein include one or more pharmaceutically acceptable carriers, diluents and/or excipients. In some aspects, pharmaceutical compositions do not include adjuvants (e.g., they do not contain adjuvants).

Suitable preservatives for the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

The term "diluent" refers to a diluent and/or a thinning agent. Furthermore, the term "diluent" includes one or more of a fluid, a liquid or solid suspension and/or a mixed medium. Examples of suitable diluents include ethanol, glycerol and water.

The term "carrier" refers to a component that can be natural, synthetic, organic, or inorganic, in which the active ingredient is combined to promote, enhance, or enable the administration of the pharmaceutical composition. The carrier used herein can be one or more compatible solid or liquid fillers, diluents, or encapsulating materials that are suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, particularly biocompatible lactide polymers, lactide/glycolide copolymers, or polyoxyethylene/polyoxypropylene copolymers. In some aspects, the pharmaceutical composition of the present disclosure includes sodium chloride.

Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In some aspects, the composition comprises an RNA molecule comprising an open reading frame encoding an immunogenic polypeptide. In some aspects, the immunogenic polypeptide comprises an E. coli antigen. In some aspects, the E. coli antigen is an E. coli FimH protein.

In some aspects, the composition comprises an RNA molecule containing an open reading frame encoding a full-length E. coli FimH protein. In some aspects, the encoded immunogenic polypeptide is a truncated E. coli FimH protein. In some aspects, the encoded immunogenic polypeptide is a variant of the E. coli FimH protein. In some aspects, the encoded immunogenic polypeptide is a fragment of the E. coli FimH protein.

A. Immunogenic Compositions Including LNPS

In some aspects, the pharmaceutical composition comprises an RNA molecule disclosed herein (e.g., polynucleotide) formulated with a lipid-based delivery system. Therefore, in some aspects, the composition includes a lipid-based delivery system (e.g., LNP) (e.g., a lipid-based vaccine), which delivers nucleic acid molecules to the interior of the cell, which can then replicate, inhibit expression of the target protein, and/or express the encoded target polypeptide in the cell. The delivery system can have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some aspects, the composition comprises at least one RNA molecule encoding the FimH polypeptide, which is compounded with one or more lipids, encapsulated in one or more lipids and/or formulated with one or more lipids, and forms lipid nanoparticles (LNP), liposomes, lipid complexes and/or nanoliposomes. In some aspects, the composition comprises lipid nanoparticles. Therefore, in some aspects, the disclosure relates to a composition comprising one or more lipids associated with a nucleic acid or polypeptide/peptide (e.g., FimH RNA-LNP).

In some cases, the immunogenic composition comprising the delivery system based on lipid can also include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents and/or excipients. In some respects, the immunogenic composition comprising the delivery system based on lipid also includes a pharmaceutically acceptable carrier (vehicle). In some respects, each of buffer, stabilizer and optional salt can be included in the immunogenic composition comprising the delivery system based on lipid. In other respects, one or more of buffer, stabilizer, salt, surfactant, preservative and excipient can be excluded from the immunogenic composition comprising the delivery system based on lipid.

In other aspects, the immunogenic composition including a lipid-based delivery system also includes a stabilizer. In some aspects, the stabilizer includes sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinyl pyrrolidone, glycine or a combination thereof. In some aspects, the stabilizer is a disaccharide or sugar. In one aspect, the stabilizer is sucrose. On the other hand, the stabilizer is trehalose. In other aspects, the stabilizer is a combination of sucrose and trehalose. In some aspects, the total concentration of the stabilizer in the composition is about 5% to about 10% w/v. For example, the total concentration of the stabilizer can be equal to at least, at most, just, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% w/v, or any range or value that can be derived therein. In some aspects, the stabilizer concentration includes but is not limited to a concentration of about 10 mg/mL to about 400 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 150 mg/mL, about 100 mg/mL to about 140 mg/mL, about 100 mg/mL to about 130 mg/mL, about 100 mg/mL to about 120 mg/mL, about 100 mg/mL to about 110 mg/mL, or about 100 mg/mL to about 105 mg/mL. In some aspects, the concentration of the stabilizer is equal to at least, at most, exactly, or between any two of 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL or more.

In other aspects, the mass amount of the stabilizer has a specific ratio to the mass amount of the RNA. In one aspect, the mass amount ratio of the stabilizer to the RNA is not greater than 5000. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 2000. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 1000. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 500. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 100. On the other hand, the mass amount ratio of the stabilizer to the drug substance is not greater than 50. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 10. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 1. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 0.5. On the other hand, the mass amount ratio of the stabilizer to the RNA is not greater than 0.1. On the other hand, the stabilizer and RNA contain a mass ratio of about 200-2000 stabilizer: 1RNA.

In some aspects, the immunogenic composition including the delivery system based on lipid also includes buffer.The example of buffer includes but is not limited to citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronic acid, calcium glucoheptonic acid, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium valeric acid, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate (calciumhydroxide phosphate), potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, sulfamate buffer (such as HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol and/or its combination.In some aspects, buffer is HEPES buffer, Tris buffer or PBS buffer. In one aspect, the buffer is a Tris buffer. On the other hand, the buffer is a HEPES buffer. In other aspects, the buffer is a PBS buffer. For example, the buffer concentration can be equal to at least, at most, just or between any two of 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM or 20mM, or any range or value that can be drawn therein. The buffer can be in neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0 or pH 7.2 to pH 7.6. For example, the buffer can be at least, at most, just, or between any two of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In a specific aspect, the buffer is at pH 7.4.

In some aspects, the immunogenic composition comprising a lipid-based delivery system may also include salts. Examples of salts include, but are not limited to, sodium salts and/or potassium salts. In one aspect, the salt is a sodium salt. In a specific aspect, the sodium salt is sodium chloride. In one aspect, the salt is a potassium salt. In some aspects, the potassium salt comprises potassium chloride. The salt concentration in the composition may be from about 70mM to about 140mM. For example, the salt concentration may be equal to at least, at most, just, or between any two of 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200mM.

In some aspects, salt concentrations include, but are not limited to, about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 15 mg/mL. In some aspects, the concentration of salt is equal to at least, at most, just, or between any two of 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL or more. The salt can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the pH of the salt can be equal to at least, at most, just, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5.

In some aspects, the immunogenic composition comprising a lipid-based delivery system further comprises a surfactant, a preservative, any other excipient, or a combination thereof. As used herein, "any other excipient" includes, but is not limited to, antioxidants, glutathione, EDTA, methionine, deferoxamine, antioxidants, metal scavengers, or free radical scavengers. In one aspect, the surfactant, preservative, excipient, or a combination thereof is sterile water for injection (sWFI), bacteriostatic water for injection (BWFI), saline, glucose solution, polysorbate, poloxamer, Triton, divalent cations, Ringer's lactate, amino acids, sugars, polyols, polymers, or cyclodextrins.

Excipients refer to ingredients that are not active ingredients in the immunogenic composition, examples of which include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, tablets, wet granulators, or colorants. Preservatives used in the compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents known to those skilled in the art (e.g., water, alcohol/water solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delay agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements, and combinations thereof. Diluents or diluents or diluents include, but are not limited to, ethanol, glycerol, water, sugars (e.g., lactose, sucrose, mannitol, and sorbitol), and starches derived from wheat, corn, rice, and potatoes; and cellulose (e.g., microcrystalline cellulose). The amount of diluent in the composition may be from about 10% to about 90%, from about 25% to about 75%, from about 30% to about 60%, or from about 12% to about 60% by weight of the total composition.

The pH and precise concentrations of various components in immunogenic compositions, including lipid-based delivery systems, are adjusted according to well-known parameters. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, it is contemplated for use in immunogenic, prophylactic and/or therapeutic compositions.

In one aspect, the pharmaceutical composition comprises a FimH RNA molecule encoding a FimH polypeptide as disclosed herein, which is complexed with one or more lipids, encapsulated in one or more lipids, and/or formulated with one or more lipids to form a FimH RNA-LNP. In some aspects, the FimH RNA-LNP composition is a liquid. In some aspects, the FimH RNA-LNP composition is frozen. In some aspects, the FimH RNA-LNP composition is lyophilized. In some aspects, the FimH RNA-LNP composition comprises a FimH RNA polynucleotide molecule encoding a FimH polypeptide as disclosed herein, which is encapsulated in a LNP of a lipid composition having a cationic lipid, a PEGylated lipid (i.e., a PEG-lipid) and one or more structural lipids (e.g., a neutral lipid).

In some aspects, the FimH RNA-LNP composition comprises a cationic lipid. The cationic lipid may comprise any one or more cationic lipids disclosed herein. In specific aspects, the cationic lipid comprises ((4-hydroxybutyl) azanediyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315). In some aspects, a cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least, at most, between any two of the following, or exactly: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 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, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55 .42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66 ,0.67,0.68 , 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.9 3, 0.94, 0.9 5. 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2 ,1.21,1.2 2. 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1 .47, 1.48, 1 .49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/μg/mg/mL. In some aspects, a cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least, at most, between any two of, or exactly 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64 , 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.5, between 0.5 and 0.6, between 0.6 and 0.7, between 0.7 and 0.8, between 0.8 and 0.9, or between 0.9 and 1. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.45, between 0.45 and 0.5, between 0.5 and 0.55, between 0.55 and 0.6, between 0.6 and 0.65, between 0.65 and 0.7, between 0.7 and 0.75, between 0.75 and 0.8, between 0.8 and 0.85, between 0.85 and 0.9, between 0.9 and 0.95, or between 0.95 and 1 mg/mL.

In a specific aspect, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8 to 0.95 mg/mL. In a specific aspect, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8 to 0.9 mg/mL. In a specific aspect, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.85 to 0.9 mg/mL. In a specific aspect, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94 or 0.95 mg/mL. The concentration of the lyophilized composition is determined after reconstruction.

In some aspects, the FimH RNA-LNP composition further comprises a PEGylated lipid (ie, a PEG-lipid). The PEGylated lipid may comprise any one or more PEGylated lipids disclosed herein. In specific aspects, the PEGylated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide (ALC-0159). In some aspects, the pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least, at most, between any two of, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 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, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.6 6, 0.67, 0.6 8. 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0. 93, 0.94, 0. 95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1. 2, 1.21, 1.2 2. 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1 .47, 1.48, 1 .49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/μg/mg/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least, at most, between any two of, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 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, 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, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55 .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 mg/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.4 mg/mL, at least 0.45 mg/mL, or at least 0.5 mg/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of between 0.01 and 0.05, between 0.05 and 0.1, between 0.1 and 0.15, between 0.15 and 0.2, or between 0.2 and 0.25 mg/mL.

In a specific aspect, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of 0.05 to 0.15 mg/mL. In a specific aspect, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of 0.10 to 0.15 mg/mL. In a specific aspect, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14 or 0.15 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.

In some aspects, the FimH RNA-LNP composition further comprises one or more structural lipids. One or more structural lipids may comprise any one or more structural lipids disclosed herein. In a specific aspect, one or more structural lipids comprise neutral lipids and steroids or steroid analogs. In a specific aspect, one or more structural lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. In some aspects, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least, at most, between any two of, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 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 ,0.42,0.43,0.44,0.45,0.46,0.47,0.48,0.49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0. 66, 0.67, 0. 68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0 .93, 0.94, 0 .95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1 .2, 1.21, 1. 22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73 ,1.74,1.75 , 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/μg/mg/mL. In some aspects, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least, at most, between any two of, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL. In some aspects, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of between 0.05 and 0.1, 0.1 and 0.15, 0.15 and 0.2, 0.2 and 0.25, 0.25 and 0.3, 0.3 and 0.35, 0.35 and 0.4, 0.4 and 0.45, 0.45 and 0.5, 0.5 and 0.55, 0.55 and 0.6, 0.6 and 0.65, 0.65 and 0.7, 0.7 and 0.75, 0.75 and 0.8, 0.8 and 0.85, 0.85 and 0.9, 0.9 and 0.95, or 0.95 and 1 mg/mL.

In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at a concentration of 0.1 to 0.25 mg/mL. In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at a concentration of 0.15 to 0.25 mg/mL. In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at a concentration 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 or 0.25 mg/mL.

In a specific aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of 0.3 to 0.45mg/mL. In a specific aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of 0.3 to 0.4. In a specific aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of 0.35 to 0.45. In a specific aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45mg/mL. The concentration of the lyophilized composition is determined after reconstruction.

In some aspects, the FimH RNA-LNP composition further comprises one or more buffers and stabilizers, and optionally salts. Thus, in some aspects, the FimH RNA-LNP composition comprises a cationic lipid, a PEGylated lipid, one or more structural lipids, one or more buffers, stabilizers, and optionally salts.

In some aspects, the FimH RNA-LNP composition comprises one or more buffers. The one or more buffers may include any one or more buffers disclosed herein. In specific aspects, the composition comprises a Tris buffer comprising at least a first buffer and a second buffer. In some aspects, the first buffer is tromethamine. In some aspects, the second buffer is Tris hydrochloride (HCl). In some aspects, the first buffer and the second buffer of the Tris buffer (e.g., tromethamine and Tris HCl) are at least, at most, between any two of the following, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.2 1. 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, 0.4 7. 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0. 72,0.73 . 98,0.99 , 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1. 26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1 .51, 1.5 2. 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1 .77, 1.7 8, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/μg/mg/mL concentration is included in the composition. The concentration of the lyophilized composition is determined after reconstitution.

In some aspects, the FimH RNA-LNP composition is a liquid composition comprising a Tris buffer. In some aspects, the Tris buffer comprises a first buffer. In some aspects, the first buffer is tromethamine. In some aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of at least, at most, between any two of the following, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 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, 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, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65 , 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 mg/mL. In some aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of at least 0.1, at least .05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL. In some aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of between 0.05 and 0.15, between 0.15 and 0.25, between 0.25 and 0.35, between 0.35 and 0.45, between 0.45 and 0.55, between 0.55 and 0.65, between 0.65 and 0.75, between 0.75 and 0.85, or between 0.85 and 0.95. In some aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of between 0.05 and 0.1, between 0.1 and 0.15, between 0.15 and 0.2, between 0.2 and 0.25, between 0.25 and 0.3, between 0.3 and 0.35, between 0.35 and 0.4, between 0.4 and 0.45, between 0.45 and 0.5, between 0.5 and 0.55, between 0.55 and 0.6, between 0.6 and 0.65, between 0.65 and 0.7, between 0.7 and 0.75, between 0.75 and 0.8, between 0.8 and 0.85, between 0.85 and 0.9, between 0.9 and 0.95, or between 0.95 and 1 mg/mL.

In a specific aspect, the first buffer (e.g., tromethamine) is included in the liquid composition with a concentration of 0.1 to 0.3 mg/mL. In a specific aspect, the first buffer (e.g., tromethamine) is included in the liquid composition with a concentration of 0.15 to 0.25 mg/mL. In a specific aspect, the first buffer (e.g., tromethamine) is included in the liquid composition with a concentration of 0.1, 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 0.3 mg/mL.

In some aspects, the FimH RNA-LNP composition is a liquid composition comprising a Tris buffer containing a second buffer. In some aspects, the second buffer comprises Tris HCl. In some aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of at least, at most, between any two of the following, or exactly 0.5, 0.55, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1 .21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49 or 1.5 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1, at least 1.05, at least 1.10, at least 1.15, at least 1.20, at least 1.25, at least 1.30, at least 1.35, at least 1.40, at least 1.45, or at least 1.50 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of between 0.5 and 0.6, between 0.6 and 0.7, between 0.7 and 0.8, between 0.8 and 0.9, between 0.9 and 1, between 1 and 1.10, between 1.10 and 1.20, between 1.20 and 1.30, between 1.30 and 1.40, or between 1.40 and 1.50 mg/mL.

In a specific aspect, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of 1.25 to 1.40 mg/mL. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of 1.30 to 1.40 mg/mL. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34 or 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg/mL.

In some aspects, the FimH RNA-LNP composition is a lyophilized composition comprising a Tris buffer. In some aspects, the Tris buffer comprises a first buffer. In some aspects, the first buffer is tromethamine. In some aspects, the first buffer (e.g., tromethamine) is at least, at most, between any two of the following, or just 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 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, 0.30, 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, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.56, 0.57, 0.58, 0.59, 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, , 0.23, 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 mg/mL concentration is included in the lyophilized composition. In some aspects, the first buffer (e.g., tromethamine) is included in the lyophilized composition with a concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45 or at least 0.5 mg/mL after reconstruction. In some aspects, the first buffer (e.g., tromethamine (Tris base)) is included in the lyophilized composition at a concentration of 0.01 to 0.05, 0.05 to 0.1, 0.1 to 0.15, 0.15 to 0.2, 0.2 to 0.25 mg/mL, 0.25 to 0.3 mg/mL, 0.3 to 0.35 mg/mL, 0.35 to 0.4 mg/mL, 0.4 to 0.45 mg/mL, or 0.45 to 0.5 mg/mL after reconstitution.

In a specific aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a concentration of 0.01 to 0.15 mg/mL after reconstruction. In a specific aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a concentration of 0.01 to 0.10 mg/mL after reconstruction. In a specific aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a concentration of 0.05 to 0.15 mg/mL after reconstruction. In a specific aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a concentration of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14 or 0.15 mg/mL after reconstruction.

In some aspects, the FimH RNA-LNP composition is a lyophilized composition comprising a Tris buffer containing a second buffer. In some aspects, the second buffer comprises Tris HCl. In some aspects, the second buffer (e.g., Tris HCl) is at least, at most, between any two of the following, or exactly 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, In some aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9 or at least 1 mg/mL after reconstitution. In some aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition after reconstitution at a concentration of between 0.1 and 0.2, between 0.2 and 0.3, between 0.3 and 0.4, between 0.4 and 0.5, between 0.5 and 0.6, between 0.6 and 0.7, between 0.7 and 0.8, between 0.8 and 0.9, or between 0.9 and 1 mg/mL.

In a specific aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of 0.5 to 0.65 mg/mL after reconstruction. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of 0.5 to 0.6 mg/mL after reconstruction. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of 0.55 to 0.65 mg/mL after reconstruction. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64 or 0.65 mg/mL after reconstruction.

In some aspects, the FimH RNA-LNP composition comprises a stabilizer. The stabilizer may comprise any one or more stabilizers disclosed herein. In some aspects, the stabilizer may also serve as a cryoprotectant. In specific aspects, the stabilizer comprises sucrose. In some aspects, the stabilizer (e.g., sucrose) is at least, at most, between any two of the following or just 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 ,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 12, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 14 2. 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 1 A concentration of 59, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 ng/μg/mg/mL is included in the composition.

In some aspects, the FimH RNA-LNP composition is a liquid composition and the stabilizer (e.g., sucrose) is present in an amount of at least, at most, between any two of, or exactly 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, 99 In some aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125 or at least 130 mg/mL. In some aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of between 70-80, between 80-90, between 90-100, between 100-110, between 110-120, or between 120-130 mg/mL.

In a specific aspect, stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of 95 to 110 mg/mL. In a specific aspect, stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of 95 to 105 mg/mL. In a specific aspect, stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of 100 to 110 mg/mL. In a specific aspect, stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg/mL.

In some aspects, the FimH RNA-LNP composition is a lyophilized composition and the stabilizer (e.g., sucrose) is included in the lyophilized composition after reconstitution at a concentration of at least, at most, between any two of, or exactly 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 mg/mL. In some aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 mg/mL after reconstitution. In some aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of between 20 and 30, between 30 and 40, between 40 and 50, between 50 and 60, between 60 and 70, or between 70 and 80 mg/mL after reconstitution.

In a specific aspect, stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of 35 to 50 mg/mL after reconstruction. In a specific aspect, stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of 35 to 45 mg/mL after reconstruction. In a specific aspect, stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of 40 to 50 mg/mL after reconstruction. In a specific aspect, stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg/mL after reconstruction.

In some aspects, the FimH RNA-LNP composition is a lyophilized composition, and the lyophilized FimH RNA-LNP composition further comprises a salt. The salt may comprise one or more salts disclosed herein. In specific aspects, the salt comprises sodium chloride (NaCl). 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 30.5, 31, 32, 33.5, 34, 35.5, 36, 37.5, 38, 39.5, 40, 41.5, 42, 43.5, 44, 45.5, 46, 47.5, 48, 49, 50.5, 51, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 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 In some aspects, salt (e.g., NaCl) is included in the lyophilized composition after reconstitution at a concentration of at least, at most, between any two of the following, or exactly 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/mL. In some aspects, salt (e.g., NaCl) is included in the lyophilized composition at a concentration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 mg/mL after reconstitution.

In a specific aspect, salt (e.g., NaCl) is included in the lyophilized composition at a concentration between 5 and 15 mg/mL after reconstruction. In some aspects, salt (e.g., NaCl) is included in the lyophilized composition at a concentration between 5 and 10 mg/mL after reconstruction. In a specific aspect, salt (e.g., NaCl) is included in the lyophilized composition at a concentration between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg/mL after reconstruction.

In some aspects, the lyophilized composition is reconstituted in a suitable carrier or diluent. The carrier or diluent may comprise any one or more of the carriers or diluents disclosed herein. In specific aspects, the carrier or diluent comprises saline, such as normal saline. The saline may comprise 0.9% saline for injection. In some aspects, the lyophilized composition is at least, at most, between any two of the following, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.2 2. 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 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, 0.45, 0.46 ,0.47,0.48, 0.49, 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, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0 .73, 0.74, 0. In some aspects, the lyophilized composition is reconstituted in at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1 mL of saline.

In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of saline. In a specific aspect, the lyophilized composition is reconstituted in 0.65 to 0.75 mL of saline. In a specific aspect, the lyophilized composition is reconstituted in 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, or 0.75 mL of saline.

The pH value of the FimH RNA-LNP composition can be at least, at most, just, or between any two of the following: pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5, or any range or value that can be derived therefrom. In some aspects, the FimH RNA-LNP composition is at a pH value of at least 6.5, at least 7.0, at least 7.5, at least 8.0 or at least 8.5. In specific aspects, the FimH RNA-LNP composition is at a pH value between 6.0 and 7.5, between 6.5 and 7.5, between 7.0 and 8.0, between 7.5 and 8.5. In specific aspects, the FimH RNA-LNP composition is between 7.0 and 8.0. In specific aspects, the FimH RNA-LNP composition is at pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

In a specific aspect, the FimH RNA-LNP composition comprises a FimH RNA polynucleotide encoding a FimH polypeptide disclosed herein, encapsulated in a LNP having a lipid composition comprising a cationic lipid at a concentration of 0.8 to 0.95 mg/mL, a PEGylated 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.

In a specific aspect, the FimH RNA-LNP composition comprises a FimH RNA polynucleotide encoding a FimH polypeptide as disclosed herein, encapsulated in a LNP having a lipid composition comprising 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.

In a specific aspect, the FimH RNA-LNP composition is a liquid FimH RNA-LNP composition, and the liquid FimH RNA-LNP composition further comprises a buffer composition, the 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 a specific aspect, the FimH RNA-LNP composition is a liquid FimH RNA-LNP composition, and the liquid FimH RNA-LNP composition further comprises a Tris buffer composition, the Tris buffer composition comprising tromethamine 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 sucrose at a concentration of 95 to 110 mg/mL.

Thus, in a specific aspect, the liquid FimH RNA-LNP composition comprises a cationic lipid at a concentration of 0.8 to 0.95 mg/mL, a PEGylated 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, a second structural lipid at a concentration of 0.3 to 0.45 mg/mL, and further comprises a first buffer at a concentration of 0.1 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.

Thus, in a specific aspect, the liquid FimH RNA-LNP composition comprises 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, cholesterol at a concentration of 0.3 to 0.45 mg/mL, and further comprises tromethamine 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 sucrose at a concentration of 95 to 110 mg/mL.

In a specific aspect, the FimH RNA-LNP composition is a lyophilized FimH RNA-LNP composition, and the lyophilized FimH RNA-LNP composition further comprises a first buffer at a concentration of 0.01 to 0.15 mg/mL, a second buffer at a concentration of 0.5 to 0.65 mg/mL, a stabilizer at a concentration of 35 to 50 mg/mL, and a salt at a concentration of 5 to 15 mg/mL.

In a specific aspect, the FimH RNA-LNP composition is a lyophilized FimH RNA-LNP composition, and the lyophilized FimH RNA-LNP composition further comprises a Tris buffer composition comprising tromethamine at a concentration of 0.01 to 0.15 mg/mL, Tris HCl at a concentration of 0.5 to 0.65 mg/mL, sucrose at a concentration of 35 to 50 mg/mL, and sodium chloride (NaCl) at a concentration of 5 to 15 mg/mL.

Therefore, in a specific aspect, the lyophilized FimH RNA-LNP composition comprises a cationic lipid at a concentration of 0.8 to 0.95 mg/mL, a PEGylated 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, a second structural lipid at a concentration of 0.3 to 0.45 mg/mL, and further comprises a first buffer at a concentration of 0.01 to 0.15 mg/mL, a second buffer at a concentration of 0.5 to 0.65 mg/mL, a stabilizer at a concentration of 35 to 50 mg/mL, and a salt at a concentration of 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in a carrier or diluent at a concentration of 0.6 to 0.75 mL.

Thus, in some aspects, the lyophilized FimH RNA-LNP composition comprises 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, cholesterol at a concentration of 0.3 to 0.45 mg/mL, and further comprises tromethamine at a concentration of 0.01 and 0.15 mg/mL, Tris HCl at a concentration of 0.5 and 0.65 mg/mL, sucrose at a concentration of 35 to 50 mg/mL, and NaCl at a concentration of 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL saline.

The concentration of the lyophilized FimH RNA-LNP composition was determined after reconstitution.

The FimH RNA-LNP composition also comprises the FimH RNA described herein encapsulated in LNP, see D. Administration section.

In a specific aspect, the FimH RNA-LNP composition is a liquid FimH RNA-LNP composition comprising a FimH RNA polynucleotide encoding a FimH polypeptide as disclosed herein, wherein the FimH The RNA polynucleotide has a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in an LNP having a lipid composition comprising a cationic lipid at a concentration of 0.8 to 0.95 mg/mL, a PEGylated 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 specific aspects, the liquid FimH RNA-LNP composition comprises a FimH RNA polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in a LNP having a lipid composition comprising 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 comprising a Tris buffer composition comprising tromethamine 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 sucrose at a concentration of 95 to 110 mg/mL.

In a specific aspect, the FimH RNA-LNP composition is a lyophilized FimH RNA-LNP composition comprising a FimH RNA polynucleotide encoding a FimH polypeptide as disclosed herein, wherein the FimH The concentration of RNA polynucleotide is at least, at most, just or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90mg/mL, encapsulated in LNP with lipid composition, the lipid composition comprises the cationic lipid that concentration is 0.8 to 0.95mg/mL, the PEGylated lipid that concentration is 0.05 to 0.15mg/mL, the first structural lipid that concentration is 0.1 to 0.25mg/mL and the second structural lipid that concentration is 0.3 to 0.45mg/mL, and also comprises the first buffer that concentration is 0.01 to 0.15mg/mL, the second buffer that concentration is 0.5 to 0.65mg/mL, the stabilizer that concentration is 35 to 50mg/mL and the salt that concentration is 5 to 15mg/mL. In a specific aspect, the lyophilized composition is reconstructed in the carrier or diluent of 0.6 to 0.75mL. The concentration of the lyophilized FimH RNA-LNP composition was determined after reconstitution.

In a specific aspect, the lyophilized FimH RNA-LNP composition comprises a FimH RNA polynucleotide encoding a FimH polypeptide disclosed herein at a concentration of at least, at most, exactly, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in a LNP having a lipid composition comprising 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 comprising tromethamine at a concentration of 0.01 to 0.15 mg/mL, Tris at a concentration of 0.5 to 0.65 mg/mL. HCl, sucrose at a concentration of 35 to 50 mg/mL, and NaCl at a concentration of 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of saline. The concentration of the lyophilized FimH RNA-LNP composition is determined after reconstitution.

B. Vaccine

In some aspects, the pharmaceutical composition described herein is an immunogenic composition for inducing an immune response. For example, in some aspects, the immunogenic composition is a vaccine. In some aspects, the composition described herein includes at least one isolated nucleic acid or polypeptide molecule as described herein. In specific aspects, the immunogenic composition comprises nucleic acid, and the immunogenic composition is a nucleic acid vaccine. In some aspects, the immunogenic composition comprises RNA (such as mRNA), and the vaccine is an RNA vaccine. In other aspects, the immunogenic composition comprises DNA, and the vaccine is a DNA vaccine. In some other aspects, the immunogenic composition comprises polypeptide, and the vaccine is a polypeptide vaccine. The conditions and/or diseases that can be treated with nucleic acid and/or peptide or polypeptide compositions include but are not limited to those conditions and/or diseases caused and/or affected by infection, cancer, rare diseases, and other diseases or conditions caused by excessive production, insufficient production or improper production of proteins or nucleic acids.

In some aspects, the composition is substantially free of one or more impurities or contaminants, and, for example, comprises a nucleic acid or polypeptide molecule that is at least, at most, exactly, or between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure or at least 99% pure.

The present disclosure includes methods for preventing, treating or improving an infection, disease or condition of a subject, comprising administering to the subject an effective amount of an RNA molecule, the RNA molecule comprising at least one open reading frame encoding a polypeptide or composition as described herein. Like this, the present disclosure contemplates vaccines for active and passive immunization. An immunogenic composition proposed to be suitable for use as a vaccine can be prepared from an RNA molecule encoding a polypeptide (e.g., an E. coli FimH polypeptide as described herein). In some aspects, the immunogenic composition is lyophilized so as to be more easily formulated into a desired carrier.

The preparation of vaccines containing nucleic acids and/or peptides or polypeptides as active ingredients is generally well known in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference in their entirety. Typically, such vaccines are prepared as injectable liquid solutions or suspensions: solid forms suitable for dissolving or suspending in liquids prior to injection can also be prepared. The preparation can also be emulsified. The active immunogenic ingredients are typically mixed with pharmaceutically acceptable excipients that are compatible with the active ingredients. Suitable excipients are, for example, water, saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the vaccine may contain a certain amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, or adjuvants that enhance the effectiveness of the vaccine. In a specific aspect, the vaccine is formulated from a combination of substances as described in US Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference in their entirety.

Vaccines can be routinely administered parenterally by injection, such as subcutaneous injection or intramuscular injection. Additional preparations suitable for other modes of administration include suppositories, and oral preparations in some cases. For suppositories, traditional adhesives and carriers can include, for example, polyalkylene glycols or triglycerides: such suppositories can be formed by a mixture containing an active ingredient in the range of about 0.5% to about 10%. In some aspects, suppositories can be formed by a mixture containing an active ingredient in the range of about 1% to about 2%. Oral preparations include such commonly used excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations or powders, and contain about 10% to about 95% active ingredients.

The nucleic acid constructs encoding polypeptides and polypeptides can be formulated into vaccines in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and those formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or such organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.).

Typically, vaccines are administered in a manner compatible with the dosage form and in a therapeutically effective and immunogenic amount. The amount to be administered depends on the subject to be treated, including the ability of the individual immune system to synthesize antibodies and the desired degree of protection. The exact amount of active ingredient to be administered depends on the judgment of the practitioner. However, a suitable dosage range is on the order of several hundred micrograms of active ingredient per vaccination. The appropriate regimen for initial administration and booster injections also varies, but typically an initial administration is followed by subsequent vaccinations or other administrations.

The mode of application may vary widely. Any conventional method of administering the vaccine is suitable. It is believed that these include oral administration, parenteral administration, injection, etc. in a physiologically acceptable solid matrix or in a physiologically acceptable dispersion. The dosage of the vaccine depends on the route of administration and varies according to the size and health of the subject.

In some aspects, it is desirable to administer a vaccine. In some aspects, it is desirable to administer the vaccine multiple times, for example, 2, 3, 4, 5, 6 or more times. The vaccination can be an interval of 1, 2, 3, 4, 5, 6, 7, 8 weeks to 5, 6, 7, 8, 9, 10, 11, 12 weeks, including all ranges therebetween. In some aspects, the vaccination can be an interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, including all ranges therebetween. It may be desirable to regularly strengthen once every 1 to 5 years to maintain the protection level of the antibody.

i. Carrier

A pharmaceutically acceptable carrier may include a liquid or non-liquid base of the composition. If the composition is provided in liquid form, the carrier may be water, such as pyrogen-free water; isotonic saline or a buffered (aqueous) solution, such as a phosphate, citrate buffer solution. Water or a buffer, such as an aqueous buffer, may be used, which contains sodium, calcium and/or potassium salts. The sodium, calcium and/or potassium salts may be present in the form of their halides (e.g., chlorides, iodides or bromides), or in the form of their hydroxides, carbonates, bicarbonates or sulfates, etc. Examples of sodium salts include, but are not limited to, NaCI, Nal, NaBr, Na ₂ CO ₃ , NaHCO ₃ , Na ₂ SO ₄ , Na ₂ HPO ₄ , Na ₂ HPO ₄ · ₂ H ₂ O; examples of potassium salts include, but are not limited to, KCI, KI, KBr, K ₂ CO ₃ , KHCO ₃ , K ₂ SO ₄ , KH ₂ PO ₄ ; examples of calcium salts include, but are not limited to, CaCl ₂ , CaI ₂ , CaBr ₂ , CaCO ₃ , CaSO ₄ , Ca(OH) ₂ . Examples of other carriers may include sugars such as lactose, glucose, trehalose, and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; tragacanth powder; malt; gelatin; beef tallow; solid glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid. Examples of other carriers may include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical excipients using them, are described in Remington's Pharmaceutical Sciences.

ii. Adjuvant

Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions. A variety of adjuvants can be used to enhance the antibody response. Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides and natural substances. Specific adjuvants that can be used include Freund's adjuvant, oils (e.g. ISA51), IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, α-interferon, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts (such as aluminum hydroxide or other aluminum compounds), MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), lipopeptides (such as Pam3Cys). RIBI contains three components extracted from bacteria, namely MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS), in a 2% squalene/Tween80 emulsion. Even MHC antigens can be used.

Various methods of achieving adjuvant effects for vaccines include the use of agents such as aluminum hydroxide or aluminum phosphate (alum) (usually used as about 0.05 to about 0.1% solutions in phosphate-buffered saline), synthetic polymers with sugars, mixture (used as about 0.25% solution), by heat treatment at a temperature between about 70° and about 101°C for 30 seconds to 2 minutes to aggregate the proteins in the vaccine. Aggregation produced by reactivation of albumin with pepsin-treated (Fab) antibodies; mixtures with bacterial cells (e.g., C. parvum), endotoxins, or lipopolysaccharide components of gram-negative bacteria; emulsions in physiologically acceptable oil carriers (e.g., mannide monooleate (Aracel A)); or with 20% perfluorocarbon used as a blood substitute (block substitute) Emulsions of solutions can also be used to produce an adjuvant effect.

In addition to adjuvants, it may be desirable to co-administer biological response modifiers (BRMs) to enhance the immune response. BRMs have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose cyclophosphamide (CYP; 300 mg/m ² ) (Johnson/Mead, NJ) and cytokines such as gamma interferon, IL-2 or IL-12 or genes encoding proteins involved in immune adjuvant functions, such as B-7.

The administration of compositions described herein can be carried out by any acceptable mode of administration of pharmaceutical agents, to achieve similar effects. In some aspects, pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In specific aspects, FimH RNA molecules and/or RNA-LNP compositions are administered intramuscularly. In some aspects, pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration can include enteral administration (which relates to absorption by the gastrointestinal tract) or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner except by the gastrointestinal tract, such as by intravenous injection. In one aspect, pharmaceutical compositions are formulated for intramuscular administration. On the other hand, pharmaceutical compositions are formulated for systemic administration, such as for intravenous administration.

Pharmaceutical compositions can be formulated into solid, semisolid, liquid, lyophilized, frozen or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical compositions described herein are formulated to allow the active ingredients contained therein to be bioavailable when the composition is administered to a patient. The composition administered to a subject or patient is in the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and a container of a compound in the form of an aerosol can accommodate multiple dosage units. In any case, the composition to be administered will contain a therapeutic and/or prophylactic effective amount of a compound within the scope of the present disclosure or a pharmaceutically acceptable salt thereof for treating a disease or condition of interest according to the teachings described herein.

The pharmaceutical compositions within the scope of the present disclosure may be in the form of a solid or liquid and may be frozen or lyophilized. In one aspect, the carrier is granular, so the composition is, for example, in the form of a tablet or powder. The carrier may be a liquid, wherein the composition is, for example, an oral syrup, an injection, or an aerosol, which may be used, for example, for inhalation administration. In some aspects, when intended for oral administration, the pharmaceutical composition is in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included in the forms considered as solid or liquid herein. As a solid composition for oral administration, the pharmaceutical composition may be formulated in the form of a powder, granules, compressed tablets, pills, capsules, chewing gum, glutinous rice paper capsules, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present or excluded: a binder, such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin; an excipient, such as starch, lactose, or dextrin; a disintegrant, such as alginic acid, sodium alginate, corn starch, etc.; lubricants such as magnesium stearate or Glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavoring agents, such as mint, methyl salicylate or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it may contain a liquid carrier, such as polyethylene glycol or oil, in addition to the above-mentioned types of materials. The pharmaceutical composition may be in the form of a liquid, such as an elixir, a syrup, a solution, an emulsion or a suspension. To give two examples, the liquid may be used for oral administration or delivered by injection. In some aspects, when intended for oral administration, the composition contains, in addition to the compound of the present invention, one or more sweeteners, preservatives, dyes/colorants and flavor enhancers. In a composition intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents may be included or excluded.

Liquid pharmaceutical compositions, whether solutions, suspensions or other similar forms, may include or exclude one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions, such as physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, polypropylene glycol or other solvents that can be used as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for adjusting tonicity, such as sodium chloride or glucose; agents used as cryoprotectants, such as sucrose or trehalose. Parenteral preparations can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In one aspect, physiological saline is an adjuvant. In one aspect, the injectable pharmaceutical composition is sterile. Liquid pharmaceutical compositions intended for parenteral or oral administration should contain an amount of the compound so that a suitable dosage will be obtained.

Pharmaceutical compositions can be prepared by methods well known in the pharmaceutical field. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining a nucleic acid or polypeptide with sterile distilled water or other carrier to form a solution. Surfactants can be added to facilitate the formation of a uniform solution or suspension. Surfactants are compounds that interact non-covalently with compounds consistent with the teachings herein to facilitate dissolution or uniform suspension of the compound in an aqueous delivery system.

The pharmaceutical composition according to the present invention or its pharmaceutically acceptable salt is usually applied in the form of "therapeutically effective amount" or "prophylactically effective amount" and "pharmaceutically acceptable formulation". The term "pharmaceutically acceptable" means that the material is non-toxic and does not interact with the action of the active ingredient of the pharmaceutical composition. The terms "therapeutically effective amount" and "prophylactically effective amount" refer to the amount that achieves the desired response or desired effect alone or together with other doses. In the case of treating a specific disease, in one aspect, the desired response involves inhibiting the progression of the disease. This includes slowing the progression of the disease, particularly interrupting or reversing the progression of the disease. The desired response in the treatment of a disease can also be to delay the onset of the disease or the condition or to prevent the onset of the disease or condition.

The compositions within the scope of the present disclosure are administered in a therapeutically and/or prophylactically effective amount, which will vary according to a variety of factors, including the activity of the specific therapeutic and/or prophylactic agent used; the metabolic stability and duration of action of the therapeutic and/or prophylactic agent; the individual parameters of the patient, including the patient's age, weight, general health, sex and diet; the mode, time and/or duration of administration; the rate of excretion; the drug combination; the severity of a particular disorder or condition; and the subject being treated. Therefore, the dosage of the compositions described herein may depend on a variety of such parameters. If the patient's response to the initial dose is insufficient, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used. In some aspects, the composition may be administered at such a dose level (e.g., FimH RNA-LNP compositions), the dosage level being sufficient to deliver 0.0001 ng/μg/mg/kg to 100 ng/μg/mg/kg, 0.001 ng/μg/mg/kg to 0.05 ng/μg/mg/kg, 0.005 ng/μg/mg/kg to 0.05 ng/μg/mg/kg, 0.001 ng/μg/mg/kg to 0.005 ng/μg/mg/kg, 0.05 ng/μg/mg/kg to 0.5 ng/μg/mg/kg, 0.01 ng/μg/mg/kg to 50 ng/μg/mg/kg, 0.1 ng/μg/mg g/kg to 40ng/μg/mg/kg, 0.5ng/μg/mg/kg to 30ng/μg/mg/kg, 0.01ng/μg/mg/kg to 10ng/μg/mg/kg, 0.1ng/μg/mg/kg to 10ng/μg/mg/kg or 1ng/μg/mg/kg to 25ng/μg/mg/kg of subject body weight/day, once or multiple times per day, per week, per month, etc. to obtain the desired treatment, diagnosis, prevention or imaging effect (see, e.g., the unit dose range described in International Publication No. WO2013/078199, which is incorporated herein by reference in its entirety). In some aspects, the composition (e.g., FimH) can be administered at such a dosage level. RNA-LNP composition), the dosage level is sufficient to deliver at least, at most, just, or between any two of the following to achieve the desired therapeutic, diagnostic, prophylactic or imaging effect: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0 .004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3 ,4,5,6,7,8,9,10,11,12,13,14 ,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 ng/μg/mg/kg subject body weight/day, administered once or multiple times a day, weekly, monthly, etc.

In some aspects, a composition (e.g., a FimH RNA-LNP composition) can be administered at a total dose or dose level sufficient to deliver a total dose of at least, at most, exactly, or between any two of the following to achieve the desired therapeutic, diagnostic, preventive, or imaging effect: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 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, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0 .9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,5 2, 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, 99 or 100 ng/μg/mg/day, once or more per day, per week, per month, etc.

In particular aspects, the composition (e.g., FimH RNA-LNP composition) can be administered at a total dose or dose level sufficient to deliver a total dose of at least, at most, exactly, or between any two of the following: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 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, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 ,6,7,8,9,10,11,12,1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 ,54,55,56,57,58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg/mL of FimH RNA encapsulated in LNPs.

In exemplary aspects, the composition (e.g., FimH RNA-LNP composition) can be administered at a dosage level of at least, at most, just, or between 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL of any two of the FimH RNAs encapsulated in LNPs. In exemplary aspects, the composition (e.g., FimH RNA-LNP composition) can be administered at a dosage level of at least, at most, just, or between 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg of any two of the FimH RNAs encapsulated in LNPs.

In particular aspects, the composition (e.g., FimH RNA-LNP composition) can be administered at a total dose or dose level sufficient to deliver a total dose of at least, at most, exactly, or between any two of the following: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 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, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 ,6,7,8,9,10,11,12,1 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 ,54,55,56,57,58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg/mL of FimH RNA encapsulated in LNPs.

In exemplary aspects, the composition (e.g., FimH RNA-LNP composition) can be administered at a dosage level of at least, at most, just, or between 1, 15, 30, 45, 60, 75, or 90 μg/mL of any two of the FimH RNAs encapsulated in the LNP. In exemplary aspects, the composition (e.g., FimH RNA-LNP composition) can be administered at a dosage level of at least, at most, just, or between 1, 15, 30, 45, 60, 75, or 90 μg of any two of the FimH RNAs encapsulated in the LNP.

The desired dosage can be delivered multiple times a day (e.g., 1, 2, 3, 4, 5 times or more a day), every other day, every three days, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc. In some aspects, the desired dosage can be delivered using a single dose administration. In some aspects, multiple administrations (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations) can be used to deliver the desired dosage. When multiple administrations are used, a split administration regimen can be used. The administration time between the initial administration of the composition and the subsequent administrations of the composition can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day ... 6 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years.

In some aspects, the composition (e.g., FimH RNA-LNP composition) can be administered in a single dose. In some aspects, the composition (e.g., FimH RNA-LNP composition) can be administered twice (e.g., 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 1 0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.01, 0.02, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0. ... 0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 ,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43, 44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100ng/μg/mg of FimH RNA encapsulated in LNP. The present disclosure encompasses higher and lower dosages and frequencies of administration. For example, the composition (e.g., FimH RNA-LNP composition) can be administered three or four times. It may be desirable to perform regular boosts at intervals of 1-5 years to maintain antibody protection levels.

In some aspects, a composition (e.g., a FimH RNA-LNP composition) is administered to a subject in a single dose of at least, at most, exactly, or between any two of the following: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0 .006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9 ,10,11,12,13,14,1 5,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55 ,56,57,58,59,60,6 1, 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, 99 or 100 ng/μg/mg of FimH RNA encapsulated in LNP. In some aspects, the composition (e.g., FimH RNA-LNP composition) is administered to a subject at a single dose of at least, at most, exactly, or between any two of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg of FimH RNA encapsulated in LNP.

In some aspects, a composition (e.g., a FimH RNA-LNP composition) is administered to a subject in two doses of at least, at most, exactly, or between any two of the following: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 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, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55 ,56,57,58,59,60,6 1, 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, 99 or 100 ng/μg/mg of FimH RNA encapsulated in LNP. In some aspects, the composition (e.g., FimH RNA-LNP composition) is administered to a subject at two doses of at least, at most, exactly, or between any two of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg of FimH RNA encapsulated in LNP.

In specific aspects, the composition (e.g., FimH RNA-LNP composition) can be administered twice (e.g., day 0 and day 28, day 0 and day 60, day 0 and day 180, day 0 and 2 months later, day 0 and 6 months later), with each administration being performed at a total dose or dose level sufficient to deliver a total dose of at least, at most, exactly, or between any two of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, or 90 μg of FimH RNA encapsulated in LNPs.

IX. How to use

Provided herein are compositions (eg, pharmaceutical compositions comprising FimH RNA molecules and/or FimH RNA-LNPs), methods, kits, and reagents for preventing and/or treating E. coli infections in humans and other mammals.

RNA (e.g., mRNA) vaccines can be used in a variety of settings, depending on the prevalence of infection or the degree or level of unmet medical needs. RNA vaccines can be used to treat and/or prevent E. coli infections of a variety of genotypes, strains, and isolates. RNA vaccines generally have superior properties because they produce higher antibody titers than commercially available antibacterial therapeutic treatments and produce responses earlier. Although not wishing to be bound by theory, it is believed that, since RNA vaccines employ natural cell mechanisms, RNA vaccines, as mRNA polynucleotides, are better designed to produce appropriate protein conformations during translation. Unlike conventional vaccines that are manufactured in vitro and may elicit unwanted cellular responses, RNA (e.g., mRNA) vaccines are presented to cell systems in a more natural manner.

In some cases, people may be at risk of infection with more than one E. coli antigen. RNA (e.g., mRNA) therapeutic vaccines are particularly suitable for combined vaccination methods due to a variety of factors, including but not limited to speed of manufacture, ability to quickly customize vaccines to adapt to perceived geographical threats, etc. In addition, because vaccines utilize the human body to produce antigenic proteins, vaccines are prone to produce larger and more complex antigenic proteins, allowing for correct folding, surface expression, antigen presentation, etc. in human subjects. In order to protect against more than one E. coli antigen, a combined vaccine can be administered, which includes RNA (e.g., mRNA) encoding a first E. coli antigen (e.g., FimH or a fragment thereof) or at least one antigenic polypeptide protein (or antigenic portion thereof) of an organism, and also includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second antigen. The RNA (e.g., mRNA) can be co-formulated in, for example, a single lipid nanoparticle (LNP), or can be formulated in a separate LNP for co-administration.

FimH RNA compositions (e.g., FimH RNA-LNP compositions) can be used as prophylactic agents. They can be used medically to prevent and/or treat infectious diseases. In exemplary aspects, FimH RNA compositions are used to provide prophylactic protection against urinary tract infections (UTIs). The FimH vaccines disclosed herein may be particularly useful for preventing and/or treating immunocompromised and elderly patients to prevent or reduce the severity and/or duration of E. coli infections.

In some aspects, a FimH RNA composition (eg, a FimH RNA-LNP composition) of the present disclosure is administered to a subject (eg, a mammalian subject, such as a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

In some aspects, the disclosed FimH RNA compositions can be used to prime immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

In some aspects, after administering FimH RNA molecules as described herein (e.g., formulated as RNA-LNP), at least a portion of RNA is delivered to a target cell. In some aspects, at least a portion of RNA is delivered to the cytosol of a target cell. In some aspects, RNA is translated by a target cell to produce a polypeptide or protein encoded by the target cell. In some aspects, the target cell is a splenocyte. In some aspects, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In some aspects, the target cell is a dendritic cell or a macrophage. RNA molecules (e.g., RNA-LNP as described herein) can be used to deliver RNA to such target cells. Therefore, the present disclosure also relates to a method for delivering RNA to a target cell of a subject, comprising administering RNA particles as described herein to the subject.

In some aspects, RNA is delivered to the cytosol of target cells. In some aspects, RNA is translated by target cells to produce polypeptides or proteins encoded by RNA." Coding " refers to the inherent properties of specific nucleotide sequences in polynucleotides (such as genes, cDNA or mRNA), to be used as templates for synthesizing other polymers and macromolecules with determined nucleotide sequences (such as, rRNA, tRNA and mRNA) or determined amino acid sequences in biological processes, and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to the gene produce protein in cells or other biological systems, the gene has just encoded protein. Coding strand (its nucleotide sequence is identical to the mRNA sequence, and is usually provided in the sequence table) and non-coding strand (as the template for gene or cDNA transcription) can be referred to as protein or other products encoding the gene or cDNA.

In some aspects, nucleic acid compositions described herein, such as compositions comprising FimH RNA-LNPs, are characterized in that (e.g., when administered to a subject) the encoded polypeptide is continuously expressed. For example, in some aspects, such compositions are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and in some aspects, such expression continues for at least 36 hours or longer, including, for example, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours or longer.

In some aspects, the present disclosure relates to a method of inducing an immune response in a subject. The method comprises administering to the subject an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

In another aspect, the present disclosure relates to a method for vaccinating a subject. The method comprises administering an effective amount of an RNA molecule, RNA-LNP and/or composition described herein to a subject in need thereof.

In another aspect, the present disclosure relates to a method for treating or preventing a bacterial disease. The method comprises administering to a subject an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

In another aspect, the present disclosure relates to a method for treating or preventing or reducing the severity of E. coli infection and/or a disease caused by E. coli. The method comprises administering to a subject an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

On the other hand, the disclosure relates to methods for treating or preventing or reducing the severity of an infection in a subject by, for example, inducing an immune response to an infectious agent (infectious agent) (e.g., E. coli) in a subject. In some aspects, the method includes administering an initiating composition comprising an effective amount of RNA molecules, RNA-LNPs, and/or compositions as described herein, and administering a booster composition comprising an effective amount of RNA molecules, RNA-LNPs, and/or compositions. In some aspects, the composition induces an immune response, including an antibody response. In some aspects, the composition induces an immune response, including a T cell response.

On the other hand, the disclosure relates to a method for treating or preventing or alleviating the severity of an E. coli infection and/or a disease caused by E. coli, for example, by inducing an immune response to E. coli FimH in a subject. In some aspects, the method comprises administering a triggering composition comprising an effective amount of an RNA molecule, RNA-LNP and/or a composition as described herein, and administering a booster composition comprising an effective amount of an RNA molecule, RNA-LNP and/or a composition as described herein. In some aspects, the composition induces an immune response, including an antibody response. In some aspects, the composition induces an immune response, including a T cell response.

The methods disclosed herein may involve administering to a subject a FimH RNA-LNP composition comprising at least one FimH RNA molecule having an open reading frame encoding at least one FimH antigenic polypeptide, thereby inducing an immune response specific to an E. coli FimH antigenic polypeptide in the subject, wherein the anti-antigen polypeptide antibody titer in the subject is increased after vaccination relative to the anti-antigen polypeptide antibody titer in a subject vaccinated with a prophylactic effective dose (e.g., a therapeutically effective dose that prevents viral infection at a clinically acceptable level) of a conventional E. coli vaccine. "Anti-antigen polypeptide antibodies" are serum antibodies that specifically bind to an antigenic polypeptide. In some aspects, the anti-antigen polypeptide antibody titer in the subject increases after administration of the FimH RNA-LNP composition by at least, at most, between any two of the following, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 logs relative to the anti-antigen polypeptide antibody titer in a subject administered a prophylactic effective dose of a conventional composition for FimH.

The methods disclosed herein may involve administering to a subject a FimH RNA-LNP composition comprising at least one FimH RNA-LNP having an open reading frame encoding at least one FimH antigenic polypeptide. RNA molecules, thereby inducing an immune response specific for a FimH antigenic polypeptide in a subject, wherein the immune response in the subject is equivalent to the immune response in a subject administered a traditional composition for FimH, wherein the dosage level of the traditional composition is at least, at most, between any two of the following, or exactly 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 times that of the RNA composition.

In some aspects, RNA molecules, RNA-LNPs and/or compositions are used as vaccines. In some aspects, RNA molecules, RNA-LNPs and/or compositions can be used in a variety of therapeutic or preventive methods for preventing, treating or ameliorating urinary tract infections, urosepsis, pyelonephritis or cystitis.

FimH RNA compositions can be administered prophylactically to healthy subjects or during the early incubation period of infection or during active infection after symptoms appear. In some aspects, the subject is immunocompetent. In some aspects, the subject is immunocompromised.

In some aspects, RNA molecules, RNA-LNPs and/or compositions are administered in a single dose. In some aspects, a second, third or fourth dose may be given. In some aspects, RNA molecules, RNA-LNPs and/or compositions are administered in multiple doses.

In some aspects, the RNA molecules, RNA-LNPs, and/or compositions are administered intramuscularly (IM) or intradermally (ID).

The present disclosure also provides kits comprising RNA molecules, RNA-LNPs and/or compositions.

In some aspects, the RNA molecules, RNA-LNPs and/or compositions described herein are administered to a subject less than about 1 year old, or about 1 year old to about 10 years old, or about 10 years old to about 20 years old, or about 20 years old to about 50 years old, or about 60 years old to about 70 years old, or older.

In some aspects, the subject is at least, at most, exactly, or between any two of the following ages: less than 1 year old, greater than 1 year old, greater than 5 years old, greater than 10 years old, greater than 20 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 60 years old, greater than 70 years old, or older. In some aspects, the subject is greater than 50 years old.

In some aspects, the subject is at least, at most, exactly, or between any two of the following ages: 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 aspects, the subject may be about 50 years old or older.

In some aspects, the subject is at least, at most, exactly, or between any two of the following ages: 1 year or older, 5 years or older, 10 years or older, 20 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older, or older. In some aspects, the subject may be 50 years or older.

In one embodiment, the mRNA vaccine of the present invention comprises lipids. The lipids and modRNA can form nanoparticles together. The lipids can encapsulate the mRNA in the form of lipid nanoparticles (LNPs) to help the RNA/lipid nanoparticles enter cells and remain stable.

Lipid nanoparticle can comprise lipid component and one or more additional components, for example therapeutic component and/or prevention component.LNP can be designed for one or more specific applications or targets.Can be based on specific applications or targets, and/or based on the effectiveness, toxicity, expense, ease of use, availability or other characteristics of one or more compositions to select the composition of LNP.Similarly, can be according to for example, the effectiveness and toxicity of specific composition combination, for specific application or target select specific formulation (formulation) of LNP.The effectiveness and tolerability of LNP preparation may be subject to the influence of formulation stability.

Lipid nanoparticles can be designed for one or more specific applications or targets. For example, LNPs can be designed to deliver therapeutic and/or prophylactic agents (e.g., RNA) to specific cells, tissues, organs, systems, or groups thereof in a mammal.

The physicochemical properties of lipid nanoparticles can be changed to increase the selectivity to specific body targets. For example, the particle size can be adjusted according to the window size of different organs. The therapeutic agent and/or preventive agent included in the LNP can also be selected according to one or more desired delivery targets. For example, a therapeutic agent and/or preventive agent can be selected for a specific indication, a patient's condition, a disease or a disorder and/or be delivered to a specific cell, tissue, organ or system or its group (such as local or specific delivery). In certain embodiments, LNP can include an mRNA encoding a polypeptide of interest, which can be translated to produce a polypeptide of interest in the cell. Such compositions can be designed to be delivered specifically to a specific organ. In some embodiments, the composition can be designed to be delivered specifically to a mammal's liver. In some embodiments, the composition can be designed to be delivered specifically to a lymph node. In some embodiments, the composition can be designed to be delivered specifically to a mammal's spleen.

LNP may include one or more components as described herein. In some embodiments, LNP formulations of the present disclosure include at least one lipid nanoparticle component. Lipid nanoparticles may include a lipid component and one or more additional components, such as a therapeutic and/or prophylactic agent, such as a nucleic acid. LNP can be designed for one or more specific applications or targets. The composition of LNP can 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, a specific formulation of LNP can be selected for a specific application or target, for example, based on the efficacy and toxicity of a specific combination of ingredients. The efficacy and tolerability of LNP formulations may be affected by formulation stability.

In some embodiments, for example, a polymer can be included in the LNP and/or used to encapsulate or partially encapsulate the LNP. The polymer can be biodegradable and/or biocompatible. The polymer can be selected from, but not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. For example, the polymer may include polycaprolactone (PCL), ethylene vinyl acetate polymer (EVA), polylactic acid (PLA), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), poly-lactic acid-glycolic acid copolymer (PLGA), poly-L-lactic acid-glycolic acid copolymer (PLLGA), poly-D,L-lactide (PDLA), poly-L-lactide (PLLA), poly-D,L-lactide-caprolactone copolymer, poly-D,L-lactide-caprolactone-glycolide copolymer, poly-D,L-lactide-PEO-D,L-lactide copolymer, poly-D,L-lactide ester-PPO-D,L-lactide copolymers, polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyethylene ethers, polyethylene esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinyl pyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic acid polymers such as poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(methacrylic acid), acrylate), poly(butyl acrylate), poly(octadecyl acrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and copolymers and mixtures thereof, polydioxanone and copolymers thereof, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, polyorthoesters, polybutyric acid, polyvaleric acid, poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, dasyphylline, bromhexine, carbocysteine, eprazone, mesna, ambroxol, sobrexol, domiol, letosteine, sitoronin, tiopronin, gelsolin, thymosin β4, dornase α, netexin, and erdosteine), and DNases (e.g., rhDNase). The surface-altering agent may be located within the nanoparticle and/or on the surface of the LNP (e.g., by coating, adsorption, covalent bonding, or other processes).

LNP can also comprise one or more functionalized lipids.For example, lipid can be functionalized with alkyne groups, and when exposed to azide under appropriate reaction conditions, cycloaddition reaction can take place.Specifically, lipid bilayer can be functionalized by one or more groups that help to promote membrane penetration, cell recognition or imaging in this way.The surface of LNP can also be put together with one or more useful antibodies.Can be used for target cell delivery, imaging and membrane penetration and conjugates are well known in the art.

In addition to these components, lipid nanoparticles may also include any substance that can be used in pharmaceutical compositions. For example, lipid nanoparticles may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, surfactants, buffers, preservatives and other types.

Surfactants and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [ 20], polyoxyethylene sorbitan[ 60], polyoxyethylene sorbitan monooleate[ 80], Sorbitan monopalmitate[ 40], Sorbitan monostearate[ 60], Sorbitan tristearate[ 65], glycerol monooleate, sorbitan monooleate[ 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [ 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [ 30]), polyvinyl pyrrolidone, diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F68, 188. Cetrimide bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and/or a combination thereof.

The example of preservative may include but is not limited to antioxidant, chelating agent, free radical scavenger, antimicrobial preservative, antifungal preservative, alcohol preservative, acidic preservative and/or other preservative. The example of antioxidant includes but is not limited to alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite and/or sodium sulfite. The example of chelating agent includes ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, ethylenediaminetetraacetic acid, fumaric acid, malic acid, phosphoric acid, sodium ethylenediaminetetraacetate, tartaric acid and/or trisodium ethylenediaminetetraacetate. The example of antimicrobial preservative includes but is not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerine, hexetidine, imidazolidinyl urea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol and/or thimerosal. The example of antifungal preservative includes but is not limited to butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and/or sorbic acid. The example of alcohol preservative includes but is not limited to ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate and/or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT Methylparaben, 115. II, NEOLONE ^™ , KATHON ^™ and/or Exemplary free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

Examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium valerate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, monopotassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, monosodium phosphate, sodium phosphate mixtures, tromethamine, sulfamate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and/or combinations thereof.

In some embodiments, the preparation comprising LNP may also include salts, such as chloride salts. In some embodiments, the preparation comprising LNP may also include sugars, such as disaccharides. In some embodiments, the preparation also includes sugars but does not include salts, such as chloride salts. In some embodiments, LNP may also include one or more small hydrophobic molecules, such as vitamins (such as vitamin A or vitamin E) or sterols. Carbohydrates may include monosaccharides (such as glucose) and polysaccharides (such as glycogen and derivatives thereof and analogs).

The characteristics of LNP may depend on its components. For example, LNPs including cholesterol as structural lipids may have different characteristics from LNPs including different structural lipids. As used herein, the term "structural lipid" refers to sterols and lipids containing sterol moieties. As defined herein, "sterols" are steroid subgroups consisting of steroids. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is alpha-tocopherol.

In some embodiments, the characteristic of LNP may depend on the absolute amount or relative amount of its components. For example, LNP comprising a higher mole fraction of phospholipids may have different characteristics from LNP comprising a lower mole fraction of phospholipids. Characteristics may also vary because of the preparation method and conditions of lipid nanoparticles. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

Phospholipid part can be selected from the non-restrictive group such as being made up of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin.Fatty acid part can be selected from the non-restrictive group such as being made up of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid.Specific phospholipid can promote the fusion with film.In some embodiments, cationic phospholipid can interact with one or more negatively charged phospholipids of film (for example, cell or intracellular membrane).Phospholipid and membrane fusion can allow one or more compositions (for example, therapeutic agent) containing lipid composition (for example, LNP) to pass through film, thereby allow for example one or more compositions to be delivered to target tissue. Non-natural phospholipid species are also contemplated, including natural species that are modified and substituted (including branching, oxidation, cyclization and alkynes). In some embodiments, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, copper-catalyzed cycloaddition reactions can occur when alkyne groups are exposed to azides. Such reactions can be used for the lipid bilayer of functionalized nanoparticle compositions to promote membrane penetration or cell recognition, or for conjugating nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes). Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid. Phospholipids also include phospho-sphingolipids, such as sphingomyelin. In some embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC.

Lipid nanoparticles 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 LNP. Zeta potential can be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Dynamic light scattering can also be used to measure particle size. Instruments such as ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure the various characteristics of LNP, such as particle size, polydispersity index, and zeta potential.

The average size of the LNPs may be between 10 nm and 100 nm, for example, as measured by dynamic light scattering (DLS). For example, the average size may be between about 40 nm and about 150 nm, for example, 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 LNP can be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In certain embodiments, the average size of LNP can be from about 70nm to about 100nm. In specific embodiments, the average size can be about 80nm. In other embodiments, the average size can be about 100nm.

LNP may be relatively uniform. Polydispersity index can be used to indicate the homogeneity of LNP, such as the particle size distribution of lipid nanoparticles. Small (e.g., less than 0.3) polydispersity index generally represents a narrow particle size distribution. LNP can have a polydispersity index of 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 LNP can be about 0.10 to about 0.20.

The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low charge (positive or negative) are generally desirable because the species with higher charge may interact undesirably with cells, tissues, and other components in the body. In some embodiments, the zeta potential of the LNP can 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 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 +5 mV to about +10 mV.

The encapsulation efficiency of therapeutic and/or prophylactic agents describes the amount of therapeutic and/or prophylactic agents encapsulated with LNP or otherwise associated with LNPs after preparation relative to the initial amount provided. It is expected that the encapsulation efficiency is high (e.g., close to 100%). For example, the encapsulation efficiency can be measured by comparing the amount of therapeutic and/or prophylactic agents in a solution containing lipid nanoparticles before and after decomposing the lipid nanoparticles with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agents (e.g., RNA) in the solution. For lipid nanoparticles as described herein, the encapsulation efficiency of therapeutic and/or prophylactic agents can be at least 50%, such as 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 can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

The LNP may optionally include one or more coatings. For example, the LNP may be formulated into a capsule, film or tablet having a coating. The capsule, film or tablet comprising the composition described herein may have any useful size, tensile strength, hardness or density.

The preparation comprising amphiphilic polymers and lipid nanoparticles can be formulated as a pharmaceutical composition in whole or in part. The pharmaceutical composition can include one or more amphiphilic polymers and one or more lipid nanoparticles. For example, the pharmaceutical composition can include one or more amphiphilic polymers and one or more lipid nanoparticles including one or more different therapeutic agents and/or preventive agents. The pharmaceutical composition can also include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the preparation and manufacture of pharmaceutical compositions and medicaments can be found in, for example, Remington's The Science and Practice of Pharmacy, 21st edition, A.R.Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, unless any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the LNP or one or more amphiphilic polymers in the preparation of the present disclosure. An excipient or adjunct ingredient may be incompatible with a component of the LNP of the formulation or the amphiphilic polymer if the combination of the excipient or adjunct ingredient with the component or amphiphilic polymer may result in any undesirable biological effect or other deleterious effect.

In some embodiments, one or more excipients or auxiliary components may account for more than 50% of the total mass or volume of the pharmaceutical composition including LNP. For example, one or more excipients or auxiliary components may account for 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration (United States Food and Drug Administration). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (United States Pharmacopoeia (USP)), the European Pharmacopoeia (European Pharmacopoeia (EP)), the British Pharmacopoeia (British Pharmacopoeia) and/or the International Pharmacopoeia (International Pharmacopoeia). According to the present disclosure, the relative amounts of one or more amphiphilic polymers, one or more lipid nanoparticles, one or more pharmaceutically acceptable excipients and/or any additional ingredients in the pharmaceutical composition will vary according to the identity, size and/or condition of the subject being treated, and also according to the route by which the composition is to be administered. For example, a pharmaceutical composition may include 0.1% to 100% (wt wt) of one or more lipid nanoparticles. As another example, a pharmaceutical composition may include 0.1% to 15% (wt/vol) of one or more amphiphilic polymers (e.g., 0.5%, 1%, 2.5%, 5%, 10% or 12.5% w/v).

In certain embodiments, the lipid nanoparticles and/or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and/or transportation (e.g., stored at a temperature of 4°C or less, such as a temperature between about -150°C and about 0°C or a temperature 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, a pharmaceutical composition comprising one or more amphiphilic polymers and one or more lipid nanoparticles is a solution or solid (e.g., by lyophilization) that is stored at, for example, about -20°C. In some embodiments, the present disclosure also relates to a method for increasing the stability of lipid nanoparticles by adding an effective amount of an amphiphilic polymer and storing the lipid nanoparticles and/or pharmaceutical compositions thereof at a temperature of 4°C or less (e.g., a temperature between about -150°C and about 0°C or a temperature between about -80°C and about -20°C, such as 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).

The chemical properties of the LNPs, LNP suspensions, lyophilized LNP compositions or LNP formulations of the present disclosure can be characterized by a variety of methods. In some embodiments, electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reversed phase liquid chromatography) can be used to check mRNA integrity.

In some embodiments, the LNP integrity of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure is about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more.

In some embodiments, the LNP integrity of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present 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, about 100% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, about 200% or more, about 210% or more, about 220% or more, about 230% or more, about 240% or more, about 250% or more, about 260% or more, about 270% or more, about 280% or more, about 290% or more, about 300% or more, about 310% or more, about 320% or more, about 330% or more, about 340% or more, about 350% or more, about 360% or more, about 370% or more, about 380% or more, about 390% or more, about 400% or more, about 410% or more, about 420% or more, about 430% or more, about 440% or more, about 450% or more, about 460% or more, about 470% or more, about 480% or more, about 490% or more, about 500% or more, about 500% or more, about 510% or more, about 520% or more, about 5 times or more, about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, about 50 times or more, about 100 times or more, about 200 times or more, about 300 times or more, about 400 times or more, about 500 times or more, about 1000 times or more, about 2000 times or more, about 3000 times or more, about 4000 times or more, about 5000 times or more, or about 10000 times or more.

In some embodiments, the Txo% of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure 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, about 120 months or longer.

In some embodiments, the Txo% of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present 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, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more longer than the Txo% of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations produced by comparable methods.

In some embodiments, the T1/2 of the LNPs, LNP suspensions, lyophilized LNP compositions, or LNP formulations of the present disclosure 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, about 120 months or longer.

In some embodiments, the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation of the present 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, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, longer than the T1/2 of the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation produced by a comparable method.

As used herein, "Tx" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation is degraded to about X of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For example, "T80%" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation is degraded to about 80% of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation. For another example, "T1/2" refers to the amount of time that the nucleic acid integrity (e.g., mRNA integrity) of a LNP, LNP suspension, lyophilized LNP composition, or LNP formulation is degraded to about 1/2 of the initial integrity of the nucleic acid (e.g., mRNA) used to prepare the LNP, LNP suspension, lyophilized LNP composition, or LNP formulation.

Lipid nanoparticle can comprise lipid component and one or more additional components, for example therapeutic component and/or prevention component, for example nucleic acid.LNP can be designed for one or more specific applications or targets.Can be based on specific applications or targets, and/or based on the effectiveness, toxicity, expense, ease of use, availability or other characteristics of one or more compositions to select the composition of LNP.Similarly, can be according to for example, the effectiveness and toxicity of specific composition combination, for specific application or target select the specific formulation of LNP.The effectiveness and tolerability of LNP preparation may be subject to the influence of formulation stability.

The lipid component of LNP can include, for example, cationic lipids, phospholipids (eg, unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The ingredients of the lipid component can be provided in specific fractions.

In some embodiments, the LNP further comprises a phospholipid, a PEG lipid, a structured lipid, or any combination thereof. Suitable phospholipids, PEG lipids, and structured lipids for use in the disclosed methods are further disclosed herein.

In some embodiments, the lipid components of LNP include cationic lipids, phospholipids, PEG lipids and structural lipids. In certain embodiments, the lipid components of lipid nanoparticles include cationic lipids of about 30mol% to about 60mol%, phospholipids of about 0mol% to about 30mol%, structural lipids of about 18.5mol% to about 48.5mol%, and PEG lipids of about 0mol% to about 10mol%, with the premise that total mol% is no more than 100%. In some embodiments, the lipid components of lipid nanoparticles include cationic lipid compounds of about 35mol% to about 55mol%, phospholipids of about 5mol% to about 25mol%, structural lipids of about 30mol% to about 40mol%, and PEG lipids of about 0mol% to about 10mol%. In a specific embodiment, lipid components include the described cationic lipids of about 50mol%, the phospholipids of about 10mol%, the structural lipids of about 38.5mol%, and the PEG lipids of about 1.5mol%. In another specific embodiment, the lipid component includes about 40mol% of the cationic lipid, about 20mol% of the phospholipid, about 38.5mol% of the structural lipid, and about 1.5mol% of the PEG lipid. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-DMG and/or the structural lipid can be cholesterol.

In some embodiments, the amount of therapeutic and/or preventive agent in LNP may depend on the size, composition/desired target and/or application or other properties and the property of therapeutic and/or preventive agent. For example, the amount of RNA useful in LNP may depend on the size, sequence and other characteristics of RNA. The relative amount of therapeutic and/or preventive agent (i.e., drug substance) and other components (e.g., lipid) in LNP may also change. In some embodiments, the wt/wt ratio of lipid component and therapeutic and/or preventive agent in LNP can be 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 prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt/wt ratio is about 20: 1. For example, the amount of the therapeutic and/or prophylactic agent in the LNP can be measured using absorption spectroscopy (e.g., UV-visible spectroscopy).

RNA (e.g., mRNA) vaccines can be used in a variety of settings, depending on the prevalence of infection or the degree or level of unmet medical needs. RNA vaccines can be used to treat and/or prevent E. coli infections of a variety of genotypes, strains, and isolates. RNA vaccines generally have superior properties because they produce higher antibody titers than commercially available antiviral or antibacterial therapeutic treatments, and produce responses earlier. Although not wishing to be bound by theory, it is believed that, since RNA vaccines employ natural cell mechanisms, RNA vaccines, as mRNA polynucleotides, are better designed to produce appropriate protein conformations during translation. Unlike conventional vaccines that are manufactured in vitro and may elicit unwanted cellular responses, RNA (e.g., mRNA) vaccines are presented to cell systems in a more natural manner.

In some cases, people may be at risk of infection with more than one E. coli antigen. RNA (e.g., mRNA) therapeutic vaccines are particularly suitable for combined vaccination methods due to a variety of factors, including but not limited to speed of manufacture, the ability to quickly customize vaccines to adapt to perceived geographical threats, etc. In addition, because the vaccine utilizes the human body to produce antigenic proteins, the vaccine is easy to produce larger and more complex antigenic proteins, thereby allowing for correct folding, surface expression, antigen presentation, etc. in human subjects. In order to produce protection against more than one E. coli antigen, a combined vaccine can be administered, which includes RNA (e.g., mRNA) encoding a first E. coli antigen (e.g., FimH or a fragment thereof) or at least one antigenic polypeptide protein (or antigenic portion thereof) of an organism, and also includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second antigen. The RNA (e.g., mRNA) can be co-formulated in, for example, a single lipid nanoparticle (LNP), or can be formulated in separate LNPs for co-administration.

Some embodiments of the present disclosure provide an E. coli vaccine (or composition or immunogenic composition) comprising at least one RNA polynucleotide having an open reading frame encoding at least one E. coli FimH antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to E. coli).

Some embodiments of the present disclosure provide an E. coli vaccine, comprising at least one RNA polynucleotide having an open reading frame, the open reading frame encoding at least one E. coli FimH polypeptide or an immunogenic fragment of the above-mentioned new FimH polypeptide sequence (e.g., an immunogenic fragment capable of inducing an immune response to E. coli). In some embodiments, the E. coli vaccine comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame, the open reading frame encoding at least one FimH polypeptide, the FimH polypeptide comprising a modified sequence having at least 75% (e.g., any number between 75% and 100%, including, for example, 70%, 80%, 85%, 90%, 95%, 99% and 100%) identity with the amino acid sequence of the above-mentioned new FimH sequence. The modified sequence may have at least 75% (e.g., any number between 75% and 100%, including, for example, 70%, 80%, 85%, 90%, 95%, 99% and 100%) identity with the amino acid sequence of the above-mentioned new FimH sequence.

Some embodiments of the present disclosure provide an isolated nucleic acid comprising a sequence encoding the above-mentioned novel E. coli FimH polypeptide sequence; an expression vector comprising the nucleic acid; and a host cell comprising the nucleic acid. The present disclosure also provides a method for producing a polypeptide of any of the above-mentioned novel E. coli FimH sequences. The method may include culturing the host cell in a culture medium under conditions that allow expression of the nucleic acid of the above-mentioned novel E. coli FimH sequence, and purifying the novel E. coli FimH polypeptide from the cultured cells or the culture medium of the cells.

In some embodiments, the RNA (eg, mRNA) vaccine further comprises an adjuvant.

In some embodiments, the at least one RNA polynucleotide encodes at least one E. coli FimH polypeptide that is not attached to a cell.

In some embodiments, at least one RNA polynucleotide encodes at least one E. coli FimH polypeptide that does not allow the bacteria to bind to the cell, wherein the cell is a bladder epithelial cell. Some embodiments of the present disclosure provide a vaccine comprising at least one ribonucleic acid (RNA) (e.g., mRNA) polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide, at least one 5' terminal cap and at least one chemical modification, formulated in a lipid nanoparticle.

In some embodiments, the 5' terminal cap is m7G(5')ppp(5')(2'OMeA)pG.

In some embodiments, at least one chemical modification is selected from pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyluridine. In some embodiments, the chemical modification is at the 5th position of uracil. In some embodiments, the chemical modification is N1-methyl pseudouridine. In some embodiments, the chemical modification is N1-ethylpseudouridine.

In some embodiments, the lipid nanoparticles include cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. In some embodiments, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol. In some embodiments, the cationic lipids are selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-ene-1-yl) 9-((4-(dimethylamino)butanoyl)oxy) heptadecanedioate (L319), (12Z, 15Z)-N, N-dimethyl-2-nonyl hexone carbon-12, 15-diene-1-amine (L608) and N, N-dimethyl-1-[(1S, 2R)-2-octylcyclopropyl] heptadecanedioate (L530).

Some embodiments of the present disclosure provide vaccines comprising at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide, wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%) of the uracils in the open reading frame have chemical modifications, optionally wherein the vaccine is formulated in a lipid nanoparticle (e.g., the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid).

In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, 100% of the uracils in the open reading frame have an N1-methylpseudouridine at the 5-position of the uracil.

In some embodiments, the open reading frame of the RNA (e.g., mRNA) polynucleotide encodes at least one E. coli polypeptide. In a preferred embodiment, the E. coli polypeptide is a pilus antigen. In a preferred embodiment, the E. coli pilus antigen is FimH. In some embodiments, the open reading frame encodes at least two, at least five, or at least ten E. coli polypeptides. In some embodiments, the open reading frame encodes at least 100 E. coli polypeptides. In some embodiments, the open reading frame encodes 1-100 E. coli polypeptides.

In some embodiments, the vaccine comprises at least two RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide. In some embodiments, the vaccine comprises at least five or at least ten RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide or an immunogenic fragment thereof. In some embodiments, the vaccine comprises at least 100 RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide. In some embodiments, the vaccine comprises 2-100 RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide.

In other aspects, the present invention provides such a multivalent vaccine, wherein the multivalent vaccine comprises at least two RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide. In some embodiments, the multivalent vaccine comprises at least five or at least ten RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide or an immunogenic fragment thereof. In some embodiments, the multivalent vaccine comprises at least 100 RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide. In some embodiments, the multivalent vaccine comprises 2-100 RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one E. coli FimH polypeptide. In other embodiments, the multivalent vaccine comprises RNA (e.g., mRNA) polynucleotides, each polynucleotide having an open reading frame encoding at least one additional polypeptide, including but not limited to E. coli FmlH, E. coli PapG, K. pneu. MrkA, E. faecalis EbpA or an immunogenic fragment thereof.

Also provided herein is an E. coli RNA (eg, mRNA) vaccine formulated in nanoparticles (eg, lipid nanoparticles) as described in any of the preceding paragraphs.

In some embodiments, the nanoparticles have an average diameter of 50-200nm. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids. In some embodiments, the lipid nanoparticles comprise a molar ratio of about 20-60% cationic lipids, 0.5-15% PEG-modified lipids, 25-55% sterols, and 25% non-cationic lipids. In some embodiments, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol.

In some embodiments, the nanoparticles have a polydispersity value less than 0.4 (eg, less than 0.3, 0.2, or 0.1).

In some embodiments, the nanoparticles have a net neutral charge at neutral pH.

Some embodiments of the present disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject any RNA (e.g., mRNA) vaccine provided herein that effectively produces an amount of an antigen-specific immune response. In some embodiments, the RNA (e.g., mRNA) vaccine is an Escherichia coli vaccine. In some embodiments, the RNA (e.g., mRNA) vaccine is a combined vaccine comprising an Escherichia coli vaccine combination (broad-spectrum Escherichia coli vaccine).

In some embodiments, the antigen-specific immune response comprises a T cell response or a B cell response.

In some embodiments, a method of generating an antigen-specific immune response comprises administering to a subject a single dose (without booster doses) of an E. coli RNA (eg, mRNA) vaccine of the present disclosure.

In some embodiments, the method further comprises administering a second dose (boosting dose) of the E. coli RNA (eg, mRNA) vaccine to the subject. Additional doses (boosting doses) of the E. coli RNA (eg, mRNA) vaccine may be administered.

In some embodiments, the subject exhibits a seroconversion rate of at least 80% (e.g., at least 85%, at least 90%, or at least 95%) after receiving the first or second (boost) dose of the vaccine. Seroconversion is the period of time during which specific antibodies are produced and become detectable in the blood. After seroconversion has occurred, the antigen can be detected in a blood test for antibodies. During infection or immunization, the antigen enters the blood and the immune system begins to produce antibodies in response. Prior to seroconversion, the antigen itself may or may not be detected, but the antibody is considered absent. During seroconversion, the antibody is present but not yet detected. Antibodies can be detected in the blood at any time after seroconversion, indicating a previous or current infection.

In some embodiments, the E. coli RNA (e.g., mRNA) vaccine is administered to the subject by intradermal injection, intramuscular injection, or intranasal administration. In some embodiments, the E. coli RNA (e.g., mRNA) vaccine is administered to the subject by intramuscular injection.

Some embodiments of the present disclosure provide a method for inducing an antigen-specific immune response in a subject, comprising administering an effective amount of an E. coli RNA (e.g., mRNA) vaccine to a subject to produce an antigen-specific immune response in the subject. In some embodiments, the antigen-specific immune response in the subject can be determined by measuring the antibody titer (the titer of antibodies bound to the E. coli FimH polypeptide) after administering any E. coli RNA (e.g., mRNA) vaccine of the present disclosure to the subject. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases by at least 1 log relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases by 1-3 log relative to the control.

In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases at least 2 times relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases at least 5 times relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases at least 10 times relative to the control. In some embodiments, the anti-antigen polypeptide antibody titer produced in the subject increases 2-10 times relative to the control.

In some embodiments, the control is the anti-antigen polypeptide antibody titer produced in a subject to which the RNA (e.g., mRNA) vaccine of the present disclosure has not been administered. In some embodiments, the control is the anti-antigen polypeptide antibody titer produced in a subject to which an E. coli FimH polypeptide or fragment thereof has been administered, or wherein the control is the anti-antigen polypeptide antibody titer produced in a subject to which a recombinant or purified E. coli FimH vaccine has been administered. In some embodiments, the control is the anti-antigen polypeptide antibody titer produced in a subject to which a recombinant or purified E. coli FimH vaccine has not been administered.

The RNA (e.g., mRNA) vaccines disclosed herein are administered to a subject in an effective amount (an amount that effectively induces an immune response). In some embodiments, the effective amount is a dose that is equivalent to a standard care dose of a recombinant E. coli vaccine reduced by at least 2 times, at least 4 times, at least 10 times, at least 100 times, at least 1000 times, wherein the anti-antigen polypeptide antibody titer produced in the subject is equivalent to the anti-antigen polypeptide antibody titer produced in the control subject of the standard care dose of the recombinant E. coli protein vaccine. In some embodiments, the effective amount is a dose that is equivalent to a standard care dose of a recombinant E. coli protein vaccine reduced by 2 to 1000 times, wherein the anti-antigen polypeptide antibody titer produced in the subject is equivalent to the anti-antigen polypeptide antibody titer produced in the control subject of the standard care dose of the recombinant E. coli protein vaccine.

In some embodiments, RNA (e.g., mRNA) vaccines are formulated in an effective amount to generate an antigen-specific immune response in a subject.

In some embodiments, the effective amount is a total dose of ≤25 μg. In some embodiments, the effective amount is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg administered to the subject, twice or more in total. In some embodiments, the effective amount is a dose of 100 μg administered to the subject, twice or more in total. In some embodiments, the effective amount is a dose of 400 μg administered to the subject, twice or more in total. In some embodiments, the effective amount is a dose of 500 μg administered to the subject, twice or more in total.

In some embodiments, the efficacy (or effectiveness) of the RNA (eg, mRNA) vaccine is greater than 60%. In some embodiments, the RNA (eg, mRNA) polynucleotide of the vaccine encodes at least one E. coli FimH polypeptide.

Vaccine efficacy can be assessed using standard analyses. For example, vaccine efficacy can be measured in double-blind, randomized, controlled clinical trials. Vaccine efficacy can be expressed as a proportional reduction in the disease attack rate (AR) between unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease in the vaccinated group using the following formula:

Efficacy = (ARU - ARV) / ARU x 100; and Efficacy = (1 - RR) x 100.

Similarly, standard analyses can be used to assess vaccine effectiveness. Vaccine effectiveness is an assessment of how well a vaccine (which may have been shown to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, rather than just the vaccine itself, under natural field conditions rather than in controlled clinical trials. Vaccine effectiveness is proportional to vaccine potency (efficacy), but is also influenced by the extent of immunity in the target group in the population, as well as other non-vaccine-related factors that affect the "real-world" outcomes of hospitalizations, outpatient visits, or costs. For example, a retrospective case-control analysis can be used, in which vaccination rates in a group of infected cases and appropriate controls are compared. Vaccine effectiveness can be expressed as a rate difference using the odds ratio (OR) of still developing an infection after vaccination:

Effectiveness = (1-OR)×100.

In some embodiments, the efficacy (or effectiveness) of the RNA (e.g., mRNA) vaccine is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

In some embodiments, the vaccine can immunize a subject against E. coli for up to 2 years. In some embodiments, the vaccine immunizes a subject against E. coli for more than 2 years, more than 3 years, more than 4 years, or 5-10 years.

In some embodiments, the subject is about 5 years old or younger. For example, the age of the subject can be between about 1 year old and about 5 years old (e.g., about 1, 2, 3, 4, or 5 years old), or between about 6 months and about 1 year old (e.g., about 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, the subject is about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months, or 1 month). In some embodiments, the subject is about 6 months or younger.

In some embodiments, the subject is born at full term (e.g., about 37-42 weeks). In some embodiments, the subject is premature, for example, about 36 weeks of pregnancy or earlier (e.g., about 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 or 25 weeks). For example, the subject may be born at about 32 weeks of pregnancy or earlier. In some embodiments, the subject is premature between about 32 weeks of pregnancy to about 36 weeks. In such subjects, RNA (e.g., mRNA) vaccines can be administered at a later age, for example, at about 6 months to about 5 years old or older.

In some embodiments, the subject is an adolescent between the ages of about 11-19 years old (e.g., about 11, 12, 13, 14, 15, 16, 17, 18, or 19 years old).

In some embodiments, the subject is an adult between the ages of about 20 and about 59 years old (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 59 years old).

In some embodiments, the subject is an elderly subject, about 60 years old, about 70 years old, or older (eg, about 60, 65, 70, 75, 80, 85, or 90 years old).

In some embodiments, the subject has been exposed to E. coli; the subject is infected with E. coli; or the subject is at risk of being infected with E. coli.

In some embodiments, the subject is immunocompromised (has a compromised immune system, eg, suffers from an immune disorder or an autoimmune disorder).

In some embodiments, the nucleic acid vaccines described herein are chemically modified. In other embodiments, the nucleic acid vaccines are unmodified.

Still other aspects provide compositions and methods for vaccinating a subject, the methods comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, wherein the RNA polynucleotides do not include a stabilizing component, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

In other aspects, the present invention is a composition or method for vaccinating a subject, the method comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the nucleic acid vaccine is administered to the subject at a dose of 10 μg/kg to 400 μg/kg. In some embodiments, the dose of the RNA polynucleotide is ≤1 μg, 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, 100 In some embodiments, the nucleic acid vaccine is administered to a subject by intradermal injection or intramuscular injection.

In some embodiments, the nucleic acid vaccine administered to the subject includes a 25 microgram dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject includes a 100 microgram dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject includes a 50 microgram dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject includes a 75 microgram dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject includes a 150 microgram dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject includes a 400 microgram dose of RNA polynucleotide. In some embodiments, the nucleic acid vaccine administered to the subject includes a 200 microgram dose of RNA polynucleotide. In some embodiments, the accumulation level of RNA polynucleotides in local lymph nodes is 100 times higher than the accumulation level in distal lymph nodes. In other embodiments, the nucleic acid vaccine is chemically modified, and in other embodiments, the nucleic acid vaccine is not chemically modified.

Aspects of the present invention provide such nucleic acid vaccines, which comprise one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotide does not include a stabilizing element, and a pharmaceutically acceptable carrier or excipient, wherein the vaccine does not include an adjuvant. In some embodiments, the stabilizing element is a histone stem loop. In some embodiments, the stabilizing element is a nucleic acid sequence with an increased GC content relative to the wild-type sequence.

Aspects of the present invention provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotide is present in a preparation for in vivo administration to a host, which confers an antibody titer that is superior to the standard for serum protection of the first antigen of an acceptable percentage of human subjects. In some embodiments, the antibody titer produced by the mRNA vaccine of the present invention is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is higher than that of a protein vaccine. In other embodiments, the neutralizing antibody titer produced by the mRNA vaccine of the present invention is higher than that of an adjuvanted protein vaccine. In still other embodiments, the neutralizing antibody titer produced by the mRNA vaccine of the present invention is 1,000-10,000, 1,200-10,000, 1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000, 2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500. Neutralization titers are typically expressed as the highest serum dilution required to achieve a 50% reduction in the number of bacteria bound to the plate.

Also provided is a nucleic acid vaccine, which comprises one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotide is present in a formulation for in vivo administration to a host, to elicit a more durable high antibody titer than the antibody titer elicited by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigen polypeptide. In some embodiments, the RNA polynucleotide is formulated to produce neutralizing antibodies within one week after a single administration. In some embodiments, the adjuvant is selected from a cationic peptide and an immunostimulatory nucleic acid. In some embodiments, the cationic peptide is protamine.

A nucleic acid vaccine is provided, comprising one or more RNA polynucleotides having an open reading frame, wherein the open reading frame comprises at least one chemically modified or optionally unmodified nucleotide, and the open reading frame encodes a first antigen polypeptide, wherein the RNA polynucleotide is present in a preparation for in vivo administration to a host, so that the antigen expression level in the host significantly exceeds the antigen expression level produced by an mRNA vaccine having a stabilizing element or formulated with an adjuvant and encoding the first antigen polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemically modified or optionally unmodified nucleotide, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10 times less RNA polynucleotides than the RNA polynucleotides required for the unmodified mRNA vaccine to produce equivalent antibody titers. In some embodiments, the RNA polynucleotides are present in a dose of 25 to 100 micrograms.

Aspects of the present invention also provide for the use of a unit vaccine comprising 10 ug to 400 ug of one or more RNA polynucleotides having an open reading frame comprising at least one chemically modified or optionally unmodified nucleotide, the open reading frame encoding a first antigenic polypeptide, and a pharmaceutically acceptable carrier or excipient, formulated for delivery to a human subject. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

Aspects of the present invention provide methods for producing, maintaining or restoring antigen memory to bacteria or viruses in an individual or a population of individuals, comprising administering an antigen memory-boosting nucleic acid vaccine to the individual or population, the vaccine comprising (a) at least one RNA polynucleotide, the polynucleotide comprising at least one chemically modified or optionally unmodified nucleotide and two or more codon-optimized open reading frames, the open reading frames encoding a set of reference antigen polypeptides, and (b) optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the vaccine is administered to an individual by a route selected from the group consisting of intramuscular administration, intradermal administration, and subcutaneous administration. In some embodiments, the administering step comprises contacting the muscle tissue of the subject with a device suitable for injecting the composition. In some embodiments, the administering step comprises contacting the muscle tissue of the subject with a device suitable for injecting the composition in combination with electroporation.

Aspects of the invention provide a method for vaccinating a subject, comprising administering to the subject a single dose of 25 ug/kg to 400 ug/kg of a nucleic acid vaccine in an effective amount to vaccinate the subject, wherein the nucleic acid vaccine comprises one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification, the open reading frame encoding a first antigenic polypeptide, wherein the vaccine has at least 10 times less RNA polynucleotides than the RNA polynucleotides required for the unmodified mRNA vaccine to produce equivalent antibody titers. In some embodiments, the RNA polynucleotides are present in a dose of 25 to 100 micrograms.

Other aspects provide a nucleic acid vaccine comprising an RNA polynucleotide formulated with LNP, the RNA polynucleotide having an open reading frame without nucleotide modification (unmodified), the open reading frame encoding a first antigen polypeptide, wherein the vaccine has at least 10 times less RNA polynucleotide than the RNA polynucleotide required for producing an equivalent antibody titer of an unmodified mRNA vaccine not formulated in LNP. In some embodiments, the RNA polynucleotide is present in a dose of 25 to 100 micrograms.

According to the present invention, both chemically modified and unmodified RNA vaccines are useful. Prior art reports indicate that it is preferred to use unmodified mRNA formulated in a carrier to produce vaccines. Both chemically modified and unmodified RNA vaccines of the present invention produce better immune responses than mRNA vaccines formulated in different lipid carriers.

In other aspects, the present invention includes a method for treating an elderly subject aged 60 years or older, comprising administering to the subject a nucleic acid vaccine in an effective amount to vaccinate the subject, wherein the nucleic acid vaccine comprises one or more RNA polynucleotides having an open reading frame encoding an Escherichia coli antigen polypeptide.

In other aspects, the invention includes a method of treating a young subject 17 years of age or younger, comprising administering to the subject a nucleic acid vaccine in an effective amount to vaccinate the subject, wherein the nucleic acid vaccine comprises one or more RNA polynucleotides having an open reading frame encoding an E. coli antigen polypeptide.

In other aspects, the present invention includes a method of treating an adult subject between the ages of about 20 and about 50 years old, comprising administering to the subject a nucleic acid vaccine in an effective amount to vaccinate the subject, wherein the nucleic acid vaccine comprises one or more RNA polynucleotides having an open reading frame encoding an E. coli antigen polypeptide.

In some aspects, the present invention is a method for vaccinating a subject with a combined vaccine, the combined vaccine comprising at least two nucleic acid sequences encoding an antigen, wherein the dose of the vaccine is a combined therapeutic dose, wherein the dose of each individual nucleic acid encoding the antigen is a subtherapeutic dose. In some embodiments, the combined dose in the nucleic acid vaccine administered to the subject is 25 micrograms of RNA polynucleotides. In some embodiments, the combined dose in the nucleic acid vaccine administered to the subject is 100 micrograms of RNA polynucleotides. In some embodiments, the combined dose in the nucleic acid vaccine administered to the subject is 50 micrograms of RNA polynucleotides. In some embodiments, the combined dose in the nucleic acid vaccine administered to the subject is 75 micrograms of RNA polynucleotides. In some embodiments, the combined dose in the nucleic acid vaccine administered to the subject is 150 micrograms of RNA polynucleotides. In some embodiments, the combined dose in the nucleic acid vaccine administered to the subject is 400 micrograms of RNA polynucleotides.

In preferred aspects, the vaccine of the present invention (e.g., LNP-encapsulated mRNA vaccine) produces antigen-specific antibodies of effective levels, concentrations and/or titers for prevention and/or treatment in the blood or serum of vaccinated subjects. As defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in a subject (e.g., a human subject). In an exemplary embodiment, the antibody titer is expressed as the reciprocal of the maximum dilution (in a serial dilution) that still produces a positive result. In an exemplary embodiment, the antibody titer is measured or measured by enzyme-linked immunosorbent assay (ELISA) or Luminex. In an exemplary embodiment, the antibody titer is measured or measured by neutralization assay (e.g., E. coli binding inhibition assay). In some aspects, the antibody titer measurement is expressed as a ratio, such as 1:40, 1:100, etc.

In an exemplary embodiment of the invention, an effective vaccine produces an antibody titer greater than 1:40, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, greater than 1:10000. In an exemplary embodiment, the antibody titer is produced or reached within 10 days after vaccination, 20 days after vaccination, 30 days after vaccination, 40 days after vaccination, or 50 days after vaccination or more. In an exemplary embodiment, the titer is produced or reached after a single dose of vaccine is administered to the subject. In other embodiments, the titer is produced or reached after multiple doses, such as after the first and second doses (such as booster doses). In an exemplary aspect of the invention, antigen-specific antibodies are measured in μg/ml, or measured in IU/L (international units per liter) or mIU/ml (milli-international units per milliliter). In an exemplary embodiment of the invention, an effective vaccine produces> 0.5 μg/ml,> 0.1 μg/ml,> 0.2 μg/ml,> 0.35 μg/ml,> 0.5 μg/ml,> 1 μg/ml,> 2 μg/ml,> 5 μg/ml or> 10 μg/ml. In an exemplary embodiment of the invention, an effective vaccine produces> 10mIU/ml,> 20mIU/ml,> 50mIU/ml,> 100mIU/ml,> 200mIU/ml,> 500mIU/ml or> 1000mIU/ml antigen-specific antibodies. In an exemplary embodiment, the antibody level or concentration is produced or reached within 10 days after vaccination, 20 days after vaccination, 30 days after vaccination, 40 days after vaccination or 50 days after vaccination or more. In an exemplary embodiment, the level or concentration is produced or reached after a single dose of vaccine is administered to a subject. In other embodiments, the level or concentration is produced or achieved after multiple doses, such as after a first and second dose (e.g., a booster dose). In an exemplary embodiment, the antibody level or concentration is determined or measured by an enzyme-linked immunosorbent assay (ELISA) or Luminex. In an exemplary embodiment, the antibody level or concentration is determined or measured by a neutralization assay (e.g., an E. coli binding inhibition assay).

Example

Below are examples of specific aspects of the present disclosure. The following examples are included to illustrate aspects of the present disclosure. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the implementation of the present disclosure. However, it will be appreciated by those skilled in the art that many changes can be made to the disclosed specific aspects and still obtain the same or similar results without departing from the spirit and scope of the present disclosure, based on the present disclosure. We have tried our best to ensure the accuracy of the numbers used (e.g., quantity, temperature, etc.), but of course some experimental errors and deviations should be allowed. The following examples illustrate some embodiments of the present invention.

Example 1

RNA-based expression of Escherichia coli FimH antigen in mammalian cells

The expression of FimH in mammalian cells has been described in International Patent Publication No. WO2021084429A1, which is incorporated herein by reference in its entirety. FimH mutations that stabilize protein conformation and improve bioprocessing properties and functional immunogenicity associated with expression and purification have been described in International Patent Publication No. WO2022090893, which is incorporated herein by reference in its entirety. Preclinical efficacy data from a cynomolgus monkey cystitis challenge model showed that recombinant full-length FimH-DSG triple mutant (G15A G16AV27A) protein had a protective effect when administered with liposome MPLA/QS21 adjuvants, and were described in International Patent Publication No. WO2022137078, which is incorporated herein by reference in its entirety. The expression of FimH from mRNA vaccines can provide the benefits of enhanced cellular immunity, as well as a lower commodity cost relative to protein antigens. It is not clear whether cell-associated constructs expressed on the cell surface or secreted FimH are more immunogenic in vivo. Therefore, different constructs with these properties were generated and the results are shown below.

In the following example, we evaluated the expression of chimeric FimH antigens designed to localize to the cell surface by anchoring to the plasma membrane. To this end, we fused the FimH lectin domain containing the stabilizing triple mutation to the C-terminal transmembrane domain of herpes simplex virus gD (HSVgD), SARS-COV2 or the C-terminal GPI anchor signal (DAFgpi) of the human decay accelerating factor (DAF) protein (also known as CD55). We also tested whether a secretory construct could be generated by modifying the construct for extracellular expression of our benchmark FimH-DSG triple mutant (G15AG16AV27A) (N-deglycosylated), which had previously been shown to be efficiently secreted from mammalian cells after transfection with a DNA expression plasmid.

Materials and methods

1. Plasmid Construction, Cloning, and In Vitro Transcription

A DNA sequence encoding the E. coli FimH protein was prepared and used in an in vitro transcription reaction to generate RNA. In vitro transcription of RNA is known in the art and described herein. The DNA template was cloned into a modRNA clone entry vector with backbone sequence elements (T7 promoter, 5' and 3' UTR, 3' 80nt poly A tail) to improve RNA stability and translation efficiency. The DNA was purified, quantified spectrophotometrically and transcribed in vitro. Using AG kit (TriLink), which simultaneously caps newly transcribed mRNA molecules with m7G(5')ppp(5')(2'OMeA)pG.

FimH RNA is produced from codon-optimized (CO) DNA to achieve stability and excellent protein expression. Table 4 shows the RNA constructs of the present disclosure and their corresponding sequences, which include a 5'UTR, an open reading frame encoding a FimH polypeptide, a 3'UTR and a poly A tail.

2. Immunofluorescence Analysis of Transfected HeLa Cells

HeLa cells (ATCC#CCL-2) were seeded in polylysine-coated 96-well plates at 1x10 ⁴ cells/well. RNA was diluted to 100 ng/well and 50 ng/well in culture medium, combined with diluted lipofectamine (MessengerMax, Invitrogen), and kept at room temperature (RT) for 5 minutes. Next, mRNA plus lipofectamine reagent mixture was added to each well in triplicate. The microplate was centrifuged at 500 g for 5 minutes at RT and incubated overnight at 37 ° C and 5% CO _2. The next day, the monolayer cells were rinsed in PBS containing Ca ^2+/ Mg ²⁺ , and 1 μg/mL anti-FimH mAb 926 (reconstructed according to International Publication No. WO2016183501, which is incorporated by reference in its entirety) in PBS containing Ca ^2+/ Mg ²⁺ was used to stain the cell surface at 4 ° C for 1 hour. For whole-cell staining, cells were fixed with 4% PFA for 20 min at RT and then permeabilized with 0.1% saponin in 0.1% goat serum. Binding of the FimH antibody was detected using a secondary goat anti-human IgG conjugated to AlexaFluor488 (Invitrogen) at a final dilution of 1:500. Cell nuclei were stained using 100 ng/mL of 4'6-diamidino-2-phenylindole dilactate (DAPI). CellMaskOrange Stain (ThermoFisher) (1:140,000 final dilution) was used as a cell delineation tool for automated high-content analysis by marking the entire cell. Confocal imaging of the stained cells was performed using the Opera Phenix Plus high-content screening platform (PerkinElmer). Imaging software automatically quantifies cell fluorescence. Complete cells in each captured image are identified by a combination of nuclear staining (DAPI) and cytoplasmic staining (CellMask). Images of 250 fields of view in each well were captured and analyzed at 40x magnification. Mock transfected cells depicted in this way are used to define baseline thresholds for quantifying protein antigen fluorescence intensity (MFI) in fully stained cells and surface stained cells.

3. Flow Cytometry and Octet Analysis of Transfected Expi293 Cells

A 25 μL volume of 5-fold serial dilutions of RNA (starting from 500 ng/well) was combined with lipofectamine reagent (MessengerMax, Invitrogen) in a 96-well deep well (2.2 mL) plate at RT for 5 minutes. Expi293 suspension cells (ThermoFisher) were diluted in 0.45 mL Opti-MEM growth medium to a final concentration of 1×10 ⁶ /well and cultured overnight with shaking at 37°C, 8% CO ₂ and 80% humidity for 24 hours. The next day, cells were equally divided into different 96-well plates for surface staining and full staining as described for HeLa cells, except that FimH mAb was used at a concentration of 5.0 μg/ml. Fixable dyes were also used. Cells were stained at 780°C to assess cell viability. Cell plates were read on an LSRII flow cytometer instrument (BD Biosciences).

The FimH secreted into the culture medium supernatant 24 hours after the Expi293 cells were transfected with FimH-DSG-TM mRNA was quantified by Octet biolayer interferometry. The transfection was evaluated using 5-fold dilutions of RNA starting from 500ng/well. The anti-human Fc biosensor was first used to bind FimH specific mAb, and then the binding reaction was carried out with the clarified FimH transfection supernatant. The biosensor was first hydrated for 10 minutes at RT with Expi293 conditioned medium (200 μL/well), and then captured for 10 minutes on an orbital oscillator at room temperature with a saturated concentration of mAb (final concentration of 10 μg/mL, 200 μL/well). Using linear regression analysis, the FimH concentration was determined by interpolating the values of the parallel titrations from the purified recombinant FimH-DSG standard.

Results and Discussion

Bacterial FimH is expressed in mammalian cells to expose the ligand binding site of its native conformation, and needs to be processed by mammalian signal peptidase in such a way that the method accurately reproduces the treatment performed by similar bacterial signal peptidase before secretion into the periplasmic space of Escherichia coli. The signal peptide for mammalian expression is the same mouse IgGκ sequence (see International Publication No. WO2022/090893) (SEQ ID NO:65) that has previously been shown to confer effective secretion of biologically active Escherichia coli FimH in mammalian cells. The gene chimera of the FimH lectin domain comprising the G15AG16AV27A triple mutation (TM) is fused to the C-terminal membrane targeting domain of three different viral glycoproteins, separated by a 7-amino acid linker sequence "GGSSGGG" (SEQ ID NO:74). The gene of secretory FimH-DSG TM is also cloned into an mRNA vector and evaluated. The structural features of these FimH proteins are highlighted in Figure 1. Gene and protein sequences are listed in Figure 2, and in Tables 1-3.

The four constructs and their key features are summarized below:

1)FimHLDTMCt60HSVgD:

FimH _LD TM is fused to a trimer C-terminal domain comprising Ct-60 amino acid residues of HSV gD, including a hydrophobic transmembrane domain (TMD) (Cocchi F, et al. Proceedings of the National Academy of Sciences of the United States of America 2004; 101: 7445);

2) FimH _LD TMCtΔ5Spike (also referred to as “FimH _LD TMCtΔ5COVID19Spike”):

FimH _LD TM is fused to the trimeric transmembrane membrane domain of the SARS-CoV2 spike protein, which spans 68 amino acid residues at the C-terminus. Five amino acids at the C-terminus are deleted because they constitute the endoplasmic reticulum (ER) retention motif (KLHYT) (SEQ ID NO: 80), which reduces intracellular transport efficiency (Xia X. Viruses 2021; 13: 109). The C-terminal domain includes eight TM proximal amino acids, which include near-membrane aromatic residues. The distal end of the hydrophobic transmembrane domain (TMD) is adjacent to a conserved cysteine-rich region (SEQ ID NO: 79 residues 1234-1254), which contains multiple palmitoylation sites. In SARS-CoV-1, palmitoylation of these sites promotes membrane distribution and cell fusion (McBride CE, Machamer CE. Virology 2010; 405: 139-48). The C-terminal cytoplasmic tail also contains a conserved charged region (1255-KFDEDDSE (SEQID NO: 81) (Xia X. Viruses 2021; 13: 109);

3)FimHLDTMCtDAFgpi:

FimH _LD TM is fused to the membrane targeting domain of the monomeric human DAF protein GPI anchor signal. It includes the C-terminal 37 residues, which are sufficient to confer heterologous membrane binding on the extracellular domain of heterologous viral glycoproteins (Lisanti MP, et al. Journal of Cell Biology 1989; 109: 2145-56). Most of the C-terminal GPI attachment signal is cleaved off in the endoplasmic reticulum, and the glycosylphosphatidylinositol (GPI, also known as "gpi" or "gpI") lipid part is added (Galian C, et al. J Biol Chem 2012; 287: 16399-409); and

4) Secretory FimH-DSG-TM (N-deglycosylated):

A secreted fully N-deglycosylated (no glycosyl) version of FimH-DSG TM with the asparagine of the pilin domain, which is usually replaced by serine N-glycosylation (N228S N235S) to prevent glycosylation. The added C-terminal donor chain G-peptide is appended to the C-terminus of the FimH protein, separated by a seven amino acid linker sequence "GGSSGGG" (SEQ ID NO: 74).

Tables 1-3 provide the sequences of the above-mentioned FimH constructs, respectively, which include the antigen/polypeptide, DNA and RNA of the present invention. The sequences may contain any stop codon.

Table 1. FimH construct polypeptides

Table 2. FimH construct DNA

Table 3. FimH construct RNA

Table 4. FimH modRNA constructs

The expression of FimH from mRNA transfected into adherent HeLa cells was evaluated by confocal fluorescence microscopy, and the results are shown in Figures 3, 4A and 4B. Microscope images of cells fixed and stained with FimH antibodies (Figure 3) show that the C-terminal GPI anchor domain of human DAF protein mediates antigen effective targeting to the outer surface of HeLa cell membrane. These 37 amino acids contain a C-terminal hydrophobic domain and a cleavage/attachment site, which directs proteolytic removal of the C-terminal 28 residues and attaches the lipidated GPI anchor to an acceptor serine, which is located at a position only 8 residues from the border, where "GGSGGGG" (SEQ ID NO: 74) joints separate this domain from FimH _LD (Moran P, Caras IW. J Cell Biol 1991; 115: 329-36). Relatively low levels of FimH were observed with SARS-COV2 C-terminal spike protein chimeras, while moderate levels were detected with C-terminal HSVgD chimeras. No background level of expression was seen in mock transfected cells. Negligible surface expression levels were observed using mRNA expressing secreted FimH-DSG antigen, which was expected based on the efficient secretion observed using plasmid DNA expression vectors (see International Publication No. WO2022/090893). Similar expression patterns of all mRNA constructs were observed in permeabilized HeLa cells compared to non-permeabilized fixed cells (Figures 4A and 4B), suggesting that most FimH was exposed on the surface of HeLa cells.

The same mRNA is transfected into Expi293 suspension cells for parallel evaluation of the targeting of FimH antigen in different mammalian cell types. The results shown in Figure 5A and Figure 5B are consistent with the results observed with transfected adherent HeLa cells. The chimeric FimH with C-terminal GPI anchor produces the highest level of expression, which can be titrated to 20ng transfected mRNA. The FimH chimera with C-terminal HSVgD transmembrane domain is only well expressed at 500ng level, while the C-terminal spike protein chimera is relatively poorly expressed. First, the secretory protein level in the culture supernatant from transfection is analyzed by Western blotting, wherein only high levels of antigen (data not shown) are detected using FimH-DSG antigen transfection. In this case, the biolayer interferometry method confirms the presence of FimH antigen proportional to the RNA concentration transfected with 20ng, 100ng and 500ng levels (Figure 6).

The results show that the FimH _LD antigen can be expressed on the surface of mammalian cells by fusing a heterologous membrane targeting signal to the C-terminus. These chimeric fusion proteins are expressed from transfected mRNA in HeLa and Epi293 cells. The membrane targeting signal can come from a viral glycoprotein or a GPI anchor signal. The membrane-targeted expression of these prototype FimH-viral glycoprotein chimeras can be further optimized, such as adding a fibritin trimerization (Foldon) motif, which is used to promote the trimerization of recombinant viral glycoprotein antigens (Vogel AB, Kanevsky I, Che Y, et al. Nature 2021; 592: 283-9; Meier S, Güthe S, Kiefhaber T, Grzesiek SJ Mol Biol 2004; 344: 1051-69). In these experiments, we also confirmed that, as previously observed using a transfected plasmid DNA expression vector, the full-length FimH-DSG protein can be effectively secreted into the cell culture medium when expressed from transfected mRNA.

Embodiment 2:

Immunogenicity of modRNA LNPs expressing Escherichia coli FimH antigen in mice

In Example 1, it was shown that the secreted form and membrane-targeted form of the Escherichia coli fimbriae protein antigen FimH can be expressed from transfected mRNA in mammalian cells in vitro. In this Example 2, we evaluated the ability of modRNA lipid nanoparticles (LNPs) to elicit functional neutralizing antibodies in mice. LNP produced FimH IgG and neutralizing antibody titers that were significantly stronger than recombinant FimH proteins formulated with LiNA-2 adjuvant. As used herein, "LiNA-2" refers to a liposomal adjuvant comprising MPLA and QS21. FimH-specific Th1 and Th2 responses were determined in the spleen cells of vaccinated animals. Intracellular cytokine staining (ICS) assays based on flow cytometry were used to measure the production and accumulation of cytokines and the surface expression of activation-induced markers (AIM) after stimulating T- cells with a FimH peptide library. Compared with protein/LiNA2 antigens, modRNA triggered a stronger CD8+ T cell response. Vaccination with membrane-targeted FimH modRNA resulted in enhanced FimH-specific Th1-biased CD4+ T cell responses compared to protein/LiNA2 antigen or modRNA expressing secreted FimH _DSG TM. These results support the use of the described FimH modRNA as a vaccine to treat UTIs.

Materials and methods

1. Animal study details

CD-1 female mice (6-8 weeks old upon arrival) from Charles River Laboratories were immunized with protein or LNP antigen at 7-9 weeks of age. The study plan was as follows:

(i) Vaccinations were administered at weeks 0, 4, and 8;

(ii) bleeding the animals at weeks 0, 3, and 6 and at week 10; and

(iii) Spleens were obtained from 5 mice in each group at week 10 (end point).

Doses and vaccine components are summarized in Tables 5 and 6.

Table 5. Mouse Study EC-088

Table 6. Vaccine components

2. ModRNA LNP Production

The construction of plasmid mRNA vectors with four FimH gene variants containing conformationally stable triple mutations (G15AG16AV27A) has been described previously in Example 1 above. The FimH gene encoding the full-length secretory FimH _DSG protein or the membrane-targeted FimH _LD chimera is cloned between 5' and 3' UTR. Due to the low expression level of FimH antigen observed in the in vitro expression (IVE) experiment of HeLa or Expi293 mammalian cells transfected with FimH mRNA, two of these surface membrane-targeted constructs did not progress to immunogenicity studies. The two best performing constructs that are preferentially used for LNP preparation are secretory full-length FimH _DSG TM (N-deglycosylation) (Fig. 1), and the membrane-associated FimH _LD gpi anchor chimera (FimH _LD -TMCtDAFgpi) (Fig. 1). The properties of the two modRNA plasmids used to produce in vitro RNA transcripts and LNPs are summarized in Table 7.

Table 7. Plasmid construct details

Using T7-polymerase and In vitro transcription of plasmid DNA templates linearized with restriction enzyme BspQ I was performed using the PCR amplification reagent (TriLink Biotechnologies). The resulting RNA contained a 5' cap structure in which pseudouridine was incorporated instead of uridine. The transcripts were capped and analyzed by a fragment analyzer to find that the purity of the three mRNAs was >92%, and the capping efficiency was determined to be >97%.

RNA was formulated into an LNP formulation containing two functional lipids, ALC-0315 and ALC-0159, and two structural lipids, DSPC (1,2 distearoyl-sn-glycero-3-phosphocholine) and cholesterol. The physicochemical properties and structures of the four lipids are shown in Table 8 below.

Lipid nanoparticles were prepared and tested according to the general procedures described in U.S. Patent 9737619 (PCT Pub. No. WO2015/199952) and U.S. Patent 10166298 (WO2017/075531) and WO2020/146805, each of which is incorporated herein by reference in its entirety. Briefly, cationic lipid (ALC-0315), cholesterol, DSPC and PEG-lipid (ALC-0159) were dissolved in ethanol at a molar ratio of about 46.3:42.7:9.4:1.6.

Table 8. Lipids in LNP Formulations

CAS = Chemical Abstracts Service; DSPC = 1,2-distearoyl-sn-glycero-3-phosphocholine

RNA integrity was assessed by fragment analyzer capillary gel electrophoresis, encapsulation efficiency was assessed by RiboGreen analysis, LNP size and polydispersity index (PDI) were assessed by dynamic light scattering (DLS) (Malvern), and endotoxin was assessed by the LAL test kit system (Endosafe). The properties of the antigens are summarized in Table 9.

Table 9. LNP quality attributes

3. Immunogenicity Assay

FimH direct Luminex immunoassay (dLIA) IgG assay can measure the binding of mouse serum antibodies to FimH _DSG antigen, which is immobilized on Luminex beads by EDC/NHS. The beads are shaken and incubated for 18 hours at 4°C with serially diluted single mouse serum or FimH control mAb. After washing, the bound FimH specific IgG is detected with PE-conjugated goat anti-mouse IgG mouse secondary antibody (90 minutes RT incubation). The microplate is read on a FlexMap 3D instrument (Biorad). Anti-FimH IgG levels are quantified using FimH specific mouse IgG mAb as an internal standard. The mAb standard curve produces a linear slope curve in 10 ³ serum dilutions (log luminescence vs log serum dilution).

4. Live Bacterial FimH Specific Neutralization Assay

For the yeast mannan assay, black microtiter 96-well plates (Maxisorb, Nunc) were coated with 20 μg/ml yeast mannan (Sigma-Aldrich) dissolved in PBS buffer. The wells were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 20 minutes. The human bladder epithelial cell line 5637 was obtained from ATCC (ATCC HTB-9). Cells were grown in RPMI 1640 (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (FBS; Sigma), 2.0 g/l sodium bicarbonate (Sigma) and 0.3 g/l l-glutamine on black tissue culture microplates (Greiner) at 37°C and 5% CO2 and used between passages 10 and 24. Surface expression of the Uroplakin 1a receptor was confirmed by immunofluorescence staining using a polyclonal antibody (Novus #NBP214694). E. coli serotype O25b UTI strain PFEEC0547 (AtlasUTI strain 1525121) was serially passaged in 10 mL static LB cultures at 37°C to induce FimH expression. Expression of FimH on the bacterial surface was confirmed by flow cytometry using rabbit immune serum against the FimH _LD antigen. The specificity of bacterial binding to mannan or bladder cells was determined by including a negative control compound, methyl α-D-pyranoside (Sigma), which reduced binding by >95% at the 50 mM level. Eight two-fold serial dilutions of test sera starting at 1:100 (in PBS, 0.1% BSA) were co-incubated with 1x10 ⁷ E. coli at 37°C for 1 hour before being added to immobilized yeast mannan or 5637 cell monolayers. Serially diluted anti-FimH _LD rabbit serum was used as an internal standard on each plate. The plates were incubated at 37°C for 1 hour and then washed to remove unbound bacteria. The bound E. coli were stained with 3 μg/ml of O25b-specific mAb conjugated to Alexafluor 488 for 45 minutes at RT. The mAb was reconstructed from the variable light and heavy chain sequences of the O25b antibody 3E9-11 as described previously (Szijarto V, et al. 2015. Antimicrob Agents Chemother 59:3109–3116). Human pTT5 IgG expression plasmid was used as a cloning vector (NRC, Canada). The fluorescence intensity of each well was read on a ClarioStar Plus instrument. IC ₅₀ inhibition values were interpolated using a sigmoidal dose-response variable slope curve fit (Graphpad Prism). The titer is the reciprocal of the serum dilution at which half of the maximum inhibition is observed. Vaccine antigen responders are defined as neutralization titers exceeding 80% inhibition at a starting serum dilution of 1:100. In addition, serum dilution titrations of binding activity had to meet variable slope sigmoidal curve fit parameters ( ^R2 > 0.95, using interpolated Log IC50 values or triggering experimental repeats at wider dilutions until resolved). Statistical significance (p-value) of differences in responses between groups was determined using an unpaired t-test with Welch's correction applied to log-transformed data.

5. Analysis of Adaptive Immune Response

The flow cytometry gating strategy and fluorophore-labeled marker-specific antibodies used in the ICS assay are shown in Figure 7 and Table 10. FimH-specific T cell responses in freshly isolated splenocytes were analyzed using a flow cytometry-based intracellular cytokine staining (ICS) assay. The ICS assay compared the response of medium-DMSO unstimulated to that observed in splenocytes after stimulation with a pool of FimH peptides.

Briefly, mouse spleens were treated using a non-enzymatic procedure (gentleMACS ^™ ) to obtain single cell suspensions. The spleens were subjected to erythrocyte lysis and passed through a cell strainer to remove erythrocytes and clots. Splenocytes (2*10 ⁶ cells/well) were cultured in vitro at 37°C for 6-7 hours in cRPMI containing medium-DMSO (unstimulated) or FimH-specific peptide pools (15aa, 11aa overlap, 1 μg/mL/peptide, JPT Peptide Technology, Berlin) in the presence of anti-CD107a APC antibodies and protein transport inhibitors GolgiPlug and GolgiStop. After stimulation, splenocytes were incubated with fluorescently conjugated antibodies against surface proteins CD19, CD3, CD4, CD8, CD44, CD40L (25 ± 5 minutes at 18-25°C), then fixed, permeabilized and stained for intracellular proteins IFN-g, TNF-α, IL-2, IL-4, IL-10, IL-17 (25 ± 5 minutes at 18-25°C). After staining, cells were washed and resuspended in staining buffer. Samples were collected on a Cytek Aurora flow cytometer. Results were background-subtracted (medium-DMSO) and presented as percentages of CD4+ T cells and CD8+ T cells expressing cytokines, respectively.

Table 10. Mouse ICS/AIM Panel

Specificity Fluorophore Purpose CD19 PerCp-Cy5.5 B-cells, exclusion CD3 BV605 T-cells CD4 BV650 CD4+ T-cells CD8 APC-H7 CD8+ T-cells CD107a APC Threshing IFNγ Pe-CF594 TH1 TNFa Pe-Cy7 TH1 IL2 APC-R700 TH1 IL4 BV711 TH2 IL17A FITC TH17 IL-10 PE Regulatory CD40L BV786 activation CD44 BV421 activation

Results and Discussion

The structural features of FimH variants expressed as modRNA in vitro and formulated into LNP for this mouse immunogenicity study are illustrated in Figure 1. The processed FimH _DSG protein expressed by modRNA LNP has the same amino acid sequence as the purified recombinant protein antigen used for comparison in this study. The sequence includes a conformationally stable triple mutation (G15AG16AV27A) and substitution mutations (N75S, N70S, N228S and N235S) that replace asparagine N-glycosylation sites. The membrane-targeted FimH _LD chimera has the same mouse IgK signal peptide, but lacks the FimH C-terminal pilin domain.

1. Expression of FimH LNPs in Primary Human Skeletal Muscle Cells

The expression of membrane-associated FimH after modRNA LNP transfection was evaluated in primary human skeletal muscle cells cultured in vitro. Before incubation with FimH detection antibodies, total cell FimH expression was evaluated by fixing and permeabilizing cells with paraformaldehyde and saponin. Surface staining was determined only after paraformaldehyde fixation. As shown in Figures 8A and 8B, regardless of the fixation procedure, transfection with RNA levels greater than 1.6ng per well using FimH _LD -gpi chimeras produced similar strong FimH expression levels, confirming that the antigen was mainly exposed on the outer membrane surface. FimH _DSG modRNA LNP was used as a negative control because previous mRNA transfection experiments (described in Example 1) showed that the antigen was effectively secreted into the culture medium. In this case, surface expression was not detected, and only very low levels of intracellular expression were observed.

2. Immunogenicity in mice

Figures 9A and 9B and Table 11 show the levels of FimH-specific IgG produced after mice were vaccinated with adjuvanted FimH _DSG protein and two LNPs. After a single vaccination with FimH _DSG modRNA LNP at any dose level, the IgG titers were significantly higher than the IgG titers after a single vaccination with a 10 μg dose of adjuvanted FimH _DSG protein. The IgG titers of modRNA LNPs at 10 μg tended to be higher than the 1 μg dose level, but there was no significant difference in the titers between the two different RNA-encoded antigens at any dose level. After the second dose of vaccine antigen, the IgG titers of all groups increased by 5-10 times. At this second dose (PD2) time point, the titer of the modRNA group encoded by 10 μg FimH _DSG was significantly higher than the compared FimH _DSG protein subunit antigen (after the second 5 μg dose), but the trend of increased titers in other modRNA LNP groups was not statistically significant.

Table 11. Summary of FimH IgG dLIA titers

Table 11. Summary of FimH IgG dLIA titers

The ability of the FimH vaccine antigen to elicit antibodies that can prevent the binding of live pilied E. coli to immobilized yeast mannan was determined by neutralization assays. The neutralization titers in this study are shown in Figure 10 and Table 12. At least two doses of FimH antigen are required to produce measurable levels of functional antibodies. After vaccination twice or three times at any dose level, the functional antibodies produced by both modRNA LNPs were significantly more than those of the adjuvanted protein subunit comparator. The titer of modRNA peaked after two doses and dropped 4-7 times after the third dose, an effect that may reflect excessive immune stimulation. The neutralization titers of the two FimH modRNA LNPs were not significantly different from each other at any time point or dose level.

Table 12. Summary of FimH neutralization titers

3. T-cell profiling

Surface detection of intracellular cytokine staining (ICS) and activation-induced markers (AIM) was used to quantitatively determine the FimH specific T cell population in the spleen of five mice killed after the third vaccine dose. During flow cytometry analysis Figure 11, the identification of cytokine or surface marker expression levels in specific cell types was achieved by selective cell gating. CD107a is a surface marker for immune cell activation and cytotoxic degranulation (Alter G, Malenfant JM, Altfeld M.2004.CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods 294:15-22).

Figure 12 shows the CD8+ T cell response in mice vaccinated after activation of the FimH peptide. Mice vaccinated with the adjuvanted protein FimH _DSG antigen failed to show CD8+CD107a expression and IFN-γ production under FimH peptide stimulation. In contrast, four of the five mice vaccinated with 10 μg FimH _DSG modRNA LNP produced CD107a and IFN-γ (double positive) responses. Similarly, FimH-specific CD8+ T cells can be detected in most mice immunized with 10 μg mRNA encoding FimH _LD -gpi LNP. For FimH _LD -gpi LNP, three of the five animals responded even at a 1 μg mRNA dose.

CD4+ T cell responses in vaccinated mice are shown in Figures 13A-13D. Interestingly, the FimH peptide pool stimulated a strong Th1-biased CD4+ T cell response in mice vaccinated with mRNA encoding the membrane-targeted FimH _LD chimera, but to a lesser extent in mice vaccinated with mRNA of the secreted FimH _DSG antigen. Mice vaccinated with FimH _DSG protein adjuvanted with LiNA-2 also generated antigen-specific CD4 T-cell responses, although the median number of cytokine-positive cells was lower than the response from mice receiving the membrane-targeted FimH modRNA construct (at any dose level).

Finally, activation of CD4 T-cell Th2 or Th17 pathway-specific markers was also assessed, and the results are shown in Figures 14A-14C. In this case, little or no FimH-specific Th2 or Th17 marker expression was detected in CD4 T-cells isolated from mice vaccinated with any FimH mRNA antigen. Therefore, modRNA-encoded FimH antigens primarily induced CD8+ and Th1-biased CD4+ T cell responses, but did not induce Th2 or Th17.

In the mouse studies described in this Example, exploratory E. coli FimH modRNA LNPs were evaluated for the first time to compare their immunogenicity relative to the recombinant protein full-length FimH _DSG subunit antigen adjuvanted with liposome QS21/MPLA (LiNA-2) described in International Publication No. WO2022/137078, which is incorporated herein by reference in its entirety. Serum neutralizing antibodies elicited by mice immunized with this benchmark protein preparation were much weaker than those elicited by mice immunized with the same protein antigen expressed by modRNA in mammalian cells. In Example 1, we demonstrated that the secreted form of FimH _DSG can be easily detected in the culture supernatant of Expi293 suspension cells transfected with FimH _DSG mRNA. modRNA LNPs from the same plasmid construct produced dose-dependent antigen-specific CD8+ T cell responses and strong CD4+ T cell responses in mice. In contrast, the recombinant subunit preparation failed to elicit a CD8+ T cell response and instead generated a weaker CD4+ T cell response.

Additional modRNA LNPs were evaluated with the goal of assessing the effects of expressing the FimH antigen on the surface of mammalian cells (rather than as a secreted antigen). In this case, the N-terminal lectin binding domain of FimH was fused to a unique membrane anchor sequence: the 37 C-terminal amino acids of GPI-anchored human CD55 (decay accelerating factor or DAF), of which only nine remain after signal cleavage and GPI attachment in the endoplasmic reticulum (Lisanti MP, Caras IW, Davitz MA, Rodriguez-Boulan E. 1989. Journal of Cell Biology 109: 2145-2156). This modRNA LNP generated similar levels of FimH neutralizing antibodies compared to secreted FimH _DSG LNPs, but resulted in a stronger upregulation of markers associated with Th1 pathway activation (CD40L, IFNγ, IL2, TNFα). None of the antigens tested activated Th2 pathway markers (IL4, IL10) or induced IL17, which is the initiator of a related pro-inflammatory signaling pathway that links T-cell activation to neutrophil mobilization and activation (Zenobia C, Hajishengallis G. 2015. Periodontology 2000 69: 142-159).

These results demonstrate that the modRNA platform is able to elicit much higher levels of neutralizing antibodies in mice than the adjuvanted recombinant protein subunit antigens described herein.

sequence

Table 13: FimH wild type and mutant sequences

Claims (66)

1. A ribonucleic acid polynucleotide (RNA) molecule comprising at least one Open Reading Frame (ORF) encoding a FimH antigen polypeptide.

2. The RNA molecule of claim 1, wherein the FimH antigen polypeptide is full length, truncated, fragment or variant thereof.

3. The RNA molecule of any one of claims 1-2, wherein the FimH antigen polypeptide comprises at least one mutation.

4. The RNA molecule of any one of claims 1-3, wherein the FimH antigen polypeptide comprises the amino acids of table 13, including but not limited to any one of SEQ ID NOs 1 to 64.

5. The RNA molecule of any one of claims 1-4, wherein the FimH antigen polypeptide comprises FimH-DSG (SEQ ID NO: 59), fimH-DSG triple mutant (G15A, G16A, V a) (SEQ ID NO: 62), fimHLD triple mutant (G15A, G16A, V a) (SEQ ID NO: 54), or an immunogenic fragment thereof.

6. The RNA molecule of any one of claims 1-5, wherein the FimH polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 64.

7. The RNA molecule of any one of claims 1-6, wherein the RNA is fused to a C-terminal membrane targeting domain.

8. The RNA molecule of claim 7, wherein the RNA molecule and the C-terminal membrane targeting domain are separated by a linker.

9. The RNA molecule of claim 8, wherein the linker has the amino acid sequence GGSSGGG (SEQ ID NO: 74).

10. The RNA molecule of any one of claims 7-9, wherein the C-terminal membrane targeting domain is derived from a viral glycoprotein.

11. The RNA molecule of claim 10, wherein the viral glycoprotein is selected from the group consisting of HSV gD, SARS-CoV2 spike protein and human DAFgpi.

12. The RNA of claim 9, wherein the C-terminal membrane targeting domain is e.

13. The RNA molecule of any one of claims 1-12, wherein the open reading frame is codon optimized.

14. The RNA molecule of claim 11, wherein the FimH antigen polypeptide comprises the amino acids of table 1, including but not limited to any one of SEQ ID No. 82, SEQ ID No. 83, and SEQ ID No. 84.

15. The RNA molecule of claim 14, wherein the FimH antigen polypeptide comprises an amino acid having SEQ ID No. 84.

16. The RNA molecule of claim 12, wherein the FimH antigen polypeptide comprises an amino acid having SEQ ID No. 85.

17. The RNA molecule of any one of claims 1-16, wherein the open reading frame is transcribed from a nucleic acid sequence of table 2, including but not limited to any one of SEQ ID No. 66, SEQ ID No. 68, SEQ ID No. 70 or SEQ ID No. 72.

18. The RNA molecule of any one of claims 1-17, wherein the open reading frame comprises a nucleic acid sequence of table 3, including but not limited to any one of SEQ ID NOs 82 to 85.

19. The RNA of claim 18, wherein each uridine of any of SEQ ID NOs 82 to 85 is replaced by 1-methyl-3' -pseudouridine (ψ).

20. The RNA molecule of any one of claims 1-19, further comprising a 5 'untranslated region (5' utr).

21. The RNA molecule of claim 21, wherein the 5' utr comprises a nucleotide with SEQ ID No. 75.

22. The RNA molecule of any one of claims 1-21, further comprising a3 'untranslated region (3' utr).

23. The RNA molecule of claim 22, wherein the 3' utr comprises a nucleotide with SEQ ID No. 76.

24. The RNA molecule of any one of claims 1 to 23, wherein the RNA molecule comprises a 5' cap moiety.

25. The RNA molecule of claim 24, wherein the 5 'cap moiety is m7G (5') ppp (5 ') (2' ome) pG.

26. The RNA molecule of any one of claims 1-25, further comprising a 3' poly a tail.

27. The RNA of claim 26, wherein the poly-a tail comprises a sequence having SEQ ID No. 86.

28. The RNA molecule of any one of claims 1-27, wherein the RNA molecule comprises a 5'utr and a 3' utr.

29. The RNA molecule of any one of claims 1-28, wherein the RNA molecule comprises a 5' cap, a 5' utr, and a 3' utr.

30. The RNA molecule of any one of claims 1-29, wherein the RNA molecule comprises a 5' cap, a 5' utr, a 3' utr, and a poly a tail.

31. The RNA molecule of any one of claims 1-30, wherein the RNA molecule comprises stabilized RNA.

32. The RNA molecule of any one of claims 1-31, wherein the RNA comprises at least one modified nucleotide.

33. The RNA molecule of claim 32, wherein the modified nucleotide is pseudouridine, 1-methyl-3 ' -pseudouridyl, N1-methyl-pseudouridine, N1-ethyl-pseudouridine, 2-thiouridine, 4' -thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, or 5-methoxy-uridine or 2' -O-methyluridine.

34. The RNA molecule of claim 33, wherein the modified nucleotide is 1-methyl-3' -pseudouridine (ψ).

35. The RNA molecule of any one of claims 1-25, wherein the RNA is mRNA.

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

37. The composition of claim 36, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEG-lipid, a neutral lipid, and a steroid or steroid analog.

38. The composition of claim 36 or 37, wherein the lipid nanoparticle comprises a cationic lipid.

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

40. The composition of any one of claims 36-39, wherein the lipid nanoparticle comprises a PEG-lipid.

41. The composition of claim 40, wherein the PEG-lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC or PEG-CerC), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, ethylene glycol-lipid including PEG-c-DOMG, PEG-c-DMA, PEG-S-DMG, N- [ (methoxypolyethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG, pegylated diacylglycerols (PEG-DAG) such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerols (PEG-S-G) such as 4-O- (2 ',3' -ditetradecyloxy) propyl-1-O-O-ethoxybutanedioic acid (PEG-D) and PEG-2000-DMG Or PEG dialkoxypropyl carbamates such as co-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate or 2, 3-di (tetradecyloxy) propyl-N- (co-methoxy (polyethoxy) ethyl) carbamate.

42. The composition of claim 40 or 41, wherein said PEG-lipid is 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (ALC-0159).

43. The composition of any one of claims 36-42, wherein the lipid nanoparticle comprises a neutral lipid.

44. The composition of claim 43, wherein the neutral lipid is distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1 carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), or 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (transDOPE).

45. The composition of claim 43 or 44, wherein said neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC).

46. The composition of any of claims 36-45, wherein the lipid nanoparticle comprises a steroid or steroid analog.

47. The composition of claim 46, wherein the steroid or steroid analog is cholesterol.

48. The composition of any one of claims 36-47, wherein the lipid nanoparticle has an average diameter of about 1 to about 500 nm.

49. The composition of any one of claims 36-48, wherein the composition is a vaccine.

50. The composition of any one of claims 36-49, wherein the lipid nanoparticle size is at least 40nm.

51. The composition of any one of claims 36-49, wherein the lipid nanoparticle is up to 180nm in size.

52. A method for (i) inducing an immune response against an enteropathogenic e.coli in a subject, or (ii) inducing production of opsonophagocytosis and/or neutralizing antibodies specific for an enteropathogenic e.coli in a subject, wherein the method comprises administering to the subject an effective amount of the RNA molecule, RNA-LNP and/or vaccine of any one of claims 1-51.

53. The method of claim 52, wherein the subject is at risk of developing a urinary tract infection.

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

55. The method of claim 52, wherein the subject is at risk of developing urosepsis.

56. The method of claim 52, wherein the subject is at risk of developing cystitis.

57. Use of an RNA molecule, an RNA-LNP and/or a composition of any of claims 1-56 in the manufacture of a medicament for (i) inducing an immune response against parenteral pathogenic escherichia coli in a subject, or (ii) inducing opsonophagocytosis specific for parenteral pathogenic escherichia coli and/or the production of neutralizing antibodies in a subject.

58. The use of claim 57, wherein the infection, disease or condition is a urinary tract infection.

59. The use of claim 57, wherein the subject is at risk of developing bacteremia.

60. The use of claim 57, wherein the subject is at risk of developing sepsis.

61. The use of claim 57, wherein the subject is at risk of developing cystitis.

62. The method or use of any one of claims 52-61, 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.

63. The method or use of any one of claims 52-61, wherein the subject is about 50 years of age or older.

64. The method or use of any one of claims 52-61, wherein said subject is a pregnant woman.

65. The method or use of any one of claims 52-64, wherein said RNA molecule or composition is administered as a vaccine.

66. The method or use of any one of claims 52-65, wherein said RNA molecule or composition is administered by intradermal injection or intramuscular injection.