Norovirus vaccines and methods of use

Abstract

Provided are Norovirus vaccines comprising an mRNA molecule encoding a Norovirus VP1 antigen, and methods of use thereof for treating or preventing a disease or disorder associated with Norovirus infection.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 272,439, filed October 27, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Human noroviruses (HuNoVs) are the most common cause of acute viral gastroenteritis worldwide, causing an estimated 200 million cases and approximately 200,000 deaths in children annually. Noroviruses are classified into 10 genogroups, five of which infect humans, with over 90% of human infections caused by strains of either genogroup I or II, and genotype 4 of genogroup II causing the majority of human norovirus infections and periodic waves of pandemic disease every few years.

[0003] Norovirus is a non-enveloped, single-stranded, positive-sense RNA virus of the Caliciviridae family. Its genome is approximately 7.5 to 7.7 kb in size and contains three open reading frames (ORFs); ORF2 encodes the major capsid protein (VP1), which determines the antigenicity of the virus and consists of a shell domain located at the base of the capsid.

[0004] Noroviruses (NoVs) are classified into 10 genogroups (GI to GX) based on the sequence of the complete major capsid protein VP1, however, only GI, GII, and GIV are capable of infecting humans.

[0005] Globally, GII.4 is the most prevalent genotype, accounting for approximately 70-80% of all norovirus outbreaks in most countries over the past two decades. Summary of the Invention [Problem to be solved by the invention]

[0006] There are no approved vaccines or specific therapeutics for treating Norovirus disease. Thus, there is a need in the art for improved Norovirus vaccines. The present invention addresses this need. [Means for solving the problem]

[0007] Summary of the Invention In one embodiment, the present invention relates to a composition for inducing an immune response against Norovirus (NoV), comprising at least one mRNA molecule encoding at least one Norovirus antigen, which in one embodiment is p48, nucleoside-triphosphatase (NTPase), p22, VPg, protease, and RNA-dependent RNA polymerase (RdRp), VP1, VP2, fragments thereof, or any combination thereof.

[0008] In one embodiment, Norovirus antigens are derived from the GI, GII, GIV, GVIII, or GIX genogroups.

[0009] In one embodiment, the composition comprises a combination of mRNA molecules encoding at least two Norovirus VP1 antigens, hi one embodiment, the composition comprises a combination of mRNA molecules encoding a GI VP1 antigen and a GII VP1 antigen.

[0010] In one embodiment, the composition comprises a combination of mRNA molecules encoding at least two of the GI.1 antigen, the GI.3VP1 antigen, the GI.5VP1 antigen, the GII.3VP1 antigen, and the GII.4VP1 antigen.

[0011] In one embodiment, the composition comprises a combination of an mRNA molecule encoding the NoV VP1 GI.1 antigen and an mRNA molecule encoding the NoV VP1 GII.4 antigen.

[0012] In one embodiment, the composition comprises a combination of mRNA molecules encoding each of the NoV VP1 GI.1 antigen, the NoV VP1 GII.4 antigen, the NoV VP1 GI.3 antigen and the NoV VP1 GII.3 antigen.

[0013] In one embodiment, the composition comprises at least one mRNA molecule encoding the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0014] In one embodiment, the composition further comprises an adjuvant.

[0015] In one embodiment, the mRNA molecule is encapsulated within a lipid nanoparticle (LNP).

[0016] In one embodiment, the composition comprises a combination of two or more LNPs encapsulating two or more mRNA molecules encoding NoV VP1 antigens.

[0017] In one embodiment, the mRNA molecule is a nucleoside modified mRNA molecule comprising at least one pseudouridine, 1-methylpseudouridine, or 5-methyl-uridine modified nucleoside.

[0018] In one embodiment, the present invention relates to a method of inducing an immune response against at least one strain of Norovirus (NoV) in a subject, comprising administering to the subject an effective amount of a composition comprising an mRNA molecule encoding at least one Norovirus antigen or fragment thereof. In one embodiment, the Norovirus antigen is p48, nucleoside-triphosphatase (NTPase), p22, VPg, protease, and RNA-dependent RNA polymerase (RdRp), VP1, VP2, fragments thereof, or any combination thereof. In one embodiment, the Norovirus antigen is derived from GI, GII, GIV, GVIII, or GIV genogroups.

[0019] In one embodiment, the method comprises administering a combination of mRNA molecules encoding at least two NoV VP1 antigens, hi one embodiment, the composition comprises a combination of mRNA molecules encoding a GP VP1 antigen and a GII VP1 antigen.

[0020] In one embodiment, the method comprises administering a combination of mRNA molecules encoding at least two of the GI.1 antigen, the GI.3 VP1 antigen, the GI.5 VP1 antigen, the GII.3 VP1 antigen, and the GII.4 VP1 antigen.

[0021] In one embodiment, the method comprises administering a combination of an mRNA molecule encoding the NoV VP1 GI.1 antigen and an mRNA molecule encoding the NoV VP1 GII.4 antigen.

[0022] In one embodiment, the method comprises administering a combination of mRNA molecules encoding each of the NoV VP1 GI.1 antigen, the NoV VP1 GII.4 antigen, the NoV VP1 GI.3 antigen, and the NoV VP1 GII.3 antigen.

[0023] In one embodiment, the method comprises administering at least one mRNA molecule encoding the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0024] In one embodiment, the method further comprises administering an adjuvant.

[0025] In one embodiment, the method comprises administering at least one mRNA molecule encapsulated within a lipid nanoparticle (LNP).

[0026] In one embodiment, the method comprises administering a combination of two or more LNPs encapsulating two or more mRNA molecules encoding the NoV VP1 antigen.

[0027] In one embodiment, the method comprises administering at least one nucleoside modified mRNA molecule comprising at least one pseudouridine, 1-methylpseudouridine, or 5-methyl-uridine modified nucleoside.

[0028] In one embodiment, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery routes.

[0029] In one embodiment, the method comprises a single administration of the composition. In one embodiment, the method comprises multiple administrations of the composition.

[0030] In one embodiment, the composition treats or prevents a disease or disorder associated with Norovirus infection, hi one embodiment, the disease or disorder associated with Norovirus infection is gastroenteritis, food poisoning, vomiting or diarrhea.

[0031] The following detailed description of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which: It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0032] [Figure 1]Figure 1 shows exemplary experimental data demonstrating that nucleoside modified mRNAs encoding the capsid protein VP1 sequences of GI.1 / Norwalk1968, GII.4 / CapeTown2012, GI.3 / Sweden2008, GI.3 / Argentina2016, GI.3 / Nashville2019, GII.3 / Canada2019, GII.2 / UK2015, GII.3 / USA2011 and GII.3 / Japan2009 viruses express the correct protein. Reducing conditions were used. Loaded 10 μg per well; GI.1-anti-norovirus antibody (VP1 GI.1 capsid protein), rabbit polyclonal Rb G1; GII.4-anti-norovirus antibody (VP1 GII.4 capsid protein), rabbit polyclonal Rb428, GI.3-anti-norovirus antibody (VP1 GI.3 capsid protein), rabbit polyclonal Rb402, GII.3-anti-norovirus antibody (VP1 GII.3 capsid protein), rabbit polyclonal Rb397, GAPDH(14C10)-rabbit monoclonal antibody, (Cell Signaling, #2118S); incubated overnight at 4°C, dilution 1:2,500; second GAR-horseradish peroxidase, 1 hour at room temperature, dilution 1:10,000; enterochromaffin-like cell positive. [Diagram 2] FIG. 2 shows an exemplary immunization scheme with a bivalent mRNA vaccine (GI.1 and GII.4). [Diagram 3] FIG. 3 shows exemplary experimental data demonstrating blocking antibody titers that persist at high levels for up to 180 days. [Figure 4] FIG. 4 shows exemplary experimental data demonstrating that the vaccine induces a potent, yet genotype-specific, blocking antibody response. [Diagram 5] FIG. 5 shows an exemplary immunization scheme for a T cell stimulation assay. [Figure 6] FIG. 6 shows the results of a T cell stimulation assay using virus-like particles (VLPs) (GI.1, GI.3, GII.3, GII.4). [Figure 7]FIG. 7 shows exemplary experimental data demonstrating human norovirus bivalent mRNA vaccine dose response. [Figure 8] FIG. 8 shows exemplary experimental data demonstrating that low doses of an mRNA vaccine induce potent Th1 responses. [Figure 9] FIG. 9 shows an exemplary immunization scheme for a tetravalent Norovirus mRNA vaccine. [Figure 10] FIG. 10 shows exemplary experimental data demonstrating human norovirus tetravalent vaccine dose response. [Figure 11] FIG. 11 shows exemplary experimental data demonstrating infection of stem cell-derived human three-dimensional enteroids with live human norovirus. [Figure 12] FIG. 12 shows exemplary images demonstrating infection of stem cell derived human three-dimensional enteroids with live human norovirus. [Figure 13] FIG. 13 shows exemplary experimental data demonstrating protection of human intestinal enteroids against GII.4 infection by serum from mice vaccinated with mRNA-LNPs vaccinated at 10 μg vaccine per mouse per intradermal (id) injection. [Figure 14] FIG. 14 shows exemplary experimental data demonstrating protection of human intestinal enteroids against GII.4 infection by serum from mRNA-LNP vaccinated mice vaccinated at 0.25 μg vaccine / mouse by intramuscular (im) injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description The present invention relates to compositions and methods for inducing an immune response against Norovirus (NoV) in a subject. In some embodiments, the present invention provides compositions comprising at least one lipid nanoparticle (LNP) comprising at least one nucleotide-modified RNA molecule encoding at least one Norovirus major capsid protein (VP1) from one or more Norovirus genogroups. In some embodiments, the present invention provides compositions comprising at least one lipid nanoparticle (LNP) comprising at least one nucleoside-modified RNA molecule encoding at least one VP1 from the GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIX, or GX genogroups. In some embodiments, the present invention provides compositions comprising a combination of lipid nanoparticles (LNPs) comprising nucleoside-modified RNA molecules encoding at least two Norovirus major capsid proteins (VP1). In some embodiments, the composition comprises mRNA molecules encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 Norovirus VP1 antigens.

[0034] In one embodiment, the at least two VP1s are derived from the same Norovirus genogroup. In one embodiment, the at least two VP1s are derived from two or more different Norovirus genogroups.

[0035] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII and at least one LNP comprising an mRNA molecule encoding VP1 from GI.

[0036] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII, at least one LNP comprising an mRNA molecule encoding VP1 from GI, and at least one LNP comprising an mRNA molecule encoding VP1 from GIV.

[0037] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII.4 and at least one LNP comprising an mRNA molecule encoding VP1 from GI.1.

[0038] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII.4, at least one LNP comprising an mRNA molecule encoding VP1 from GI.1, at least one LNP comprising an mRNA molecule encoding VP1 from GI.3, and at least one LNP comprising an mRNA molecule encoding VP1 from GII.3.

[0039] The present invention also relates to methods of treating Norovirus infection or treating or preventing diseases or disorders associated with Norovirus infection, such as vomiting and diarrhea, using the compositions of the present invention.

[0040] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] As used herein, each of the following terms has the meaning associated with it in this section.

[0042] The words "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0043] As used herein, "about" when referring to a measurable value such as an amount, duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the particular value, as such variations are appropriate for practicing the disclosed methods.

[0044] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present invention can exist in a wide variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0045] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0046] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0047] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation. Kappa light chain and lambda light chain refer to the two main isotypes of antibody light chains.

[0048] By the term "synthetic antibody" as used herein is meant an antibody that is generated using recombinant DNA technology. The term should also be taken to mean an antibody that is made by synthesis of a DNA molecule that encodes the antibody, which expresses the antibody protein or the amino acid sequence that specifies the antibody, where the DNA sequence or amino acid sequence was obtained using synthetic DNA or amino acid sequence techniques available and well known in the art. The term should also be taken to mean an antibody that is made by synthesis of an RNA molecule that encodes the antibody, which expresses the antibody protein or the amino acid sequence that specifies the antibody, where the RNA was obtained by transcribing DNA (synthetic or cloned), synthesizing RNA, or by other techniques available and well known in the art.

[0049] By the term "specifically bind" as used herein with respect to an antibody, we mean an antibody that recognizes a particular antigen in a sample but does not substantially recognize or bind other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. However, such cross-species reactivity does not, in and of itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in and of itself, change the classification of the antibody as specific. In some cases, the term "specific binding" or "specifically binding" may be used in relation to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure, rather than to the protein as a whole. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (i.e., free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0050] The term "immunogen" as used herein is intended to denote an entity capable of inducing an adaptive immune response in an individual, where said adaptive immune response is capable of inducing an immune response that significantly engages a pathogenic agent, which shares immunological characteristics with the immunogen. "Immunogen" refers to any substance introduced into the body to generate an immune response. The substance may be a physical molecule, such as a protein, or may be encoded by a vector, such as DNA, mRNA, or a virus.

[0051] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an adaptive immune response. This immune response may include either the production of antibodies or the activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA or RNA. Thus, those skilled in the art will appreciate that any DNA or RNA that contains a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an adaptive immune response will encode an "antigen" as that term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not simply be encoded by a full-length nucleotide sequence of a gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and it will be readily understood that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen may be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0052] "Immune response", as the term is used herein, refers to a process involving activation and / or induction of effector functions, for example, in non-limiting examples, T cells, B cells, natural killer (NK) cells and / or antigen presenting cells (APCs). Thus, immune response includes, but is not limited to, any detectable antigen-specific activation and / or induction of helper T cell or cytotoxic T cell activity or response, production of antibodies, activity or infiltration of antigen presenting cells, activity or infiltration of macrophages, activity or infiltration of neutrophils, etc., as would be understood by one of skill in the art.

[0053] As used herein, an "immunogenic composition" may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding the antigen, a cell expressing or presenting an antigen or a cellular component, a virus expressing or presenting an antigen or a cellular component, or a combination thereof. In certain embodiments, the composition comprises or encodes all or a portion of any peptide antigen described herein, or an immunologically functional equivalent thereof. In other embodiments, the composition is in a mixture that includes additional immunostimulatory substances or nucleic acids encoding such substances. Immunostimulatory substances include, but are not limited to, additional antigens, immune modulators, antigen-presenting cells, lipid nanoparticles, or adjuvants. In other embodiments, one or more of the additional substances(s) are covalently attached to the antigen or immunostimulatory substance in any combination.

[0054] As used herein, the term "vaccine" refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.

[0055] "Encode" refers to the inherent property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand (the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding the protein or other product of that gene or cDNA.

[0056] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, ionic or amphiphilic compound-associated polynucleotides, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be taken to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0057] "Expression vector" refers to a vector containing a recombinant polynucleotide, including expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting sequences for expression; other sequences for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0058] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if the position in each of the two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in the two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed by aligning the two sequences to obtain the maximum homology.

[0059] As used herein, a nucleotide sequence is "substantially homologous" to any of the nucleotide sequences described herein if the nucleotide sequence has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to the original nucleotide sequence.

[0060] As used herein, an amino acid sequence is "substantially homologous" to any of the amino acid sequences described herein if the amino acid sequence has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to the original amino acid sequence. The identity between two amino acid sequences can be determined by using the BLASTN algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0061] The term "variant" as used herein with respect to nucleic acids refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes to the referenced nucleic acid under stringent conditions, its complement, or a sequence that is substantially identical thereto. A variant can be a nucleic acid sequence that is substantially identical over the full length of a complete gene sequence or a fragment thereof. A nucleic acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of a gene sequence or a fragment thereof.

[0062] The term "variant" as used in reference to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Variant may also refer to a protein having an amino acid sequence that is substantially identical to a reference protein, with the amino acid sequence retaining at least one biological activity. Conservative substitutions of amino acids, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophobicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes may be identified, in part, by considering the hydrophobicity index of an amino acid, as understood in the art (Kyte et al., 1982, J. Mol. Biol. 157:105-132). The hydrophobicity index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indexes can be substituted and still retain the function of the protein. In one embodiment, amino acids with hydrophobicity indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that will result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values ​​may result in peptides that retain biological activity, e.g., immunogenicity, as understood in the art. Substitutions may be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of the amino acid. Consistent with that observation, it is understood that substitution of amino acids that is compatible with biological function depends on the relative similarity of the amino acids, particularly the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0063] The term "fragment" or "functional fragment" as used herein refers to a fragment of an antigen or a nucleic acid sequence encoding an antigen that confers an increased immune response when administered to a subject. A fragment is generally 10 or more amino acids or nucleic acids in length. A "fragment" may refer to a polypeptide fragment of an antigen that is capable of eliciting an immune response in a subject. A fragment of an antigen may be 100% identical to the full length, except for the deletion of at least one amino acid from the N-terminus and / or C-terminus, and may or may not include the signal peptide and / or methionine at position 1, in each case. Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of the specified full-length antigen, excluding any added heterologous signal peptide. The fragments may include fragments of a polypeptide that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antigen, and further include an N-terminal methionine or a heterologous signal peptide that are not included when calculating identity.

[0064] A fragment of a nucleic acid sequence encoding an antigen can be 100% identical to the full length but for the deletion of at least one nucleotide from the 5' and / or 3' end, and in each case may or may not include sequences encoding a signal peptide and / or a methionine at position 1. A fragment can comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of the particular full length coding sequence, excluding any added heterologous signal peptide. The fragments may include fragments that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identical to the antigen, and may optionally further include sequences encoding an N-terminal methionine or a heterologous signal peptide, which are not included when calculating identity.

[0065] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living subject is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially pure form, or can exist in a non-native environment, such as, for example, a host cell.

[0066] In the context of the present invention, the following abbreviations for commonly occurring nucleosides (nucleobases linked to a ribose or deoxyribose sugar via an N-glycosidic bond) are used: "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0067] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Protein-encoding nucleotide sequences and RNAs may contain introns. The phrase nucleotide sequence or RNA encoding a protein may also include introns, to the extent that a protein-encoding nucleotide sequence may, in some forms, include intron(s). Additionally, the nucleotide sequence may contain modified nucleosides that can be translated by the translation machinery in a cell. Exemplary modified nucleosides are described elsewhere herein. For example, in an mRNA, some or all of the uridines are replaced with pseudouridine, 1-methylpseudouridine, 5-methyl-uridine, or another modified nucleoside, such as those described elsewhere herein. In some embodiments, the nucleotide sequence may include a sequence in which some or all of the cytidines are replaced with methylated cytidine, or another modified nucleoside, such as those described elsewhere herein.

[0068] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, so that the latter is expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous, and if necessary, two protein coding regions are connected in the same reading frame.

[0069] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, and by synthetic means.

[0070] In some cases, the polynucleotide or nucleic acid of the present invention is a "nucleoside modified nucleic acid", which refers to a nucleic acid that contains at least one modified nucleoside. "Modified nucleoside" refers to a nucleoside that has a modification. For example, more than 100 different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0071] As used herein, the terms "peptide", "polypeptide" and "protein" are used synonymously and refer to compounds that contain amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes any peptide or protein that contains two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, e.g., commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0072] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell, or introduced synthetic machinery, required to initiate specific transcription of a polynucleotide sequence. As a non-limiting example, promoters recognized by bacteriophage RNA polymerase are used to generate mRNA by in vitro transcription.

[0073] The term "adjuvant" as used herein is defined as any molecule for enhancing an antigen-specific adaptive immune response.

[0074] In some embodiments, "pseudouridine" is 1 acp 3 (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the term refers to m 1In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 In another embodiment, the term refers to m 3 Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that has not been further modified. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the pseudouridines listed above. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0075] The term "lipid nanoparticle" refers to a particle having at least one dimension in the nanometer dimension (eg, 1-1,000 nm) that comprises one or more lipids.

[0076] The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0077] The term "cationic lipid" as used herein refers to a lipid that is cationic or becomes cationic (protonated) below the pK of the ionizable group of the lipid, but becomes progressively more neutral at higher pH values. At pH values ​​below the pK, the lipid can then associate with negatively charged nucleic acid. In some embodiments, cationic lipids include zwitterionic lipids, which assume a positive charge when the pH is decreased.

[0078] The term "neutral lipid" refers to any one of a number of lipid species that exist in either uncharged or neutral zwitterionic form at physiological pH. Exemplary neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0079] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH.

[0080] The term "polymer-conjugated lipid" refers to a molecule that includes both a lipid portion and a polymer portion. One example of a polymer-conjugated lipid is a PEGylated lipid.

[0081] The term "PEGylated lipid" refers to a molecule that contains both a lipid moiety and a polyethylene glycol moiety. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), and the like.

[0082] "Liposome" is a general term that encompasses a wide variety of unilamellar and multilamellar lipid vesicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before the formation of the encapsulated structure, entrapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0083] The terms "subject," "patient," "individual," and the like are used synonymously herein and refer to any animal or cells thereof that are amenable to the methods described herein, whether in vitro or in situ. In some non-limiting embodiments, the patient, subject, or individual is a mammal, bird, poultry, cow, pig, horse, sheep, ferret, primate, dog, cat, guinea pig, rabbit, bat, or human.

[0084] A "disease" is a state of a subject's health in which the subject is unable to maintain homeostasis and where the subject's health will continue to deteriorate if the disease is not remitted.

[0085] In contrast, a "disorder" in a subject is a state in which the subject is able to maintain homeostasis, but the subject's health status is less than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the subject's health status.

[0086] By the term "modulate" as used herein is meant to mediate a detectable increase or decrease in the level of a subject's response compared to the level of the subject's response in the absence of a treatment or compound and / or compared to the level of the response of an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, such as a human.

[0087] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0088] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0089] As used herein, the term "therapy" refers to treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, reduction, amelioration, prevention, or eradication of at least one sign or symptom of a disease or disorder.

[0090] The term "therapeutically effective amount" refers to an amount of a compound of interest that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent or reduce to some extent the onset of one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0091] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0092] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0093] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavorings; colorants; preservatives; biodegradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifiers, softeners; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pennsylvania), which is incorporated herein by reference.

[0094] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be taken as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0095] description The present invention relates to compositions and methods for inducing an immune response against one or more Norovirus strains in a subject. In some embodiments, the present invention provides compositions comprising at least one RNA molecule encoding at least one VP1 from the GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIX, or GX gene group. In some embodiments, the mRNA molecule is a nucleoside modified mRNA molecule.

[0096] In some embodiments, the present invention provides compositions comprising a combination of mRNA molecules (e.g., nucleoside-modified mRNA molecules) encoding at least two Norovirus major capsid proteins (VP1). In some embodiments, the compositions comprise mRNA molecules (e.g., nucleoside-modified mRNA molecules) encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 Norovirus VP1 antigens.

[0097] In one embodiment, the at least two VP1s are derived from different strains within the same Norovirus genogroup. In one embodiment, the at least two VP1s are derived from two or more different Norovirus genogroups.

[0098] In one embodiment, the composition comprises a combination of at least one mRNA molecule encoding VP1 from at least one GII strain and at least one mRNA molecule encoding VP1 from at least one GI strain.

[0099] In one embodiment, the composition comprises a combination of at least one mRNA molecule encoding VP1 from GII, at least one LNP comprising an mRNA molecule encoding VP1 from GI, and at least one mRNA molecule encoding VP1 from GIV.

[0100] In one embodiment, the composition comprises a combination of at least one mRNA molecule encoding VP1 from GII.4 and at least one mRNA molecule encoding VP1 from GI.1.

[0101] In one embodiment, the composition comprises a combination of at least one mRNA molecule encoding VP1 from GII.4, at least one mRNA molecule encoding VP1 from GI.1, at least one mRNA molecule encoding VP1 from GI.3, and at least one mRNA molecule encoding VP1 from GII.3.

[0102] In some embodiments, the composition further comprises an mRNA molecule encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 additional viral antigens. In some embodiments, the one or more additional viral antigens are selected from the group consisting of human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papillomaviruses, such as human papillomaviruses (HPV), poliovirus, hepatitis viruses, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Volavirus Major), and the like. and variola minor), vaccinia virus, rhinovirus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-1), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), and type 1 simple The viral antigens may be derived from herpes simplex (oral herpes), herpes simplex type 2 (genital herpes), varicella zoster (varicella zoster, also known as chicken pox), cytomegalovirus (CMV), such as human CMV, Epstein-Barr virus (EBV), flavivirus, hand, foot and mouth disease virus, Lassa virus, arenavirus, Merkel cell polyomavirus (MCV), influenza virus, coronaviruses including but not limited to severe acute respiratory syndrome-coronavirus 2, cancer-causing viruses, or any combination thereof. In one embodiment, the one or more additional viral antigens are Norovirus antigens. Exemplary additional Norovirus antigens include, but are not limited to, p48, nucleoside-triphosphatase (NTPase), p22, VPg, protease, and RNA-dependent RNA polymerase (RdRp), VP1, VP2, fragments thereof, or any combination thereof.

[0103] In some embodiments, the nucleoside-modified mRNA molecules (e.g., nucleotide-modified mRNA molecules) are encapsulated in one or more LNPs. In some embodiments, the composition comprises at least one LNP comprising at least one nucleoside-modified mRNA molecule encoding at least one VP1 antigen from the GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIX, or GX gene group. In some embodiments, the composition comprises a combination of LNPs comprising nucleoside-modified RNA molecules encoding a combination of at least two VP1 antigens from the GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIX, or GX gene group.

[0104] In some embodiments, the compositions comprise a combination of LNPs comprising mRNA molecules encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 Norovirus VP1 antigens.

[0105] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from at least one GII strain and at least one LNP comprising an mRNA molecule encoding VP1 from at least one GI strain.

[0106] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII, at least one LNP comprising an mRNA molecule encoding VP1 from GI, and at least one LNP comprising an mRNA molecule encoding VP1 from GIV.

[0107] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII.4 and at least one LNP comprising an mRNA molecule encoding VP1 from GI.1.

[0108] In one embodiment, the composition comprises a combination of at least one LNP comprising an mRNA molecule encoding VP1 from GII.4, at least one LNP comprising an mRNA molecule encoding VP1 from GI.1, at least one LNP comprising an mRNA molecule encoding VP1 from GI.3, and at least one LNP comprising an mRNA molecule encoding VP1 from GII.3.

[0109] vaccine In one embodiment, the present invention provides an immunogenic composition for inducing an immune response against Norovirus (NoV) in a subject. For example, in one embodiment, the immunogenic composition is a vaccine. For a composition to be useful as a vaccine, the composition must induce an immune response against Norovirus antigens in a cell, tissue, or subject. In some embodiments, the composition induces a broad immune response against multiple Norovirus strains in a cell, tissue, or subject. In some cases, the vaccine induces a protective immune response in a subject.

[0110] The vaccines of the present invention may vary in their composition of nucleic acids and / or cellular components. In one embodiment, the vaccine comprises a nucleic acid molecule encoding a NoV VP1 antigen. In one embodiment, the vaccine comprises a combination of nucleic acid molecules encoding Norovirus VP1 antigens from two or more Norovirus genogroups. In one embodiment, the vaccine comprises a combination of nucleic acid molecules encoding Norovirus VP1 antigens from one or more Norovirus GII strains and one or more Norovirus GI strains.

[0111] In a non-limiting example, one or more nucleic acid molecules encoding a NoV VP1 antigen may also be formulated with an adjuvant. In another non-limiting example, the vaccine may include one or more adjuvants. The vaccine of the present invention, and its various components, may be prepared and / or administered by any method disclosed herein or as would be known to one of skill in the art in light of the present disclosure.

[0112] In various embodiments, induction of immunity by expression of a NoV VP1 antigen can be detected in vivo or in vitro by observing the response of all or any part of the host's immune system to one or more NoV VP1 antigens.

[0113] For example, methods for detecting induction of cytotoxic T lymphocytes are well known. Foreign substances entering the body are presented to T cells and B cells by the action of antigen-presenting cells (APCs). Some T cells that respond specifically to the antigens presented by APCs differentiate into cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs) due to stimulation by the antigen. These antigen-stimulated cells then proliferate. This process is referred to herein as "activation" of T cells. Therefore, induction of CTLs by epitopes of polypeptides or peptides or combinations thereof can be evaluated by presenting epitopes of polypeptides or peptides or combinations thereof to T cells by antigen-presenting cells and detecting induction of CTLs. Furthermore, antigen-presenting cells have the action of activating B cells, CD4 positive T cells, CD8 positive T cells, macrophages, eosinophils and natural killer cells.

[0114] Methods for evaluating the induction of CTL action using dendritic cells (DC) as antigen-presenting cells are well known in the art. Dendritic cells are representative antigen-presenting cells with robust CTL-inducing action among antigen-presenting cells. In the method of the present invention, a polypeptide or peptide epitope or combination thereof is first expressed by a dendritic cell, and then the dendritic cell is contacted with a T cell. Detection of T cells having cytotoxic action against a target cell after contact with a dendritic cell indicates that the polypeptide or peptide epitope or combination thereof has the activity of inducing cytotoxic T cells. Furthermore, the induced immune response can also be examined by measuring the interferon gamma produced and released by CTL in the presence of antigen-presenting cells with fixed peptide or peptide combination, by visualization using an anti-interferon gamma antibody, such as ELISPOT assay.

[0115] Apart from dendritic cells, peripheral blood mononuclear cells (PBMCs) can also be used as antigen-presenting cells. It has been reported that the induction of CTLs is enhanced by culturing PBMCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Similarly, it has been shown that CTLs are induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and interleukin-7.

[0116] Antigens confirmed to have CTL-inducing activity by these methods are antigens that have dendritic cell activating activity and subsequent CTL-inducing activity. Furthermore, CTLs having cytotoxicity acquired as a result of antigen presentation by antigen-presenting cells can also be used as vaccines against antigen-associated disorders.

[0117] Induction of immunity by expression of a Norovirus antigen can be further confirmed by observing induction of production of antibodies against the Norovirus antigen, for example, if antibodies against the antigen are induced in experimental subjects immunized with a composition encoding the antigen, and if pathology associated with the antigen is suppressed by such antibodies, the composition is determined to induce immunity.

[0118] The specificity of the antibody response induced in a subject can include binding to multiple regions of the delivered antigen, as well as the induction of neutralizing antibodies that prevent infection or reduce the severity of disease.

[0119] The induction of immunity by the expression of Norovirus antigens can be further confirmed by observing the induction of T cells, such as CD4 positive T cells, CD8 positive T cells, or a combination thereof. For example, CD4 positive T cells can also lyse target cells, but may primarily provide support for the induction of other types of immune responses, including the generation of CTLs and antibodies. The types of support of CD4 positive T cells can be characterized as Th1, Th2, Th9, Th17, regulatory T cells (Treg), or follicular helper T cells (Tfh). Each subtype of CD4 positive T cells provides support for a particular type of immune response. In one embodiment, the composition selectively induces follicular helper T cells, which drive a strong antibody response.

[0120] The therapeutic compounds or compositions of the present invention may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from or at risk of (or susceptible to) developing a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration is performed before the manifestation of overt clinical symptoms of the disease, thereby preventing the disease or disorder or, alternatively, slowing its progression. In the context of the medical field, the term "preventing" includes any activity that reduces the burden of mortality or morbidity from a disease. Prevention may be performed at primary, secondary, and tertiary prevention levels. Primary prevention avoids the onset of the disease, while secondary and tertiary levels of prevention include activities aimed at preventing the progression of the disease and the appearance of symptoms, as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0121] antigen The present invention provides a composition for inducing an immune response in a subject. In one embodiment, the composition comprises at least one mRNA molecule encoding at least one Norovirus (NoV) antigen. The Norovirus antigens that can be included in the composition of the present invention include, but are not limited to, p48, nucleoside-triphosphatase (NTPase), p22, VPg, protease, and RNA-dependent RNA polymerase (RdRp), VP1, VP2, fragments thereof, or any combination thereof.

[0122] In one embodiment, the antigen is a VP1 antigen. In one embodiment, the VP1 antigen is derived from the GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIX or GX gene group. In one embodiment, the VP1 antigen is derived from the GI, GII, GIV, GVIII or GIX gene group. In one embodiment, the VP1 antigen is derived from GII.3, GII.4, GI.1, GI.3, GI.5 or any combination thereof.

[0123] In some embodiments, the vaccine further comprises one or more additional nucleic acid molecules encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 additional viral antigens. In some embodiments, the one or more additional viral antigens are selected from the group consisting of human immunodeficiency virus (HIV), chikungunya virus (CHIKV), dengue virus, papillomaviruses, such as human papillomavirus (HPV), poliovirus, hepatitis viruses, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (variola major and variola minor), vaccinia virus, rhinovirus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-1), hairy cell leukemia virus (HTLV-II), calphovirus, leukemia virus (LEV), ... The antigens may be derived from a virus such as Lunia encephalitis virus, Hantavirus (hemorrhagic fever), Rabies virus, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex type 1 (oral herpes), herpes simplex type 2 (genital herpes), varicella zoster (varicella zoster, also known as chicken pox), cytomegalovirus (CMV), such as human CMV, Epstein-Barr virus (EBV), flaviviruses, hand, foot and mouth disease virus, Lassa virus, Arenaviruses, Merkel cell polyomavirus (MCV), influenza virus, coronaviruses including but not limited to severe acute respiratory syndrome-coronavirus 2, cancer-causing viruses, or any combination thereof. In one embodiment, the one or more additional viral antigens are Norovirus antigens.

[0124] For example, in some embodiments, the compositions comprise nucleoside-modified RNA, or fragments or variants thereof, encoding a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 NoV VP1 antigens from the NoV GI, GII, and GIV genetic groups.

[0125] In some embodiments, the NoV VP1 antigen comprises a full-length VP1 antigen, or a fragment or variant thereof.

[0126] In one embodiment, the mRNA molecule encoding the NoV VP1 antigen encodes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0127] In one embodiment, the nucleic acid molecule encoding the NoV VP1 antigen includes a sequence encoding a tag or signal peptide (SP). Other signal peptides that can be used include, but are not limited to, IL-2, tPA (tissue plasminogen activator), mouse and human IgG, and optimized synthetic signal sequences. In some cases, the nucleic acid sequence includes additional sequences encoding a linker or tag sequence linked to the antigen by a peptide bond.

[0128] In some embodiments, the NoV VP1 antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of a NoV VP1 antigen described herein and retains the immunogenic function of the original amino acid sequence. For example, in some embodiments, the nucleotide sequence of a nucleic acid molecule encoding the NoV VP1 antigen has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0129] Adjuvants In one embodiment, the composition comprises an adjuvant. In one embodiment, the composition comprises a nucleic acid molecule encoding the adjuvant. In one embodiment, the nucleic acid molecule encoding the adjuvant is an in vitro transcribed RNA. In one embodiment, the nucleic acid molecule encoding the adjuvant is a nucleoside modified RNA. In one embodiment, the nucleic acid molecule encoding the adjuvant is a nucleoside modified mRNA.

[0130] Exemplary adjuvants include, but are not limited to, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), tumor necrosis factor (TNF) α, TNF β, GM-SCF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, major histocompatibility complex, CD80, CD86. Other genes that may be useful adjuvants include monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-I, lymphocyte function-associated antigen (LFA)-1, very late antigen (VLA)-1, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, and suddenly Mutant IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Re1, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFκβ, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, anti-CTLA4-sc antibody, anti-LAG3-Ig antibody, anti-TIM3-Ig antibody, and those encoding functional fragments thereof.

[0131] In some embodiments, the composition comprises LNPs, wherein the LNPs act as an adjuvant.

[0132] nucleic acid In one embodiment, the present invention includes at least one mRNA molecule (e.g., a nucleoside-modified mRNA molecule) encoding at least one Norovirus antigen. Norovirus antigens that can be encoded by the mRNA molecules of the present invention include, but are not limited to, p48, nucleoside-triphosphatase (NTPase), p22, VPg, protease, and RNA-dependent RNA polymerase (RdRp), VP1, VP2, fragments thereof, or any combination thereof.

[0133] In one embodiment, the mRNA molecule encodes the VP1 antigen. In one embodiment, the VP1 antigen is derived from the GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIX or GX gene group. In one embodiment, the VP1 antigen is derived from the GI, GII, GIV, GVIII or GIX gene group. In one embodiment, the VP1 antigen is derived from GII.3, GII.4, GI.1, GI.3 or GI.5, or any combination thereof.

[0134] Nucleic acid molecules can be made using any method in the art, including but not limited to in vitro transcription, chemical synthesis, and the like.

[0135] Nucleotide sequences encoding at least one Norovirus antigen as described herein may alternatively include sequence variations, such as, for example, substitutions, insertions, and / or deletions of one or more nucleotides, relative to the original nucleotide sequence, provided that the resulting polynucleotide encodes a polypeptide as described in the present invention. Thus, the scope of the present invention includes nucleotide sequences that are substantially homologous or substantially identical to the nucleotide sequences recited herein and that encode a Norovirus antigen of the present invention.

[0136] Nucleotide sequences that are substantially homologous to the nucleotide sequence encoding the antigen can typically be isolated from the antigen producer organism based on the information contained in the nucleotide sequence, for example by introducing conservative or non-conservative substitutions. Other examples of possible modifications include the insertion of one or more nucleotides into the sequence, the addition of one or more nucleotides to either end of the sequence, or the deletion of one or more nucleotides at either end or within the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods well known to those skilled in the art.

[0137] Additionally, the scope of the present invention includes nucleotide sequences that encode amino acid sequences that are substantially homologous to the amino acid sequences recited herein and retain the immunogenic function of the original amino acid sequence.

[0138] In one embodiment, the invention relates to a construct comprising a nucleotide sequence encoding a NoV VP1 antigen. In one embodiment, the invention comprises a plurality of nucleotide sequences encoding a plurality of Norovirus antigens. For example, in some embodiments, the invention comprises a plurality of nucleotide sequences encoding one or more, two or more, three or more, or four or more NoV VP1 antigens. In one embodiment, the invention relates to a construct comprising a nucleotide sequence encoding an adjuvant. In one embodiment, the invention comprises a combination of a plurality of nucleotide sequences encoding one or more, two or more, three or more, or four or more NoV VP1 antigens and an adjuvant.

[0139] In one embodiment, the composition comprises a plurality of constructs, each construct encoding a NoV VP1 antigen. In one embodiment, the plurality of NoV VP1 antigens are derived from Norovirus genogroups GI, GII, GIV, GVIII, or GIX, or any combination thereof. In one embodiment, the composition comprises a first construct comprising a nucleotide sequence encoding a NoV GII.4 VP1 antigen; and a second construct comprising a nucleotide sequence encoding a NoV GI.1 VP1 antigen. In one embodiment, the composition comprises a first construct comprising a nucleotide sequence encoding a NoV GII.4 VP1 antigen; a second construct comprising a nucleotide sequence encoding a NoV GI.1 VP1 antigen, a third construct comprising a nucleotide sequence encoding a NoV GII.3 VP1 antigen, and a fourth construct comprising a nucleotide sequence encoding a NoV GI.3 VP1 antigen. In one embodiment, the mRNA molecule encoding the NoV GI.1 antigen encodes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In one embodiment, an mRNA molecule encoding the NoV GII.4 antigen encodes the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, an mRNA molecule encoding the NoV GI.3 antigen encodes the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In one embodiment, an mRNA molecule encoding the NoV GII.3 antigen encodes the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In one embodiment, an mRNA molecule encoding the NoV GI antigen encodes the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0140] In another particular embodiment, the construct is operably linked to a translation control sequence. The construct may incorporate operably linked regulatory sequences for expression of the nucleotide sequence of the present invention, thereby forming an expression cassette.

[0141] vector The nucleic acid sequence(s) encoding the Norovirus antigen(s) of the invention can be obtained using recombinant methods known in the art, for example, by screening libraries derived from cells expressing the gene, by deriving the gene from a vector known to contain it, or by isolating it directly from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest may be made synthetically.

[0142] Nucleic acid molecules can be cloned into many kinds of vectors.For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, PCR-generated linear DNA sequences, and cosmids.Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequence vectors, and vectors optimized for in vitro transcription.

[0143] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, carbohydrates, peptides, cationic polymers, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0144] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous portion of the liposome interior, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, encapsulated within the liposome, complexed with the liposome, dispersed in a solution containing a liquid, mixed with a lipid, combined with a lipid, contained as a suspension in a liquid, contained with or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / RNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as a micelle, or in a "collapsed" structure. They may also simply be scattered in the solution, forming aggregates that are possibly not uniform in size or shape. Lipids are fatty substances, which may be natural lipids or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0145] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories, Plainview, NY; cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Birmingham, AL. Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used because it evaporates more easily than methanol.

[0146] Regardless of the method used to introduce exogenous nucleic acid into a host cell or otherwise expose the cell to a composition of the invention, a wide variety of assays can be performed to confirm the presence of an mRNA sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Northern blots and reverse transcription PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blots), or the assays described herein to identify substances that fall within the scope of the invention.

[0147] In vitro transcribed RNA In one embodiment, the composition of the invention comprises a combination of in vitro transcribed (IVT) RNA molecules encoding the Norovirus antigens of the invention. In one embodiment, the in vitro transcribed RNA may be introduced into cells as a form of transient transfection. The RNA is produced by in vitro transcription using a synthetically produced plasmid DNA template. The DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. In one embodiment, the desired template for in vitro transcription is a Norovirus antigen capable of inducing an adaptive immune response. In one embodiment, the desired template for in vitro transcription is an adjuvant capable of enhancing the adaptive immune response.

[0148] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA may be derived from a natural DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene or a portion of a gene of interest. The gene may include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene may include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene, including 5' and / or 3' untranslated regions. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic organism or symbiont, including bacteria, viruses, parasites and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic organism or symbiont, including bacteria, viruses, parasites and fungi, including 5' and 3' untranslated regions. The DNA may alternatively be an artificial DNA sequence that is not normally expressed in a natural organism. An exemplary artificial DNA sequence is one in which portions of genes are ligated together to form an open reading frame that together encodes a fusion protein. The portions of DNA that are ligated together may be from one organism or from more than one organism.

[0149] Genes that can be used as DNA sources for PCR include genes that code for polypeptides that induce or enhance adaptive immune responses in organisms.In some cases, the genes are useful for short-term treatment.In some cases, the genes have fewer safety concerns regarding the dose of expressed genes.

[0150] In various embodiments, the plasmid is used to generate a template for in vitro transcription of mRNA used for transfection.

[0151] Chemical structures capable of promoting stability and / or translation efficiency may also be used. In some embodiments, the RNA has 5' and 3' untranslated regions. In one embodiment, the 5' untranslated region is between zero and 3000 nucleotides in length. The length of the 5' and 3' untranslated region sequences to be added to the coding region may be varied by a variety of methods, including, but not limited to, designing primers for PCR that anneal to different regions of the untranslated region. Using this approach, one skilled in the art can modify the length of the 5' and 3' untranslated regions required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0152] The 5' and 3' untranslated regions may be the naturally occurring endogenous 5' and 3' untranslated regions for the gene of interest. Alternatively, untranslated region sequences that are not endogenous to the gene of interest may be added by incorporating untranslated region sequences into the forward and reverse primers, or by any other template modification. The use of untranslated region sequences that are not endogenous to the gene of interest may be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich sequences in the 3' untranslated region sequence may reduce mRNA stability. Therefore, the 3' untranslated region may be selected or designed to increase the stability of the transcribed RNA based on the properties of untranslated region sequences that are well known in the art.

[0153] In one embodiment, the 5' untranslated region may contain the Kozak sequence of the endogenous gene. Alternatively, if a 5' untranslated region that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence may be redesigned by adding a 5' untranslated region sequence. The Kozak sequence may increase the translation efficiency of some RNA transcripts, but it appears that it is not required for all RNAs to allow efficient translation. The requirement for the Kozak sequence in many mRNAs is known in the art. In another embodiment, the 5' untranslated region may be derived from an RNA virus whose RNA genome is stable in the cell. In another embodiment, various nucleotide analogs may be used in the 3' or 5' untranslated region to prevent the degradation of the mRNA by exonucleases.

[0154] To allow synthesis of RNA from a DNA template, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0155] In one embodiment, the mRNA, which determines ribosome binding, initiation of translation, and stability of the mRNA in the cell, has both a cap on the 5' end and a 3' poly(A) tail. On a circular DNA template, such as plasmid DNA, RNA polymerase produces long products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA that is chained at the 3' untranslated end results in a normal-sized mRNA that is effective for transfection in eukaryotic cells when polyadenylated after transcription.

[0156] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0157] The traditional method of incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which can be restored through the use of recombination-incompetent bacterial cells for amplification of the plasmid.

[0158] The poly(A) tail of the RNA may be further extended after in vitro transcription with the use of a poly(A) polymerase, such as Escherichia coli poly(A) polymerase (E-PAP) or yeast poly(A) polymerase. In one embodiment, extending the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the translation efficiency of the RNA by about 2-fold. Furthermore, attachment of various chemical groups to the 3' end may also increase the stability of the mRNA. Such attachments may include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs may be incorporated into the poly(A) tail using poly(A) polymerase. The ATP analogs may further increase the stability of the RNA.

[0159] The 5' cap also confers stability to the mRNA molecule. In one embodiment, the RNA is produced by a method to include a 5'cap1 structure. Such a cap1 structure can be generated using Vaccinia Capping Enzyme and 2'-O-Methyltransferase enzyme (CellScript, Madison, Wis.). Alternatively, the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0160] RNA can be introduced into target cells using any of a number of different methods, such as commercially available methods including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, Massachusetts), or Gene Pulser II (Bio-Rad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems, such as a "gene gun" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)). In some embodiments, the RNA of the present invention is introduced into cells using methods including the use of the TransIT®-mRNA Transfection Kit (Mirus, Madison, Wis.), which in some cases results in highly efficient, low toxicity, transfection.

[0161] Nucleoside-modified RNA In one embodiment, a composition of the invention comprises a nucleotide-modified nucleic acid encoding a Norovirus antigen as described herein. In one embodiment, a composition of the invention comprises a plurality of nucleoside-modified nucleic acid molecules encoding a plurality of Norovirus antigens as described herein. In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid encoding an adjuvant as described herein. In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid encoding one or more Norovirus antigens and one or more adjuvants.

[0162] For example, in one embodiment, the composition comprises nucleoside-modified RNA. In one embodiment, the composition comprises nucleoside-modified mRNA. Nucleoside-modified mRNA has certain advantages over unmodified mRNA, including, for example, increased stability, low or non-existent natural immunogenicity, and enhanced translation. Nucleoside-modified mRNA that is useful in the present invention is further described in U.S. Patent Nos. 8,278,036, 8,691,966, and 8,835,108, each of which is incorporated herein by reference in its entirety.

[0163] In some embodiments, nucleoside-modified mRNAs do not activate any pathophysiological pathways, translate very efficiently and almost immediately after delivery, and serve as templates for sustained protein production that lasts for days to weeks in vivo (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). Small amounts of mRNA are required to achieve a physiological effect, making them applicable to human therapy. For example, as described herein, nucleoside-modified mRNAs encoding Norovirus antigens have demonstrated the ability to induce antigen-specific antibody production. For example, in some cases, antigens encoded by nucleoside-modified mRNAs induce greater production of antigen-specific antibody production compared to antigens encoded by unmodified mRNAs.

[0164] In some cases, expressing proteins by delivering coding mRNA has many advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only material delivered to the cell, thus avoiding all side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA-based and viral-based vectors, mRNA does not have the risk of being incorporated into the genome, and protein production begins immediately after delivery of the mRNA. For example, high levels of circulating protein are measured within 15 to 30 minutes of in vivo injection of coding mRNA. In some embodiments, using mRNA rather than protein also has many advantages. The half-life of proteins in circulation or tissues is often short, and thus treatment with proteins would require frequent dosing, whereas mRNA provides a template for continuous protein production that can last for days to weeks. Purification of proteins is problematic and they may contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0165] In some embodiments, the nucleoside modified RNA comprises the natural modified nucleoside pseudouridine. In some embodiments, the inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0166] It has been demonstrated that the presence of modified nucleosides, including pseudouridine, in RNA suppresses their natural immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Furthermore, in vitro transcribed RNAs encoding proteins containing pseudouridine can be translated more efficiently than RNAs that contain no modified nucleosides or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, the presence of pseudouridine has been shown to improve RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and reduce both RNA-dependent protein kinase activation and translation inhibition (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892).

[0167] Similar effects as described for pseudouridine have also been observed for RNA containing 1-methyl-1-pseudouridine.

[0168] In some embodiments, the nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule. For example, in some embodiments, the composition is purified to remove double-stranded contaminants. In some cases, a preparative high performance liquid chromatography (HPLC) purification procedure is used to obtain pseudouridine-containing RNA with excellent translational capacity and no natural immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). Administration of HPLC-purified pseudouridine-containing RNA encoding erythropoietin to mice and macaques significantly increases serum erythropoietin levels (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy. In some embodiments, the nucleoside-modified nucleic acid molecule is purified using a method other than HPLC. In some cases, the nucleoside modified nucleic acid molecules are purified using chromatographic methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC). An exemplary FPLC-based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. An exemplary purification procedure is also described in US Patent Publication No. 2016 / 0032316, which is incorporated herein by reference in its entirety.

[0169] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules that comprise pseudouridine or modified nucleosides. In some embodiments, the compositions comprise an isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises a pseudouridine or modified nucleoside. In some embodiments, the compositions comprise a vector comprising an isolated nucleic acid encoding an antigen, an adjuvant, or a combination thereof, wherein the nucleic acid comprises a pseudouridine or modified nucleoside.

[0170] In one embodiment, the nucleoside modified RNA of the present invention is an in vitro transcribed RNA as described elsewhere herein.For example, in some embodiments, the nucleoside modified RNA is synthesized by T7 phage RNA polymerase.In another embodiment, the nucleoside modified mRNA is synthesized by SP6 phage RNA polymerase.In another embodiment, the nucleoside modified RNA is synthesized by T3 phage.In another embodiment, the nucleoside modified RNA is synthesized by T3 phage RNA polymerase.

[0171] In one embodiment, the modified nucleoside is m 1 acp 3 In another embodiment, the modified nucleoside is m 1 In another embodiment, the modified nucleoside is Ψm (2'-O-methylpseudouridine). In another embodiment, the modified nucleoside is m 5 D (5-methyldihydrouridine). In another embodiment, the modified nucleoside is m 3 Ψ (3-methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a pseudouridine monophosphate, diphosphate, or triphosphate of any of the pseudouridine moieties described above. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0172] In another embodiment, the modified nucleoside in the nucleoside-modified RNA of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[0173] In another embodiment, the modified nucleoside of the present invention is 5In another embodiment, the modified nucleoside is m 5 U (5-methyluridine). In another embodiment, the modified nucleoside is 6 A(N 6 In another embodiment, the modified nucleoside is s 2 In another embodiment, the modified nucleoside is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0174] In other embodiments, the modified nucleoside is 1 A(1-methyladenosine);m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N 6 -Methyladenosine);i 6 A(N 6 -Isopentenyladenosine);ms 2 i6A(2-methylthio-N 6 Isopentenyladenosine;io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine);ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine);g 6 A(N 6 -glycinylcarbamoyl adenosine);t 6 A(N 6 -Threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -Threonylcarbamoyl adenosine);m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyl adenosine);hn 6 A(N 6 -Hydroxynorvalylcarbamoyl adenosine);ms 2hn 6 A(2-methylthio-N 6 -Hydroxynorvalylcarbamoyl adenosine; Ar(p)(2'-O-ribosyladenosine (phosphate); I(inosine); m 1 I(1-methylinosine);m 1 Im(1,2'-O-dimethylinosine);m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine);ac 4 C(N 4 -Acetylcytidine);f 5 C(5-formylcytidine);m 5 Cm(5,2'-O-dimethylcytidine);ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lysine);m 1 G(1-methylguanosine);m 2 G(N 2 -Methylguanosine);m 7 G(7-methylguanosine);Gm(2'-O-methylguanosine);m 2 2G(N 2 ,N 2 -Dimethylguanosine);m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine(phosphate));yW(uybutosine);o2yW(peroxyuybutosine);OHyW(hydroxyuybutosine);OHyW * (less modified hydroxyubutosine);imG (ubutosine);mimG (methylubutosine);Q (queuosine);oQ (epoxyqueuosine);galQ (galactosyl-queuosine);manQ (mannosyl-queuosine);preQ0 (7-cyano-7-deazaguanosine);preQ1 (7-aminomethyl-7-deazaguanosine);G + (Archaeosine);D(Dihydrouridine);m 5Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine);m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine);ho 5 U(5-hydroxyuridine); 5 U(5-methoxyuridine); cmo 5 U(uridine 5-oxyacetic acid);mcmo 5 U(uridine 5-oxyacetic acid methyl ester);chm 5 U(5-(carboxyhydroxymethyl)uridine);mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm 5 U(5-Methoxycarbonylmethyluridine);mcm 5 Um(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine); nm 5 s 2 U(5-aminomethyl-2-thiouridine);mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine);mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine);ncm 5 U(5-carbamoylmethyluridine);ncm 5 Um(5-carbamoylmethyl-2'-O-methyluridine); cmnm 5 U(5-carboxymethylaminomethyluridine);cmnm 5 Um(5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine);m 6 2A(N 6 ,N6 -Dimethyladenosine; Im (2'-O-methylinosine); m 4 C(N 4 -Methylcytidine);m 4 Cm(N 4 ,2'-O-dimethylcytidine);hm 5 C(5-hydroxymethylcytidine);m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine);m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine);m 2,7 G(N 2 ,7-Dimethylguanosine);m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 U(5-methyldihydrouridine);f 5 Cm(5-formyl-2'-O-methylcytidine); 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine); τm 5 U(5-taurinomethyluridine); τm 5 s 2 U(5-taurinomethyl-2-thiouridine);imG-14(4-demethyluiocin);imG2(isouiocin);orac 6 A(N 6 -acetyladenosine).

[0175] In another embodiment, the nucleoside modified RNA of the present invention comprises a combination of two or more of the above modifications. In another embodiment, the nucleoside modified RNA comprises a combination of three or more of the above modifications. In another embodiment, the nucleoside modified RNA comprises a combination of more than three of the above modifications.

[0176] In various embodiments, between 0.1% and 100% of the residues in the nucleoside modified RNA of the invention are modified (e.g., by the presence of either pseudouridine, 1-methyl-pseudouridine, 5-methyl-uridine, or another modified nucleoside base). In one embodiment, the percentage of modified residues is 0.1%. In another embodiment, the percentage is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.7%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 0.9%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 7%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 9%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 55%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 65%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 75%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 85%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 91%. In another embodiment, the percentage is 92%. In another embodiment, the percentage is 93%. In another embodiment, the percentage is 94%. In another embodiment, the percentage is 95%.In another embodiment, the percentage is 96%. In another embodiment, the percentage is 97%. In another embodiment, the percentage is 98%. In another embodiment, the percentage is 99%. In another embodiment, the percentage is 100%.

[0177] In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0178] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the percentage of modified residues is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.7%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 0.9%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 7%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 9%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 55%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 65%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 75%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 85%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 91%. In another embodiment, the percentage is 92%. In another embodiment, the percentage is 93%. In another embodiment, the percentage is 94%. In another embodiment, the percentage is 95%. In another embodiment, the percentage is 96%. In another embodiment, the percentage is 97%. In another embodiment, the percentage is 98%. In another embodiment, the percentage is 99%.In another embodiment, the percentage is 100%. In another embodiment, the percentage of a given nucleotide that is modified is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0179] In some embodiments, the composition comprises a purified preparation of single-stranded nucleoside-modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside-modified RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0180] In another embodiment, the nucleoside modified RNA of the invention is translated in cells more efficiently than an unmodified RNA molecule having the same sequence. In another embodiment, the nucleoside modified RNA exhibits an enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced 2-fold compared to its unmodified counterpart. In another embodiment, translation is enhanced 3-fold. In another embodiment, translation is enhanced 4-fold. In another embodiment, translation is enhanced 5-fold. In another embodiment, translation is enhanced 6-fold. In another embodiment, translation is enhanced 7-fold. In another embodiment, translation is enhanced 8-fold. In another embodiment, translation is enhanced 9-fold. In another embodiment, translation is enhanced 10-fold. In another embodiment, translation is enhanced 15-fold. In another embodiment, translation is enhanced 20-fold. In another embodiment, translation is enhanced 50-fold. In another embodiment, translation is enhanced 100-fold. In another embodiment, translation is enhanced 200-fold. In another embodiment, translation is enhanced 500-fold. In another embodiment, translation is enhanced 1000-fold. In another embodiment, translation is enhanced 2000-fold. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000-fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0181] In another embodiment, RNA encoding the nucleoside modified antigens of the invention induces a significantly more robust adaptive immune response compared to unmodified in vitro synthesized RNA molecules having the same sequence. In another embodiment, the modified RNA molecule induces an adaptive immune response that is 2-fold higher than its unmodified counterpart. In another embodiment, the adaptive immune response is increased 3-fold. In another embodiment, the adaptive immune response is increased 4-fold. In another embodiment, the adaptive immune response is increased 5-fold. In another embodiment, the adaptive immune response is increased 6-fold. In another embodiment, the adaptive immune response is increased 7-fold. In another embodiment, the adaptive immune response is increased 8-fold. In another embodiment, the adaptive immune response is increased 9-fold. In another embodiment, the adaptive immune response is increased 10-fold. In another embodiment, the adaptive immune response is increased 15-fold. In another embodiment, the adaptive immune response is increased 20-fold. In another embodiment, the adaptive immune response is increased 50-fold. In another embodiment, the adaptive immune response is increased 100-fold. In another embodiment, the adaptive immune response is increased by 200 fold. In another embodiment, the adaptive immune response is increased by 500 fold. In another embodiment, the adaptive immune response is increased by 1000 fold. In another embodiment, the adaptive immune response is increased by 2000 fold. In another embodiment, the adaptive immune response is increased by another fold difference.

[0182] In another embodiment, "inducing a more robust adaptive immune response" refers to a detectable increase in the adaptive immune response. In another embodiment, the term refers to a fold increase in the adaptive immune response (e.g., the fold increase listed above is 1). In another embodiment, the term refers to an increase such that the nucleoside modified RNA can be administered in a lower dose or less frequently than an unmodified RNA molecule while still inducing a similarly effective adaptive immune response. In another embodiment, the increase is such that the nucleoside modified RNA can be administered using a single dose to induce an effective adaptive immune response.

[0183] In another embodiment, the nucleoside modified RNA of the present invention exhibits significantly lower natural immunogenicity than unmodified in vitro synthesized RNA molecules having the same sequence. In another embodiment, the modified RNA molecule exhibits a 2-fold lower natural immune response than its unmodified counterpart. In another embodiment, the natural immunogenicity is reduced 3-fold. In another embodiment, the natural immunogenicity is reduced 4-fold. In another embodiment, the natural immunogenicity is reduced 5-fold. In another embodiment, the natural immunogenicity is reduced 6-fold. In another embodiment, the natural immunogenicity is reduced 7-fold. In another embodiment, the natural immunogenicity is reduced 8-fold. In another embodiment, the natural immunogenicity is reduced 9-fold. In another embodiment, the natural immunogenicity is reduced 10-fold. In another embodiment, the natural immunogenicity is reduced 15-fold. In another embodiment, the natural immunogenicity is reduced 20-fold. In another embodiment, the natural immunogenicity is reduced 50-fold. In another embodiment, the natural immunogenicity is reduced 100-fold. In another embodiment, the natural immunogenicity is reduced 200-fold. In another embodiment, the natural immunogenicity is reduced by 500 fold. In another embodiment, the natural immunogenicity is reduced by 1000 fold. In another embodiment, the natural immunogenicity is reduced by 2000 fold. In another embodiment, the natural immunogenicity is reduced by another fold difference.

[0184] In another embodiment, "exhibiting significantly lower natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In another embodiment, the term refers to a fold reduction in natural immunogenicity (e.g., the fold reduction listed above is 1). In another embodiment, the term refers to a reduction such that an effective amount of nucleoside modified RNA can be administered without triggering a detectable natural immune response. In another embodiment, the term refers to a reduction such that nucleoside modified RNA can be administered repeatedly without eliciting a sufficient natural immune response to detectably reduce the production of the protein encoded by the modified RNA. In another embodiment, the reduction is such that nucleoside modified RNA can be administered repeatedly without eliciting a sufficient natural immune response to eliminate the detectable production of the protein encoded by the modified RNA.

[0185] Lipid Nanoparticles In one embodiment, the delivery of nucleoside-modified RNA comprises any suitable delivery method, including the exemplary RNA transfection method described elsewhere herein. In some embodiments, the delivery of nucleoside-modified RNA to a subject comprises mixing the nucleoside-modified RNA with a transfection reagent before the contacting step. In another embodiment, the method of the present invention further comprises administering the nucleoside-modified RNA together with a transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent. In another embodiment, the transfection reagent is a cationic polymer reagent.

[0186] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a carbohydrate-based transfection reagent. In another embodiment, the transfection reagent is a cationic lipid-based transfection reagent. In another embodiment, the transfection reagent is a cationic polymer-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0187] In another embodiment, the transfection reagent forms a liposome. In another embodiment, the liposome enhances intracellular stability, enhances uptake efficiency, and improves biological activity. In another embodiment, the liposome is a hollow, spherical vesicle composed of lipids arranged in a manner similar to that of the lipids that compose the cell membrane. They have an internal aqueous space for encapsulating water-soluble compounds, and the size of the diameter ranges from 0.05 to several microns. In another embodiment, the liposome can deliver RNA to cells in a biologically active form.

[0188] In one embodiment, the composition comprises a lipid nanoparticle (LNP) and one or more nucleic acid molecules described herein. For example, in one embodiment, the composition comprises a LNP and one or more nucleoside-modified RNA molecules encoding one or more antigens, adjuvants, or a combination thereof.

[0189] In some embodiments, lipid nanoparticles are particles having at least one dimension in the nanometer dimension (e.g., 1-1,000 nm). In some embodiments, lipid nanoparticles comprise one or more lipids. For example, in some embodiments, the lipid comprises a lipid of formula (I), (II), or (III).

[0190] In some embodiments, lipid nanoparticles are included in the formulations that contain nucleoside-modified RNA as described herein. In some embodiments, such lipid nanoparticles contain cationic lipids (e.g., lipids of formula (I), (II) or (III)) and one or more excipients selected from neutral lipids, charged lipids, steroids, and lipids conjugated to polymers (e.g., PEGylated lipids, e.g., PEGylated lipids of structure (IV)). In some embodiments, nucleoside-modified RNA is encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space that is enveloped by some or all of the liquid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism or cellular mechanisms, such as harmful immune responses.

[0191] In various embodiments, the lipid nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or have an average diameter of about 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. In some embodiments, the nucleoside modified RNA, when present in the lipid nanoparticle, is resistant in aqueous solution to degradation by nucleases.

[0192] LNPs can include any lipid capable of forming a particle to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are encapsulated.

[0193] In one embodiment, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.

[0194] In one embodiment, the LNP comprises a cationic lipid. In some embodiments, the cationic lipid comprises any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1 ... Examples of cationic lipids that may be used include, but are not limited to, N-(1,2-dimyristyloxypropyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercial preparations of cationic lipids are available that may be used in the present invention. These include, for example, Lipofectin® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), Gibco / BRL, Grand Island, NY); Lipofectamine® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), Gibco / BRL); Transfectam® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, Promega, Madison, WI).The following lipids are cationic and have a positive charge at sub-physiological pH; DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0195] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the present invention include those described in WO 2012 / 016184, which is incorporated herein by reference in its entirety. Representative amino lipids include 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl -3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0196] Suitable amino lipids have the following formula: [ka] (In the formula, R1 and R2 are either the same or different and independently represent optionally substituted C 10 ~C 24 Alkyl, optionally substituted C 10 ~C 24 Alkenyl, optionally substituted C 10 ~C 24 Alkynyl or optionally substituted C 10 ~C 24 is acyl; R3 and R4 are either the same or different and independently an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, or R3 and R4 join together to form an optionally substituted heterocycle having 4-6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R5 is either absent or present, and when present, is hydrogen or C1-C6 alkyl; m, n and p are either the same or different and independently either 0 or 1, with the proviso that m, n and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and are independently O, S, or NH. In one embodiment, R1 and R2 are each linoleyl and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0197] Representative useful dilinoleyl amino lipids have the following formula: [ka] (wherein n is 0, 1, 2, 3 or 4). has.

[0198] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA as above, where n is 2).

[0199] In one embodiment, the cationic lipid component of the LNP has the following formula (I): [ka] (In the formula, L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a carbon-carbon double bond; R 1a and R 1b is, independently at each location, (a) H or C1-C 12 alkyl, or (b) R 1a is H or C1~C 12 is alkyl, R 1b together with the carbon atom to which it is attached, the adjacent R 1b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 2a and R 2b is, independently at each position, (a) H or C1 to C 12 alkyl, or (b) R 2a is H or C1~C 12 is alkyl, R 2b together with the carbon atom to which it is attached, the adjacent R 2b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 3a and R 3b is, independently at each position, (a) H or C1 to C 12 alkyl, or (b) R 3a is H or C1~C 12 is alkyl, R 3b together with the carbon atom to which it is attached, the adjacent R 3band together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 4a and R 4b is, independently at each position, (a) H or C1 to C 12 alkyl, or (b) R 4a is H or C1~C 12 is alkyl, R 4b together with the carbon atom to which it is attached, the adjacent R 4b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is independently H or C1 to C 12 is alkyl; R 8 and R 9 are each independently C1 to C 12 alkyl; or R 8 and R 9 forms together with the nitrogen atom to which it is attached a 5-, 6-, or 7-membered heterocycle containing one nitrogen atom; a and d each independently represent an integer of 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2) or a pharma- ceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0200] In some embodiments of Formula (I), R 1a , R 2a , R 3a Or R 4a At least one of C1~C 12 alkyl or L 1 Or L 2 At least one of R is -O(C=O)- or -(C=O)O-. 1a and R 1bis not isopropyl when a is 6 and is not n-butyl when a is 8.

[0201] In yet a further embodiment of formula (I), R 1a , R 2a , R 3a Or R 4a At least one of C1~C 12 alkyl or L 1 Or L 2 at least one of is -O(C=O)- or -(C=O)O-; and R 1a and R 1b is not isopropyl when a is 6 and is not n-butyl when a is 8.

[0202] In another embodiment of formula (I), R 8 and R 9 are each independently an unsubstituted C1 to C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle containing one nitrogen atom.

[0203] In some embodiments of Formula (I), L 1 Or L 2 Either one of L can be -O(C=O)- or a carbon-carbon double bond. 1 and L 2 may each be -O(C=O)- or each be a carbon-carbon double bond.

[0204] In some embodiments of Formula (I), L 1 Or L 2 In another embodiment, L 1 and L 2 Both of these are -O(C=O)-.

[0205] In some embodiments of Formula (I), L 1 Or L 2In another embodiment, L 1 and L 2 Both of these are -(C=O)O-.

[0206] In some other embodiments of Formula (I), L 1 Or L 2 In another embodiment, L 1 and L 2 Both of these are carbon-carbon double bonds.

[0207] In yet another embodiment of Formula (I), L 1 Or L 2 One of the groups is -O(C=O)-, and L 1 Or L 2 and the other is -(C=O)O-. 1 Or L 2 One of the groups is -O(C=O)-, and L 1 Or L 2 The other of L is a carbon-carbon double bond. 1 Or L 2 One of the groups is -(C=O)O-, and L 1 Or L 2 The other is a carbon-carbon double bond.

[0208] As used throughout this specification, a "carbon-carbon" double bond has the following structure: [ka] (In the formula, R a and R b is, at each occurrence, independently H or a substituent. For example, in some embodiments, R a and R b are independently H, C1 to C 12 alkyl or cycloalkyl, for example H or C1-C 12 It is an alkyl.

[0209] In another embodiment, the lipid compound of formula (I) has the following structure (Ia): [ka] has.

[0210] In another embodiment, the lipid compound of formula (I) has the following structure (Ib): [ka] has.

[0211] In yet another embodiment, the lipid compound of formula (I) has the following structure (Ic): [ka] has.

[0212] In some embodiments of the lipid compound of formula (I), a, b, c, and d are each independently In some embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0213] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0214] In some further embodiments of Formula (I), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0215] In some other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0216] In some other various embodiments of formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0217] The sum of a and b, and the sum of c and d in formula (I) are coefficients that may be modified to obtain lipids of formula (I) having desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected such that their sum is an integer in the range of 14 to 24. In further embodiments, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer, which may be in the range of 14 to 24. In yet further embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or more.

[0218] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0219] R in formula (I) 1a , R 2a , R 3a and R 4a The substituents in are not particularly limited. In some embodiments, R 1a , R 2a , R 3a and R 4a is H at each occurrence. In some other embodiments, R 1a , R 2a , R 3a and R 4a At least one of C1~C 12 In some other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C1-C8 alkyl. 1a , R 2a , R3a and R 4a At least one of is C1-C6 alkyl. In some of the above embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0220] In some embodiments of Formula (I), R 1a , R 1b , R 4a and R 4b At each location, C1~C 12 It is an alkyl.

[0221] In a further embodiment of formula (I), R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H at each location.

[0222] In some embodiments of Formula (I), R 1b is attached to the carbon atom adjacent to 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond. 4b is attached to the carbon atom adjacent to 4b and together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0223] R in formula (I) 5 and R 6 The substituents in R are not particularly limited in the above embodiments. 5 or R 6 In some other embodiments, one or both of R 5 or R 6One or both of is cycloalkyl, such as cyclohexyl. In these embodiments, the cycloalkyl can be substituted or unsubstituted. In some other embodiments, the cycloalkyl is a C1-C 12 It is substituted with alkyl, for example tert-butyl.

[0224] R 7 The substituents in are not particularly limited in the above embodiments of formula (I). In some embodiments, at least one R 7 is H. In some embodiments, R 7 is H at each occurrence. In some other embodiments, R 7 is C1~C 12 It is an alkyl.

[0225] In some other such embodiments of formula (I), R 8 or R 9 In another embodiment, R 8 and R 9 Both are methyl.

[0226] In some different embodiments of Formula (I), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle. 8 and R 9 together with the nitrogen atom to which they are attached form a five-membered heterocycle, such as a pyrrolidinyl ring.

[0227] In various different embodiments, the lipid of formula (I) has one of the structures shown in Table 1 below.

[0228] [Table 1] TIFF2024540061000010.tif216169 TIFF2024540061000011.tif216169 TIFF2024540061000012.tif216169 TIFF2024540061000013.tif205169 TIFF2024540061000014.tif216169 TIFF2024540061000015.tif200169

[0229] In some embodiments, the LNP comprises a lipid of formula (I), a nucleoside-modified RNA, and one or more excipients selected from a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (I) is compound I-5. In some embodiments, the lipid of formula (I) is compound I-6.

[0230] In some other embodiments, the cationic lipid component of the LNP has formula (II): [ka] (In the formula, L 1 and L 2 each independently represents -O(C=O)-, -(C=O)O-, -C(=O)-, -O―, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O- or a direct bond; G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond; G 2 are -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a or a direct bond; G 3 is C1-C6 alkylene; R a is H or C1-C 12 is alkyl; R 1a and R 1b is, independently at each location, (a) H or C1-C 12 or (b) R 1a is H or C1~C 12 is alkyl, R 1b is attached to the carbon atom adjacent to 1b and together with the carbon atom to which they are attached form a carbon-carbon double bond; R 2a and R 2b is, independently at each location, (a) H or C1-C 12 or (b) R 2a is H or C1~C 12 is alkyl, R 2b is attached to the carbon atom adjacent to 2b and together with the carbon atom to which they are attached form a carbon-carbon double bond; R 3a and R 3b is, independently at each location, (a) H or C1-C 12 or (b) R 3a is H or C1~C 12 is alkyl, R 3b is attached to the carbon atom adjacent to 3b and together with the carbon atom to which they are attached form a carbon-carbon double bond; R 4a and R 4b is, independently at each location, (a) H or C1-C 12 or (b) R 4a is H or C1~C 12 is alkyl, R 4b is attached to the carbon atom adjacent to 4b and together with the carbon atom to which it is attached forms a carbon-carbon double bond; R5 and R 6 are each independently H or methyl; R 7 is C4~C 20 is alkyl; R 8 and R 9 are each independently C1 to C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocycle; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0231] In some embodiments of Formula (II), L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond. 1 and G 2 are each independently -(C=O)- or a direct bond. 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond; G 1 and G 2 are each independently -(C=O)- or a direct bond.

[0232] In some different embodiments of Formula (II), L 1 and L 2 each independently represents -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)-, or -NR a -, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, NR a C(=O)O-, -NRa S(O) x NR a -, -NR a S(O) x - or -S(O) x NR a -It is.

[0233] In other embodiments of formula (II), the lipid compound has the following structure (IIA) or (IIB): [ka] It has one of the following.

[0234] In some embodiments of formula (II), the lipid compound has the structure (IIA). In other embodiments, the lipid compound has the structure (IIB).

[0235] In any of the above embodiments of Formula (II), L 1 Or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is -O(C=O)-.

[0236] In some different embodiments of Formula (II), L 1 Or L 2 One of L is -(C=O)O-. For example, in some embodiments, L 1 and L 2 Each of is -(C=O)O-.

[0237] In a different embodiment of formula (II), L 1 Or L 2 As used herein, a "direct bond" refers to a bond that is bonded to a group (e.g., L 1 Or L 2 For example, in some embodiments, L 1 and L 2 Each of is a direct bond.

[0238] In another alternative embodiment of formula (II), R 1a and R 1b For at least one occurrence of R 1a is H or C1~C 12 is alkyl, R 1b is attached to the carbon atom adjacent to 1b and together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0239] In yet another alternative embodiment of formula (II), R 4a and R 4b For at least one occurrence of R 4a is H or C1-C 12 is alkyl, R 4b is attached to the carbon atom adjacent to 4b and together with the carbon atom to which they are attached form a carbon-carbon double bond.

[0240] In a further embodiment of formula (II), R 2a and R 2b For at least one occurrence of R 2a is H or C1-C 12 is alkyl, R 2b is attached to the carbon atom adjacent to 2b and together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0241] In another alternative embodiment of formula (II), R 3a and R 3b For at least one occurrence of R 3a is H or C1-C 12 is alkyl, R 3b is attached to the carbon atom adjacent to 3b and together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0242] In various other embodiments of formula (II), the lipid compound has the following structure (IIC) or (IID): [ka] (In the formula, e, f, g, and h each independently represent an integer of 1 to 12. It has one of the following.

[0243] In some embodiments of formula (II), the lipid compound has the structure (IIC). In other embodiments, the lipid compound has the structure (IID).

[0244] In various embodiments of structure (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0245] In some embodiments of Formula (II), a, b, c, and d are each independently an integer from 2 to 12, or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12, or an integer from 5 to 9. In some embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In still other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In still other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In still other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0246] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In still other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In still other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In still other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In still other embodiments, b is 16.

[0247] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In still other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In still other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In still other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In still other embodiments, c is 16.

[0248] In some embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In still other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In still other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In still other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In still other embodiments, d is 16.

[0249] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In still other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In further embodiments, e is 7. In still other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In further embodiments, e is 11. In still other embodiments, e is 12.

[0250] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In still other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In still other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In still other embodiments, f is 12.

[0251] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In still other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In still other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In still other embodiments, g is 12.

[0252] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In still other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In still other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In still other embodiments, h is 12.

[0253] In some other various embodiments of formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0254] The sum of a and b, and the sum of c and d in formula (II) are coefficients that may be modified to obtain lipids having desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected such that their sum is an integer in the range of 14 to 24. In further embodiments, the sum of a and b, and the sum of c and d are the same. For example, in some embodiments, the sum of a and b, and the sum of c and d are both the same integer, which may be in the range of 14 to 24. In yet further embodiments, a, b, c, and d are selected such that the sum of a and b, and the sum of c and d are 12 or more.

[0255] R in formula (II) 1a , R 2a , R 3a and R 4a The substituents in are not particularly limited. In some embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is H. In some embodiments, R 1a , R 2a , R 3a and R 4ais H at each occurrence. In some embodiments, R 1a , R 2a , R 3a and R 4a At least one of C1~C 12 In some other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C1-C8 alkyl. 1a , R 2a , R 3a and R 4a At least one of is C1-C6 alkyl. In some of the above embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0256] In some embodiments of Formula (II), R 1a , R 1b , R 4a and R 4b At each location, C1~C 12 It is an alkyl.

[0257] In a further embodiment of formula (II), R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H at each location.

[0258] In some embodiments of Formula (II), R 1b is attached to the carbon atom adjacent to 1b and together with the carbon atom to which it is attached form a carbon-carbon double bond. 4b is attached to the carbon atom adjacent to 4band together with the carbon atom to which it is attached forms a carbon-carbon double bond.

[0259] R in formula (II) 5 and R 6 The substituents in R are not particularly limited in the above embodiments. 5 or R 6 In another embodiment, R 5 or R 6 One of the groups is methyl.

[0260] R in formula (II) 7 The substituents in R are not particularly limited in the above embodiments. 7 is C6~C 16 In some other embodiments, R 7 is C6-C9 alkyl. In some of these embodiments, R 7 is -(C=O)OR b , -O(C=O)R b , -C(=O)R b , -OR b , -S(O) x R b , -S-SR b , -C(=O)SR b , -SC(=O)R b , -NR a R b , -NR a C(=O)R b , -C(=O)NR a R b , -NR a C(=O)NR a R b , -OC(=O)NR a R b , -NR a C(=O)OR b , -NR a S(O) x R a R b , -NR a S(O) x R b , or -S(O) x Ra R b where R a is H or C1-C 12 R is alkyl; b is C1~C 15 alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 is -C(=O)OR b or -O(C=O)R b is replaced with

[0261] In various of the above embodiments of formula (II), R b is the branch C1~C 15 For example, in some embodiments, R b has the following structure: [ka] It has one of the following.

[0262] In some other embodiments of formula (II), R 8 or R 9 In another embodiment, R 8 and R 9 Both are methyl.

[0263] In some different embodiments of Formula (II), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle. 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocycle, e.g., a pyrrolidinyl ring. In some different embodiments of the above, R 8 and R 9 together with the nitrogen atom to which they are attached form a six-membered heterocycle, such as a piperazinyl ring.

[0264] In yet another embodiment of the lipid of formula (II), 3is C2 to C4 alkylene, for example, C3 alkylene.

[0265] In various different embodiments, the lipid compound has one of the structures shown in Table 2 below.

[0266] [Table 2] TIFF2024540061000021.tif230169 TIFF2024540061000022.tif204169 TIFF2024540061000023.tif225169 TIFF2024540061000024.tif230169 TIFF2024540061000025.tif216169 TIFF2024540061000026.tif61169

[0267] In some embodiments, the LNP comprises a lipid of formula (II), a nucleoside-modified RNA, and one or more excipients selected from a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (II) is compound II-9. In some embodiments, the lipid of formula (II) is compound II-10. In some embodiments, the lipid of formula (II) is compound II-11. In some embodiments, the lipid of formula (II) is compound II-12. In some embodiments, the lipid of formula (II) is compound II-32.

[0268] In some other embodiments, the cationic lipid component of the LNP has the following formula (III): (III) (In the formula, L 1 Or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR aC(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- and L 1 Or L 2 The other is -O(=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a - or -NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C1 to C 12 Alkylene or C1-C 12 alkenylene; G 3 is C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl; R 1 and R 2 are each independently C6 to C 24 Alkyl or C6-C 24 alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and; R 4 is C1~C 12 is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1 or 2. or a pharma- ceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0269] In some of the above embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] (In the formula, A is a 3- to 8-membered cycloalkyl ring or cycloalkylene ring; R 6 is, at each occurrence, independently H, OH, or C1-C 24 is alkyl; n is an integer ranging from 1 to 15. It has one of the following.

[0270] In some of the above embodiments of formula (III), the lipid has the structure (IIIA), and in other embodiments, the lipid has the structure (IIIB).

[0271] In another embodiment of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] (In the formula, Each of y and z is independently an integer ranging from 1 to 12. It has one of the following.

[0272] In any of the above embodiments of Formula (III), L 1 Or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of L is -O(C=O)-. In some different embodiments of any of the above, L 1 and L 2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is -(C=O)O-.

[0273] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] It has one of the following.

[0274] In some of the above embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII), or (IIIJ): [ka] It has one of the following.

[0275] In some of the above embodiments of Formula (III), n is an integer ranging from 2 to 12, such as from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0276] In some other embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0277] In some of the above embodiments of Formula (III), R 6 is H. In other such embodiments, R 6 is C1~C 24 In another embodiment, R 6 is OH.

[0278] In some embodiments of Formula (III), G 3 is unsubstituted. In other embodiments, G is substituted. In various different embodiments, G 3 is a chain C1~C 24 Alkylene or chain C1-C 24 It is alkenylene.

[0279] In some other embodiments of formula (III), R 1 Or R 2 Or both are C6~C 24 For example, in some embodiments, R 1 and R 2 each independently have the structure: [ka] (In the formula, R 7a and R 7b is, at each occurrence, independently, H or C1-C 12 is alkyl; a is an integer from 2 to 12; R 7a , R 7b and a are each R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0280] In some of the above embodiments of Formula (III), R 7a At least one occurrence of R is H. For example, in some embodiments, 7a is H at each occurrence. In another alternative embodiment of the above, R 7b At least one occurrence of is C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0281] In a different embodiment of formula (III), R 1 Or R 2 or both have the following structure: [ka] It has one of the following.

[0282] In some of the above embodiments of Formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0283] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in Table 3 below.

[0284] [Table 3] TIFF2024540061000034.tif234169 TIFF2024540061000035.tif225169 TIFF2024540061000036.tif222169 TIFF2024540061000037.tif230169

[0285] In some embodiments, the LNP comprises a lipid of formula (III), a nucleoside-modified RNA, and one or more excipients selected from a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the lipid of formula (III) is compound III-7.

[0286] In some embodiments, the cationic lipid is present in the LNP in an amount of about 30 to about 95 mol %. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 mol %. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 to about 60 mol %. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mol %. In one embodiment, the LNP comprises only cationic lipid.

[0287] In some embodiments, the LNPs contain one or more additional lipids that stabilize the formation of the particles during their formation.

[0288] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0289] Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0290] Exemplary neutral lipids include, for example, diesteroylphosphatidylcholine (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), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0291] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipid (e.g., a lipid of formula (I)) to neutral lipid ranges from about 2:1 to 8:1.

[0292] In various embodiments, the LNP further comprises a steroid or a steroid analog. A "steroid" is a compound having the following carbon skeleton: [ka] It is a compound comprising:

[0293] In some embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., a lipid of Formula (I)) to cholesterol ranges from about 2:1 to 1:1.

[0294] In some embodiments, the LNPs comprise a glycolipid (eg, monosialoganglioside GM1). In some embodiments, the LNPs comprise a sterol, such as cholesterol.

[0295] In some embodiments, the LNP comprises a lipid conjugated to a polymer.

[0296] In some embodiments, the LNP comprises an additional stabilizing lipid that is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. Exemplary polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000)carbamyl]-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In another embodiment, the LNP is a PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butane. diate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0297] In some embodiments, the LNP has the following structure (IV): [ka] (In the formula, R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has an average value in the range of 30 to 60. or a pharma- ceutically acceptable salt, tautomer or stereoisomer thereof.

[0298] In some of the above embodiments of the PEGylated lipid (IV), R 10 and R 11When z is 42, neither of R is n-octadecyl. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. 10 is a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms; R 11 is a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0299] In various embodiments, z is selected so that the PEG moiety of (II) has an average molecular weight of about 400 to about 6000 g / mol. In some embodiments, the average value of z is about 45.

[0300] In other embodiments, the PEGylated lipid has the following structure: [ka] (In the formula, n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2500 g / mol. It has one of the following.

[0301] In some embodiments, the additional lipid is present in the LNP in an amount of about 1 to about 10 mol %. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 to about 5 mol %. In one embodiment, the additional lipid is present in the LNP at about 1 mol % or about 1.5 mol %.

[0302] In some embodiments, the LNP comprises a lipid of formula (I), a nucleoside-modified RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (I) is compound I-6. In different embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In yet another embodiment, the PEGylated lipid is compound IVa.

[0303] In some embodiments, the LNPs comprise one or more targeting moieties, which can target the LNPs to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand, which directs the LNPs to a receptor found on the surface of a cell.

[0304] In some embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains that bind to cells and induce internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to receptors found on the cell surface and induce receptor-mediated uptake of the LNP. In some embodiments, the LNP can bind to biomolecules in vivo, where the biomolecules bound to the LNP can then be recognized by cell surface receptors to induce internalization. For example, in one embodiment, the LNP binds to systemic apolipoprotein E, which causes uptake of the LNP and associated cargo.

[0305] Other exemplary LNPs and their preparation are described in the art, e.g., in U.S. Patent Publication No. 20120276209; Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37; Jayaraman et al. al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which is incorporated by reference in its entirety.

[0306] The following reaction schemes show how to make lipids of formula (I), (II) or (III). [ka]

[0307]

[0033] Embodiments of lipids of formula (I) (e.g., compound A-5) can be prepared according to general reaction scheme I ("Method A"), where R is a saturated or unsaturated C1-C 24alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. Referring to general reaction scheme 1, compounds of structure A-1 may be purchased from commercial sources or prepared according to methods well known to those skilled in the art. Treatment of a mixture of A-1, A-2, and DMAP with DCC provides bromide A-3. Heating a mixture of bromide A-3 with a base (e.g., N,N-diisopropylethylamine) and N,N-dimethyldiamine A-4 at a sufficient temperature and time produces A-5 after any necessary work-up or purification steps.

[0308] [ka]

[0309] Other embodiments of compounds of formula (I) (e.g., compound B-5) can be prepared according to General Reaction Scheme 2 ("Method B"), where R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. Compounds of structure B-1 may be purchased from commercial sources or prepared according to methods well known to those skilled in the art, as shown in General Reaction Scheme 2. A solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinium chlorochromate) to recover intermediate product B-3. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to provide B-5 after any necessary workup and / or purification.

[0310] It should be noted that although starting materials A-1 and B-1 are shown above as containing only saturated methylene carbons, starting materials containing carbon-carbon double bonds can also be used to prepare compounds containing carbon-carbon double bonds.

[0311] [ka]

[0312] Various embodiments of lipids of formula (I) (e.g., C-7 or C9 compounds) can be prepared according to General Reaction Scheme 3 ("Method C"), where R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-1 may be purchased from commercial sources or prepared according to methods well known to those skilled in the art.

[0313] [ka]

[0314] Embodiments of compounds of formula (II) (e.g., compounds D-5 and D-7) can be prepared according to general reaction scheme 4 ("Method D"), where R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 , a, b, c and d are as defined herein; R 7’ is R 7 Or C3~C 19cycloalkyl. Referring to general reaction scheme 1, compounds of structure D-1 and D-2 may be purchased from commercial sources or prepared according to methods well known to those skilled in the art. Treatment of a solution of D-1 and D-2 with a reducing agent (e.g., sodium triacetoxyborohydride) gives D-3 after any necessary workup. Treatment of a solution of D-3 and a base (e.g., trimethylamine, DMAP) with an acyl chloride D-4 (or a carboxylic acid and DCC) gives D-5 after any necessary workup and / or purification. Reduction of D-5 with LiAlH4 of D-6 gives D-7 after any necessary workup and / or purification.

[0315] [ka]

[0316] Embodiments of lipids of formula (II) (e.g., compounds E-5) can be prepared according to General Reaction Scheme 5 ("Method E"), where R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 8 , R 9 , L 1 , L 2 , G 3 , a, b, c and d are as defined herein. Referring to General Reaction Scheme 2, compounds of structure E-1 and E-2 may be purchased from commercial sources or prepared according to methods well known to those skilled in the art. Heating a mixture of E-1 (in excess), E-2 and a base (e.g. potassium carbonate) gives E-3 after any necessary work-up. Treating a solution of E-3 and a base (e.g. trimethylamine, DMAP) with the acid chloride of E-4 (or a carboxylic acid and DCC) gives E-5 after any necessary work-up and / or purification.

[0317] [ka]

[0318] General Reaction Scheme 6 provides an exemplary method (Method F) for the preparation of lipids of formula (III). 1 , G 3 , R 1 and R 3 is as defined herein for formula (III), and G1' refers to a homologue that is one carbon shorter than G1. Compounds of the structure F-1 are purchased or prepared according to methods known in the art. Reaction of F-1 with a diol of F-2 under suitable condensation conditions (e.g. DCC) gives an ester / alcohol of F3, which can then be oxidized (e.g. PCC) to an aldehyde of F-4. Reaction of F-4 with an amine F-5 under reductive amination conditions gives lipids of formula (III).

[0319] It should be noted that various alternative strategies for the preparation of lipids of formula (III) are available to those skilled in the art. For example, other lipids of formula (III) (herein L 1 and L 2 In addition, general reaction scheme 6 shows that compounds of formula (III) (wherein G is other than an ester) can be prepared according to a similar method using appropriate starting materials. 1 and G 2 is the same as G; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible, which would result in the preparation of a lipid of 1 and G 2 Compounds with different

[0320] It will be appreciated by those skilled in the art that in the processes described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. Those skilled in the art will appreciate that the protecting group may be a polymeric resin, such as a Wang resin, a Rink resin, or a 2-chlorotrityl-chloride resin.

[0321] Pharmaceutical Compositions The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the field of pharmacology. In general, such preparation methods include the step of combining the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into the desired single- or multi-dose unit.

[0322] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to all types of subjects. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various subjects are well understood, and veterinary pharmacologists of ordinary skill can design and implement such modifications with no more than routine experimentation, if at all. Subjects to which the pharmaceutical compositions of the present invention may be administered include, but are not limited to, humans and other primates, mammals, including commercially relevant mammals, such as non-human primates, cows, pigs, horses, sheep, cats, and dogs.

[0323] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in a formulation suitable for intraocular, buccal, rectal, vaginal, parenteral, topical, pulmonary, nasal, buccal, intravenous, intraventricular, intradermal, intramuscular, or another route of administration. Other possible formulations include ejected nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunogenicity-based formulations.

[0324] The pharmaceutical composition of the present invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equivalent to the dose of active ingredient that would be administered to a subject, or a convenient fraction of such a dose, such as half or one third of such a dose.

[0325] The relative amounts of active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, and further depending on the route by which the composition is intended to be administered. For example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

[0326] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharma- ceutical active substances.

[0327] Sustained or extended release formulations of the pharmaceutical compositions of the invention may be made using conventional techniques.

[0328] As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically breaking the tissue of a subject, and administration of the pharmaceutical composition through a crack in the tissue. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is considered to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and renal dialysis infusion techniques.

[0329] A formulation of a pharmaceutical composition suitable for parenteral administration comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release formulations or biodegradable formulations. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0330] The pharmaceutical composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients described herein, such as dispersing agents, wetting agents, or suspending agents. Such sterile injectable preparations may be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include formulations containing the active ingredient in microcrystalline form, in liposomal preparations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0331] The pharmaceutical compositions of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising the active ingredient and having a diameter in the range of about 0.5 to about 7 nm. In some embodiments, the formulations may comprise dry particles comprising the active ingredient and having a diameter in the range of about 1 to about 6 nm. Such compositions are conveniently in a dry powder form for administration using a device comprising a dry powder reservoir capable of directing a propellant stream to disperse the powder, or using an automatic propellant / powder dispenser, such as a device comprising the active ingredient dissolved or suspended in a low boiling propellant in a sealed container. In some embodiments, such powders comprise particles, wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm, and at least 95% of the particles by number have a diameter less than 7 nm. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nm, and at least 90% of the particles by number have a diameter less than 6 nm. In some embodiments, dry powder compositions include a solid fine powder diluent, such as sugar, which is conveniently provided in a unit dosage form.

[0332] Low boiling propellants generally include liquid propellants having a boiling point below 65° F. at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant, or a solid diluent (optionally having a particle size of the same order of magnitude as the particles containing the active ingredient).

[0333] A formulation of a pharmaceutical composition suitable for parenteral administration comprises the active ingredient combined with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0334] The pharmaceutical composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients described herein, such as dispersing agents, wetting agents, or suspending agents. Such sterile injectable preparations may be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include formulations containing the active ingredient in microcrystalline form, in liposomal preparations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0335] Treatment or prevention method The present invention provides a method for inducing an adaptive immune response against Norovirus in a subject, comprising administering an effective amount of at least one mRNA molecule (e.g., a nucleoside-modified mRNA molecule) encoding a Norovirus VP1 antigen, or a composition (e.g., an LNP) comprising at least one mRNA molecule (e.g., a nucleoside-modified mRNA molecule) encoding a Norovirus VP1 antigen.

[0336] In one embodiment, the method provides immunity in a subject against multiple Norovirus strains, Norovirus infection, or a Norovirus-associated disease or disorder, including, but not limited to, gastroenteritis, food poisoning, vomiting, and diarrhea. Thus, the present invention provides methods of treating or preventing a Norovirus-associated infection, disease, or disorder.

[0337] In one embodiment, the composition is administered to a subject who has an infection, disease, or disorder associated with Norovirus. In one embodiment, the composition is administered to a subject who is at risk of developing an infection, disease, or disorder associated with Norovirus. For example, the composition may be administered to a subject who is at risk of contacting Norovirus. In one embodiment, the composition is administered to a subject who lives in, has traveled to, or is expected to travel to a geographic area where Norovirus is prevalent. Populations of interest for vaccination include, but are not limited to, children (e.g., 5 years old or younger), military personnel, cruise ship staff and passengers, staff and residents of long-term care facilities (elderly care facilities, child protection facilities, schools), food handlers, and traveling subjects. In one embodiment, the composition is administered to a subject who has contact or is expected to contact others who live in, have traveled to, or are expected to travel to a geographic area where Norovirus is prevalent. In one embodiment, the composition is administered to a subject who is known to be exposed to Norovirus through occupation or other contacts.

[0338] In one embodiment, the method comprises administering a composition comprising one or more nucleoside modified nucleic acid molecules encoding one or more Norovirus antigens. In one embodiment, the method comprises administering a composition comprising a first nucleoside modified nucleic acid molecule encoding one or more Norovirus antigens and a second nucleoside modified nucleic acid molecule encoding one or more Norovirus antigens. In one embodiment, the method comprises administering a composition comprising one or more nucleoside modified nucleic acid molecules encoding multiple Norovirus antigens as described herein.

[0339] In one embodiment, the method comprises administering one or more compositions, each of which comprises one or more nucleoside modified nucleic acid molecules encoding one or more Norovirus antigens. In one embodiment, the method comprises administering a first composition comprising one or more nucleoside modified nucleic acid molecules encoding one or more Norovirus antigens, and administering a second composition comprising one or more nucleoside modified nucleic acid molecules encoding one or more Norovirus antigens. In one embodiment, the method comprises administering multiple compositions, each of which comprises one or more nucleoside modified nucleic acid molecules encoding one or more Norovirus antigens as described herein. In some embodiments, the method comprises staggered administration of multiple compositions.

[0340] In some embodiments, the methods include administering to the subject a plurality of nucleotide-modified nucleic acid molecules encoding a plurality of Norovirus antigens, adjuvants, or combinations thereof.

[0341] In some embodiments, the methods of the invention allow for sustained expression of a Norovirus antigen or adjuvant described herein for at least several days after administration. In some embodiments, the methods of the invention allow for sustained expression of a Norovirus antigen or adjuvant described herein for at least two weeks after administration. In some embodiments, the methods of the invention allow for sustained expression of a Norovirus antigen or adjuvant described herein for at least one month after administration. However, the methods also provide for transient expression in some embodiments, since in some embodiments the nucleic acid is not integrated into the genome of the subject.

[0342] In some embodiments, the methods include administering a nucleoside modified RNA that results in stable expression of a Norovirus antigen or adjuvant described herein. In some embodiments, administration of the nucleoside modified RNA induces an effective adaptive immune response, but little or no innate immune response.

[0343] In some embodiments, the method provides persistent protection against Norovirus. For example, in some embodiments, the method provides persistent protection against Norovirus for 2 weeks or more. In some embodiments, the method provides persistent protection against Norovirus for 1 month or more. In some embodiments, the method provides persistent protection against Norovirus for 2 months or more. In some embodiments, the method provides persistent protection against Norovirus for 3 months or more. In some embodiments, the method provides persistent protection against Norovirus for 4 months or more. In some embodiments, the method provides persistent protection against Norovirus for 5 months or more. In some embodiments, the method provides persistent protection against Norovirus for 6 months or more. In some embodiments, the method provides persistent protection against Norovirus for 1 year or more.

[0344] In one embodiment, a single immunization with the composition induces persistent protection against Norovirus for one month or more, two months or more, three months or more, four months or more, five months or more, six months or more, or one year or more.

[0345] The administration of the composition of the present invention in the treatment method can be accomplished in many different ways using methods known in the art.In one embodiment, the method of the present invention includes systemic administration of the subject, including, for example, enteral or parenteral administration.In some embodiments, the method includes intradermal delivery of the composition.In another embodiment, the method includes intravenous delivery of the composition.In some embodiments, the method includes intramuscular delivery of the composition.In one embodiment, the method includes subcutaneous administration of the composition.In one embodiment, the method includes inhalation of the composition.In one embodiment, the method includes intranasal delivery of the composition.

[0346] It is understood that the compositions of the present invention can be administered to a subject either alone or in combination with another agent.

[0347] Thus, the therapeutic and prophylactic methods of the present invention encompass the use of pharmaceutical compositions comprising at least one mRNA molecule (e.g., a nucleoside modified mRNA molecule) encoding a Norovirus antigen, adjuvant, or combination thereof described herein to practice the methods of the present invention. Pharmaceutical compositions useful for practicing the present invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates administration of a dose that results in a concentration of the compound of the present invention in a mammal of 10 nM to 10 μM.

[0348] Typically, the dosage that can be administered to a mammal, such as a human, in the method of the present invention ranges from 0.01 μg / kg to about 50 mg / kg (body weight of the mammal), but the exact dosage administered varies depending on any number of factors, including, but not limited to, the type of mammal and the type of disease state being treated, the age of the mammal, and the route of administration. In some embodiments, the dosage of the compound varies from about 0.1 μg / kg to about 10 mg / kg (body weight of the mammal). In some embodiments, the dosage varies from about 1 μg / kg to about 1 mg / kg (body weight of the mammal).

[0349] The compositions may be administered to the mammal as frequently as several times per day, or less frequently, such as once per day, once per week, once every two weeks, once per month, or even less frequently, such as once per few months, once per few years, or even less frequently, such as once per 10-20 years, once per 15-30 years, or even less frequently, such as once per 50-100 years. The frequency of dosage will be readily apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the species and age of the mammal, etc.

[0350] In some embodiments, administration of the immunogenic compositions or vaccines of the invention may be performed by a single dose or may be boosted by multiple doses. EXAMPLES

[0351] Experimental Examples The present invention is further described by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited in any way to the following examples, but rather should be understood to encompass any and all modifications that become evident as a result of the teachings provided herein.

[0352] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed methods. Thus, the following data supported examples are not to be construed as limiting in any way the remainder of the disclosure.

[0353] Example 1: Nucleoside-modified mRNA-LNP vaccine for Norovirus A vaccine for norovirus was developed using a vaccine platform of nucleoside-modified mRNA complexed with lipid nanoparticles (LNPs). The norovirus vaccine encodes the norovirus capsid protein VP1 derived from multiple genetic groups (GI.1, GI.3, GII.3 and GII.4). Encapsulation into LNPs allows efficient delivery and expression of the nucleoside-modified mRNA in vivo, and the 1-methylpseudouridine nucleoside modification instead of uridine is important for the potency of the antibody response induced by the mRNA platform through its induction of follicular helper T cells (Tfh). The nucleoside-modified mRNA-LNP vaccine demonstrated high HBGA (histo-blood group antigen)-blocking antibody titers, protected human enteroids from norovirus infection, and induced strong CD4 and CD8 positive responses even at low doses (0.25 μg). These data provide proof of concept that a modified nucleoside mRNA-LNP vaccine based on the norovirus VP1 sequence is immunogenic in vivo and generates neutralizing antibodies that block viral infection in vitro. The mRNA-LNP vaccine is a non-replicating vector that is highly effective at low doses and amenable to highly scalable manufacturing; it is therefore ideally suited for use in global public health campaigns to immunize people against norovirus.

[0354] The nucleoside-modified mRNA-LNP norovirus vaccine demonstrated high titers of blocking antibodies, good protection of human enteroids from norovirus infection, and elicited strong CD4+ and CD8+ T cell responses. These data provide proof of concept that a modified nucleoside mRNA-LNP vaccine based on the norovirus VP1 sequence is immunogenic in vivo and generates neutralizing antibodies that block viral infection in vitro.

[0355] To confirm that the mRNA molecules expressed the correct protein, 293T cells were transfected with mRNAs encoded by Norwalk1968, CapeTown2012, Sweden2008, Argentina2016, Nashville2016, Canada2019, UK2015, USA2011, or Japan2009. Cells were harvested 24 hours later and analyzed by Western blot for expression of GI.1, GII.4, GI.3, or GII.3 capsids (Figure 1).

[0356] Balb / c mice (6-8 weeks old) were immunized twice on days 0 and 28 by intradermal injection with 10 μg of empty LNP or mRNA bivalent vaccine: Norwalk / 2011 / GI.1 represents genogroup I strains and CapeTown / 2012 / GII.4 represents genogroup II strains. Altogether, genogroup I and II strains account for more than 90% of human norovirus infections. Figure 2 details the immunization scheme.

[0357] Human norovirus blocking antibody responses following a bivalent mRNA vaccine Sera against Norwalk1968 / GI.1 and Sydney2012 / GII.4 virus-like particles (VLPs) were screened for their ability to block VLP-binding ligand interactions in a surrogate neutralization assay (Figure 3). Sera that did not block VLP-ligand binding by at least 50% were assigned a titer of 0.5xLLD (ID50=25), placing the marker below the limit of detection (dotted line).

[0358] Human norovirus blocking antibody responses following a bivalent mRNA vaccine Figure 4 demonstrates that blocking antibody responses to additional VLPs demonstrated that the vaccine response was genotype specific with no cross-reactivity between the genogroups tested (GI.3, GI.5, GII.3). Among the GII.4 genotypes, blocking antibody titers for GII.4 / 2009 and GII.4 / 2002 were approximately 20% and 5% of those for GII.4 / 2012, respectively, consistent with known antigenic drift within the globally predominant GII.4 pandemic genotype.

[0359] T cell stimulation assay Balb / c mice (6-8 weeks old) were immunized on day 0 by intramuscular injection with 10 μg per mouse of mRNA bivalent vaccine (GII.4 / CapeTown2012, GI.1 / Nowalk2012) or with mRNA-Luc as a control. n=5 / group (Figure 5). T cell stimulation assays were performed with VLPs (GI.1, GI.3, GII.3, GII.4) (Figure 6).

[0360] Dose response of a bivalent human norovirus mRNA vaccine To determine the optimal amount of bivalent mRNA vaccine, four doses (0.25, 0.5, 1 or 3 μg of each vaccine per mouse) or mRNA-Luc (12 μg / mouse) as a control were administered to mice by intramuscular (im) injection twice at 28-day intervals (Figure 7). Sera were collected on days 0, 28, 56 and 150 after immunization and evaluated in surrogate neutralization assays. n=10 mice / group at the indicated time points. Blocking antibody titers are high even at doses as low as 0.25 μg / mouse.

[0361] Low-dose mRNA vaccine induces strong Th1 responses To evaluate Th1 responses, Balb / c mice (6-8 weeks old) were immunized twice on days 0 and 28 by intramuscular injection with 0.25 μg of mRNA vaccine (GII.4) per mouse or with mRNA-Luc as a control (Figure 8). Two weeks after the boost, spleen single cell suspensions were stimulated with GII.4 peptide pools (15 amino acids long, overlapping by 11 amino acids) for 5 h at 37°C with 5% CO2 and analyzed for T cell activation by flow cytometry. n=10 per group. Spleen cells isolated from mice vaccinated with GII.4 mRNA-LNP and stimulated with GII.4 peptide pools demonstrated that the mRNA vaccine elicited strong CD4+ and CD8+ T cell responses.

[0362] Quadrivalent norovirus mRNA vaccine We developed a quadrivalent norovirus mRNA vaccine containing the GI.3 and GII.3 genotypes because these are the most prevalent GI and GII genotypes in norovirus outbreaks of pediatric norovirus acute gastroenteritis. The capsid protein VP1 sequences of GI.3 / Argentina2016 and GII.3 / Canada2019 were identified in GenBank, plasmids were designed, and nucleoside-modified mRNAs were produced. The mRNAs encoding GI.1, GII.4, GI.3, and GII.3 were checked for endotoxin levels and interferon-alpha activation before encapsulation into LNPs.

[0363] Balb / c and C57BL / 6 mice (6-8 weeks of age) were immunized twice on days 0 and 28 by intramuscular injection with 0.125, 0.25, 0.5, 1 μg per mouse of mRNA tetravalent vaccine (GII.4 / CapeTown2012, GI.1 / Nowalk2012, GI.3 / Argentina2016, GII.3 / Canada2019) or with empty LNP as control (n=5 per group) (Figure 9).

[0364] To determine the optimal amount of the tetravalent mRNA vaccine, four doses (0.125, 0.25, 0.5, or 1 μg of each vaccine per mouse) or empty LNP (4 μg / mouse) as a control were administered to mice by intramuscular injection twice with an interval of 28 days ( FIG. 10 ). Empty LNP was below the limit of detection (not shown).

[0365] Human intestinal enteroids A major barrier to the development of effective interventions for human noroviruses is the lack of robust and reproducible in vitro culture systems. Recently, researchers have developed human intestinal enteroids (HIE), three-dimensional in vitro cultures derived from proliferating stem cells in crypts isolated from human gastrointestinal biopsies or surgical specimens. HIE cultures have the multicellular complexity and organizational organization of the intestinal epithelium. HIE are physiologically active, contain all epithelial cell types of the normal gastrointestinal tract, and retain segment-specific properties.

[0366] Figures 11 and 12 demonstrate the effect of infection of stem cell-derived human 3D enteroids with live human norovirus derived from a patient fecal sample infected with GII.4. HIE can be used to assay for human norovirus infection, as human norovirus-infected enteroids show the presence of VP1 capsid protein and a lack of Z01 staining, a marker of tight junction integrity (Figure 12).

[0367] C57BL / 6 mice (6-8 weeks old) were immunized twice on days 0 and 28 by intramuscular injection with 0.25 μg of mRNA vaccine (GII.4) per mouse or with mRNA-Luc as a control. Four weeks after the boost, serum was collected, diluted at the indicated dilutions, pre-incubated with human norovirus-positive fecal samples, and analyzed for neutralization of human norovirus infection in human small intestinal organoids derived from biopsies. Figures 13 and 14 demonstrate that serum from mice vaccinated with mRNA-LNP protects human intestinal enteroids against GII.4 infection.

[0368] These data demonstrate that blocking antibody titers 30 days after priming were higher (GI.1) or equivalent (GII.4) to those from patients infected with human norovirus, blocking antibodies were sustained at high titers out to at least day 240, low-dose mRNA vaccine (0.25 μg) elicited strong CD4+ and CD8+ T cell responses, and sera from vaccinated mice neutralized human norovirus and protected human intestinal enteroids from GII.4 infection. Thus, the data support that modified nucleoside mRNA-LNP vaccines based on norovirus VP1 sequences are immunogenic in vivo and generate neutralizing antibodies that block viral infection in vitro.

[0369] Example 2: Sequences SEQ ID NO:1 Capsid protein VP1 [human norovirus / GI.1 / 8FIIa / 1968 / USA] AFJ38516.1 [ka]

[0370] SEQ ID NO:2 [Human norovirus / GI.1 / CHA9A004_20110419 / 2011 / USA] AGM33223.1 [ka]

[0371] SEQ ID NO:3 VP1 [Human norovirus / GII.4 / Sydney / NSW0514 / 2012 / AU] AFV08795.1 [ka]

[0372] SEQ ID NO:4 [Norovirus GII / human / ZAF / 2012 / GII.P4_GII.4 / CapeTown / 9772] ALX87357.1 [ka]

[0373] SEQ ID NO:5 Capsid protein [Norovirus GI.3] (China 2015) GenBank:ARI71147.1 [ka]

[0374] SEQ ID NO:6 VP1 [Norovirus GI.3] (Human / USA / GI.Pd-GI.3 / Nashville-0047) GenBank:AZJ17766.1 [ka]

[0375] SEQ ID NO:7 VP1 [Norovirus GII.3] UK 2015 GenBank:AWR17549.1 [ka]

[0376] SEQ ID NO:8 Major capsid protein VP1 [Norovirus GII] (Human / GII.P16-GII.3 / RUS / Novosibirsk / NS17-A996 / 2017) GenBank:AVO62572.1 [ka]

[0377] SEQ ID NO:9 VP1 [Norovirus GI] (Argentina 2016) GenBank:QPJ58837.1 [ka]

[0378] SEQ ID NO:10 VP1 [Norovirus GII] (Canada 2019) GenBank:QSD58385.1 [ka]

[0379] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to cover all such embodiments and equivalent modifications.

Claims

1. A composition for inducing an immune response against norovirus (NoV) in a subject, the composition comprising at least one mRNA molecule encoding at least one NoV antigen.

2. 2. The composition of claim 1, wherein the NoV antigen is selected from the group consisting of p48, nucleoside-triphosphatase (NTPase), p22, VPg, protease, and RNA-dependent RNA polymerase (RdRp), VP1, VP2, fragments thereof, and any combination thereof.

3. 2. The composition of claim 1, wherein the NoV antigen is selected from the gene group consisting of GI, GII, GIV, GVIII, and GIX.

4. 2. The composition of claim 1, comprising a combination of mRNA molecules encoding at least two NoV VP1 antigens, wherein the VP1 antigens are selected from the NoV gene group consisting of GI and GII.

5. 5. The composition of claim 4, comprising a combination of mRNA molecules encoding at least two NoV VP1 antigens, wherein the VP1 antigens are selected from the group consisting of GI.1, GI.3, GI.5, GII.3, and GII.

4.

6. 5. The composition of claim 4, comprising a combination of an mRNA molecule encoding the NoV VP1 GI.1 antigen and an mRNA molecule encoding the NoV VP1 GII.4 antigen.

7. 5. The composition of claim 4, comprising a combination of mRNA molecules encoding each of the NoV VP1 GI.1 antigen, the NoV VP1 GII.4 antigen, the NoV VP1 GI.3 antigen, and the NoV VP1 GII.3 antigen.

8. The composition of claim 1, comprising at least one mRNA molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

9. The composition of claim 1 further comprising an adjuvant.

10. The composition of claim 1 , wherein the mRNA molecule is encapsulated in a lipid nanoparticle (LNP).

11. 2. The composition of claim 1, wherein the mRNA molecule is a nucleoside-modified mRNA molecule comprising at least one modified nucleoside selected from the group consisting of pseudouridine, 1-methylpseudouridine, and 5-methyluridine.

12. 12. The composition of any one of claims 1 to 11 for use in inducing an immune response against at least one strain of Norovirus (NoV) in a subject.

13. The composition of claim 12 , wherein the composition further comprises an adjuvant.

14. The composition of claim 12, wherein the mRNA molecule is encapsulated in a lipid nanoparticle (LNP).

15. 13. The composition of claim 12, wherein the mRNA molecule is a nucleoside-modified mRNA molecule comprising at least one modified nucleoside selected from the group consisting of pseudouridine, 1-methylpseudouridine, and 5-methyluridine.

16. 13. The composition of claim 12, wherein the composition is formulated for administration by a delivery route selected from the group consisting of intravenous, intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

17. The composition of claim 12 , formulated for a single administration of the composition.

18. 13. The composition of claim 12, formulated for multiple administration of the composition.

19. The composition of claim 12, wherein the composition treats or prevents a disease or disorder associated with NoV infection.

20. 20. The composition of claim 19, wherein the disease or disorder associated with NoV infection is selected from the group consisting of gastroenteritis, food poisoning, vomiting, and diarrhea.

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