NUCLEOSIDE-MODIFIED mRNA-LIPID NANOPARTICLE LINEAGE VACCINE FOR HEPATITIS C VIRUS

Abstract

To provide compositions and methods for inducing an adaptive immune response against Hepatitis C virus (HCV) in a subject.SOLUTION: In some embodiments, the present invention provides a composition comprising a nucleoside-modified nucleic acid molecule encoding a HCV antigen, adjuvant, or a combination thereof. For example, in some embodiments, the composition comprises a vaccine comprising a nucleoside-modified nucleic acid molecule encoding a HCV antigen, an adjuvant, or a combination thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 490,828, filed April 27, 2017, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under NIH U19 AI088791, K08 AI102761, P30 AI094189, RO1-A1050484, and RO1-A1084860 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Hepatitis C virus (HCV) infection poses a significant clinical burden in the United States alone, affecting over 160 million people worldwide and 4.6 million Americans, and is the leading cause of liver transplantation in North America. Untreated, chronic HCV infection can lead to cirrhosis, portal hypertension, and hepatocellular carcinoma. Conventional treatments, including interferon-based therapies, have had low success rates and significant side effects. Although the emergence of new-generation oral antiviral therapies has significantly improved efficacy and tolerability, disease prevention remains an important strategy for managing the disease burden. For these reasons, there is an urgent need to develop a prophylactic HCV vaccine and to determine whether a therapeutic vaccine can be useful in treating chronically infected patients.

[0004] Current standard treatments include combinations of direct-acting antivirals (DAAs) and pharmacological inhibitors of the viral NS3 / 4A protease and NS5A or NS5B polymerase, with an overall therapeutic efficacy of >90%. Despite these advances, viral resistance to these treatments has been observed clinically and is associated with treatment failure. Most infected individuals worldwide are unaware of their infection status, potentially continuing to infect others, and treatment does not prevent reinfection after recovery. The high cost of these new therapies and the large number of HCV-infected individuals mean that healthcare systems, even in developed countries, cannot afford to treat all patients. These limitations are even more pronounced in developing countries. Therefore, the development of a vaccine to prevent acute or chronic HCV infection is essential.

[0005] Thus, there is a need in the art for improved hepatitis C virus (HCV) vaccines. The present invention addresses this need.

[0006] The details of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, it being understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0007] [Figure 1A] Figure 1A shows the results of an example experiment analyzing mutant strains of HCV E1E2 over time since infection and the ability of these proteins to bind HEPC3 and mutant antibodies. The area under the curve (AUC) for binding of serial dilutions of mAbs to the self-E1E2 protein is shown. [Figure 1B]Figure 1B shows the results of an example. Figure 1B shows a dendrogram illustrating the phenotypic relationships among E1E2 clones, as determined by the hierarchy of mAb binding to each E1E2 clone. The binding rankings of all mAbs to each E1E2 clone were compared pairwise by Spearman correlation (r), and these r values ​​were used to cluster associated E1E2 clones. Red numbers are approximately unbiased (AU) values ​​indicating the strength of the cluster, with values ​​above 95 considered highly significant. Blue clones represent each phenotypic cluster.

[0008] [Figure 2A] Figure 2A shows the results of an additional experiment analyzing mutant strains of HCV E1E2 over time since infection and the ability of these proteins to bind HEPC3 and mutant antibodies. The area under the curve (AUC) for binding of serial dilutions of mAbs to the self-E1E2 protein is shown. [Figure 2B] Figure 2B shows the results of additional experiments. Figure 2B shows a dendrogram depicting the phenotypic relationships among E1E2 clones, as determined by the hierarchy of mAb binding to each E1E2 clone. The binding rankings of all mAbs to each E1E2 clone were compared pairwise by Spearman correlation (r), and these r values ​​were used to cluster associated E1E2 clones. Red numbers are approximately unbiased (AU) values ​​indicating the strength of the cluster, with values ​​above 95 considered highly significant. Blue clones represent each phenotypic cluster.

[0009] [Figure 3A] FIG. 3A shows the phylogenetic tree of E1E2 clones from patient 117. [Figure 3B] Figure 3B shows the highlighted plot.

[0010] [Figure 4]FIG. 4 shows the results of an exemplary experiment demonstrating the presence of all proteins following transfection of 293T cells with HCVA and B mRNA, along with a Luc control. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to compositions and methods for inducing an immune response against hepatitis C virus (HCV) in a subject. In some embodiments, the present invention provides a composition comprising at least one nucleoside-modified RNA encoding an HCV antigen. For example, in one embodiment, the vaccine comprises at least one nucleoside-modified RNA encoding at least one HCV antigen, wherein the vaccine induces an immune response in a subject against the at least one HCV antigen, thereby inducing an immune response against hepatitis C virus or a pathology associated with hepatitis C virus in the subject. In some embodiments, the at least one nucleoside-modified RNA encodes an HCV core protein, an HCV envelope E1 protein, an HCV envelope E2 protein, or a combination thereof. In one embodiment, the nucleoside-modified RNA encodes a sequential lineage of envelope proteins encoded in mRNA as a single protein comprising core and envelope 1 and 2 proteins (C-E1-E2). In some embodiments, the at least one nucleoside-modified RNA is encapsulated in a lipid nanoparticle (LNP).

[0012] 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.

[0013] As used herein, each of the following terms has the meaning associated with it in this section.

[0014] The articles "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. By way of example, "an element" means one element or more than one element.

[0015] As used herein, "about," when referring to a measurable value such as an amount, duration, etc., is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1 from the stated value as is suitable for carrying out the disclosed methods within that range.

[0016] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the present invention may exist in various 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).

[0017] 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.

[0018] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0019] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0020] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology. This term should also be interpreted to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein or amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art. This term should also be interpreted to mean an antibody produced by synthesis of an RNA molecule encoding the antibody, where the RNA molecule expresses the antibody protein or amino acid sequence that specifies the antibody, where the RNA is obtained by transcription of (synthetic or cloned) DNA, synthesis of RNA, or other techniques available and well known in the art.

[0021] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an adaptive immune response. This immune response may include either antibody production or activation of specific immunogenic cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. Those skilled in the art will understand that DNA or RNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an adaptive immune response encodes an "antigen" as that term is defined herein. Furthermore, those skilled in the art will understand that an antigen need not necessarily be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that antigens can be synthetically produced or derived from biological samples, including, but not limited to, tissue samples, tumor samples, cells, or body fluids.

[0022] The term "adjuvant" as used herein is defined as a molecule that enhances the antigen-specific adaptive immune response.

[0023] A "disease" refers to a state of health in an animal in which the animal is unable to maintain homeostasis, and if the disease is not corrected, the animal's health will continue to deteriorate. In contrast, an animal "disorder" refers to a state of health in which the animal is able to maintain homeostasis, but the animal's health is not better than it would be in the absence of the disorder. Ignoring the disorder does not necessarily result in a further decline in the animal's health.

[0024] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0025] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of polymers and macromolecules in biological processes and the biological properties that result therefrom, either with a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene encodes a protein when transcription and translation of its corresponding mRNA produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually set forth in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, are said to encode the protein or other product of that gene or cDNA.

[0026] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors containing sufficient cis-acting elements for expression, as well as other elements for expression, can be supplied by a 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 the recombinant polynucleotide.

[0027] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If two sequences both contain the same base or amino acid monomer subunit at a position, e.g., if two DNA molecules contain adenine at each position, the molecules are homologous at that position. The percentage of homology between two sequences is calculated by dividing the number of positions where the two sequences match or are homologous by the total number of positions in the comparison, and multiplying by 100. For example, if 6 out of 10 positions in two sequences match or are different, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, two sequences are aligned to maximize homology.

[0028] "Immunogen" refers to any substance that is introduced into the body to generate an immune response. The substance can be a physical molecule such as a protein, or can be encoded by a vector such as DNA, mRNA, or a virus.

[0029] As used herein, the term "immune response" refers to a process involving the activation and / or induction of effector functions in, by way of non-limiting example, T cells, B cells, natural killer (NK) cells, and / or antigen-presenting cells (APCs). Thus, as will be understood by those skilled in the art, an immune response includes, but is not limited to, any detectable antigen-specific activation and / or induction, such as helper T cell or cytotoxic T cell activity or response, antibody production, antigen-presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, etc.

[0030] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0031] In the context of the present invention, the following abbreviations are used for commonly occurring nucleosides (nucleobases linked to a ribose or deoxyribose sugar via an N-glycosidic bond): "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0032] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all degenerate versions of each other nucleotide sequences that encode the same amino acid sequence. Also, the term nucleotide sequence encoding a protein or RNA may include introns to the extent that some versions of the nucleotide sequence encoding the protein may contain introns.

[0033] As used herein, the term "modulating" means detectably increasing or decreasing the level of a response in a subject 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 in an otherwise identical but untreated subject. The term encompasses disrupting and / or affecting the natural signal or response to produce a beneficial therapeutic response in a subject, such as a human.

[0034] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all degenerate versions of each nucleotide sequence that encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may include introns. Furthermore, a nucleotide sequence may include modified nucleosides that are translatable by the translational machinery in a cell. Exemplary modified nucleosides are described elsewhere herein. For example, mRNAs in which some or all of the uridines are substituted with pseudouridine, 1-methylpseudouridine, or other modified nucleosides (such as those described elsewhere herein) are included. In some embodiments, a nucleotide sequence may include a sequence in which some or all of the cytodines are substituted with methylated cytidine or other modified nucleosides (such as those described elsewhere herein).

[0035] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, thereby resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous, and, where necessary, join two protein-coding regions in the same reading frame.

[0036] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal, or cells thereof in vitro or in situ, suitable for the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human.

[0037] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides as used herein include those that can be synthesized by recombinant means, i.e., conventional cloning techniques and PCR. (商標) This includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, cloning nucleic acid sequences from recombinant libraries or cell genomes, using synthetic means, etc.

[0038] In some instances, a polynucleotide or nucleic acid of the present invention is a "nucleoside-modified nucleic acid," which refers to a nucleic acid containing at least one modified nucleoside. A "modified nucleoside" refers to a modified nucleoside. For example, over 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).

[0039] In some embodiments, "pseudouridine" is m 1 acp 3 (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the term refers to m 1 In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 D (5-methyldihydrouridine). In another embodiment, the term refers to m 3Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not 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 other pseudouridines known in the art. Each possibility represents a separate embodiment of the present invention.

[0040] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which many types exist. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0041] The term "promoter," as used herein, is defined as a DNA sequence recognized by the cellular or introduced synthetic machinery necessary to initiate specific transcription of a polynucleotide sequence. One non-limiting example is the promoter recognized by bacteriophage RNA polymerase and used to generate mRNA by in vitro transcription.

[0042] The term "specifically binds" as used herein with respect to antibodies refers to an antibody that recognizes a specific antigen in a sample but does not substantially recognize or bind other molecules. For example, an antibody that specifically binds to an antigen of one species may also bind to one or more other antigens. However, such cross-species reactivity does not, in itself, change the classification of the antibody's specificity. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody's specificity. In some instances, the terms "specific binding" or "specific binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is due to the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species. For example, antibodies generally recognize and bind to specific protein structures rather than proteins. If an antibody is specific for epitope "A," then in a reaction involving labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0043] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, reducing, ameliorating, preventing, or eradicating at least one sign or symptom of a disease or disorder.

[0044] The term "therapeutically effective amount" refers to an amount of a compound of interest that elicits the biological or medical response in a tissue, system, or subject that is desired 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 alleviate to some extent 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 being treated and its severity, age, weight, etc.

[0045] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0046] 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. The cell includes the primary subject cell and its progeny.

[0047] As used herein, the terms "under transcriptional control" or "operably linked" mean that the promoter is correctly positioned and oriented relative to the polynucleotide to control initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0048] A "vector" is a complex of substances that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer 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 viral vectors, retroviral vectors, etc.

[0049] Ranges: In the disclosure herein, various aspects of the invention may be expressed in a range format. It should be understood that the description in range format is merely for convenience and ease of presentation and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges within that range and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 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 is true regardless of the breadth of the range.

[0050] explanation The present invention relates to compositions and methods for inducing an immune response against HCV in a subject. In some embodiments, the present invention relates to HCV sequence lineage antigens that can be used as immunogens to induce a prophylactic or therapeutic immune response in a subject. In some embodiments, the composition comprises a protein or peptide comprising one or more HCV antigens described herein. In some embodiments, the composition comprises a virus, including, for example, an inactivated or attenuated virus, that expresses one or more HCV antigens described herein. In some embodiments, the composition comprises a nucleic acid molecule, including, for example, DNA, cDNA, RNA, etc., that encodes one or more HCV antigens described herein.

[0051] In some embodiments, the present invention provides a composition comprising a nucleic acid molecule encoding an HCV antigen, wherein the HCV antigen induces an immune response against HCV in a subject. In some embodiments, the induced immune response is an adaptive immune response. For example, in some embodiments, the composition comprises a vaccine comprising a nucleic acid molecule encoding an HCV antigen. In some embodiments, the HCV antigen induces the expression of protective antibodies. In some embodiments, the HCV antigen provides an adjuvant function.

[0052] In one embodiment, a composition of the invention comprises in vitro transcribed (IVT) RNA. For example, in some embodiments, a composition of the invention comprises IVT RNA encoding an HCV antigen, wherein the HCV antigen induces an adaptive immune response. In some embodiments, the HCV antigen is at least one of an envelope (E1 and / or E2) protein or an HCV core protein (C), or a fragment or variant thereof.

[0053] In some embodiments, the HCV antigens are based on lineage immunogens that can initiate and mature an immune response against the rapidly mutating HCV virus, hi some embodiments, the HCV antigens are selected to be maintained within the HCV genome even as the HCV genome mutates to evade immune surveillance.

[0054] In some embodiments, the nucleic acid encoding the antigen of the composition of the present invention is nucleoside-modified RNA. The present invention is based, in part, on the discovery that nucleoside-modified RNA encoding an HCV antigen can induce a robust and durable immune response against HCV. Furthermore, nucleoside-modified RNA encoding an HCV antigen induces antigen-specific antibody production. Furthermore, nucleoside-modified RNA encoding an HCV antigen induces a protective T cell response. Nucleoside-modified RNA can induce an adaptive immune response comparable to or superior to current HCV vaccine strategies.

[0055] In some embodiments, the nucleic acid encoding the antigen of the compositions of the invention is purified nucleoside-modified RNA, e.g., in some embodiments, the compositions are purified to be free of double-stranded contaminants.

[0056] In some embodiments, the composition comprises a lipid nanoparticle (LNP). For example, in one embodiment, the composition comprises a nucleic acid molecule encoding an HCV antigen encapsulated within the LNP. In some cases, the LNP facilitates the uptake of the nucleic acid molecule into cells.

[0057] In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition comprises a nucleic acid molecule encoding the adjuvant. For example, in one embodiment, the composition comprises a nucleoside-modified RNA encoding the adjuvant. In one embodiment, the composition comprises an HCV antigen and a nucleoside-modified RNA encoding the adjuvant. In one embodiment, the composition comprises a first nucleoside-modified RNA encoding the HCV antigen and a second nucleoside-modified RNA encoding the adjuvant. In one embodiment, the composition comprises a nucleoside-modified RNA encoding the adjuvant and an LNP, wherein the LNP has adjuvant activity.

[0058] In one embodiment, the present invention provides a method for inducing an immune response to HCV in a subject. In some embodiments, the method comprises administering to the subject a composition comprising one or more nucleoside-modified RNAs encoding an HCV antigen, an adjuvant, or a combination thereof.

[0059] In one embodiment, the method comprises administering the composition to a subject, such as intradermally or intramuscularly. In some embodiments, the method comprises administering multiple doses to the subject. In another embodiment, the method comprises administering a single dose of the composition to the subject, wherein the single dose is effective to induce an adaptive immune response. In one embodiment, the method provides a sustained or long-lasting immune response.

[0060] vaccine In one embodiment, the present invention provides an immunogenic composition for inducing an immune response against HCV in a subject. For example, in one embodiment, the immunogenic composition is a vaccine. For a composition to be useful as a vaccine, it must induce an immune response against an HCV antigen in a cell, tissue, or mammal (e.g., a human). As used herein, an "immunogenic composition" may include an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding the antigen, a cell expressing or presenting the antigen or cellular component, a virus expressing or presenting the antigen or cellular component, or a combination thereof. In certain embodiments, the composition comprises or encodes all or part of any peptide antigen described herein, or an immunogenic functional equivalent thereof. In another embodiment, the composition is in the form of a mixture that includes an additional immunostimulatory substance or a nucleic acid encoding such a substance. Immune stimulatory substances include, but are not limited to, additional antigens, immunomodulators, antigen-presenting cells, lipid nanoparticles, or adjuvants. In another embodiment, one or more of such additional substances are covalently attached to the antigen or immunostimulatory substance in any combination.

[0061] In the context of the present invention, the term "vaccine" refers to a composition that induces an immune response upon inoculation into an animal. In some embodiments, the induced immune response provides protective immunity.

[0062] The vaccines of the present invention may vary in the composition of nucleic acids and / or cellular components. In a non-limiting example, a nucleic acid encoding an HCV antigen may be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein may further comprise additional components. For example, one or more vaccine components may be contained within a lipid, liposome, or lipid nanoparticle. In another non-limiting example, the vaccine may include one or more adjuvants. The vaccines of the present invention and their various components may be prepared and / or administered by the methods described herein or by any method known to those of skill in the art in light of this specification.

[0063] In various embodiments, induction of immunity by expression of an HCV antigen can be detected by observing the response of all or part of the host's immune system to the HCV antigen in vivo or in vitro.

[0064] For example, methods for detecting the 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). A portion of T cells that respond to antigens presented by APCs in an antigen-specific manner differentiate into cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs) upon stimulation with the antigen. These cells stimulated by the antigen proliferate. This process is referred to herein as "activation" of T cells. Therefore, CTL induction by epitopes of polypeptides, peptides, or combinations thereof can be evaluated by presenting epitopes of polypeptides, peptides, or combinations thereof to T cells using APCs and detecting the induction of CTLs. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0065] Methods for evaluating the CTL induction effect using dendritic cells (DCs) as APCs are well known in the art. DCs are a representative APC with robust CTL induction activity among various APCs. In the method of the present invention, epitopes of a polypeptide, peptide, or combination thereof are first expressed by DCs, and then the DCs are contacted with T cells. After contact with DCs, detection of T cells that have a cytotoxic effect on the target cells indicates that the epitopes of the polypeptide, peptide, or combination thereof have the effect of inducing cytotoxic T cells. Furthermore, the induced immune response can also be examined by measuring IFN-γ produced and released by CTLs in the presence of antigen-presenting cells bearing immobilized peptides or peptide combinations, as visualized using an anti-IFN-γ antibody, such as in an ELISPOT assay.

[0066] Apart from DCs, peripheral blood mononuclear cells (PBMCs) can also be used as APCs. CTL induction has been reported to be enhanced by culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, CTL induction has been shown to be enhanced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0067] Antigens confirmed to have CTL-inducing activity by these methods are antigens that have DC activation effects and subsequent CTL-inducing activity. Furthermore, CTLs that have acquired cytotoxicity through antigen presentation by APCs can also be used as vaccines against antigen-associated disorders.

[0068] The induction of immunity by expression of an HCV antigen can be further confirmed by observing the induction of antibody production against the HCV antigen. For example, if antibodies against the antigen are induced in an experimental animal immunized with a composition encoding the antigen and antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.

[0069] The specificity of the antibody response induced in the animal can include binding to multiple regions of the delivered antigen as well as inducing neutralizing antibodies that prevent infection or reduce the severity of disease.

[0070] The induction of immunity through HCV antigen expression can be further confirmed by observing the induction of CD4+ T cells. Although CD4+ T cells can also lyse target cells, they primarily contribute to the induction of other types of immune responses, including CTL and antibody production. Types of CD4+ T cell help can be characterized as Th1, Th2, Th9, Th17, T regulatory (Treg), or T follicular helper (Tfh) cells. Each subtype of CD4+ T cell contributes to a specific type of immune response. In one embodiment, the composition selectively induces T follicular helper cells and promotes a potent antibody response.

[0071] Therapeutic compounds or compositions of the 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 who have or are at risk of (or susceptible to) developing a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of the present invention, prophylactic administration, such as preventing or slowing the progression of a disease or disorder, occurs before overt clinical symptoms of the disease appear. In the medical field, the term "preventing" includes any activity that reduces the mortality or morbidity burden due to disease. Prevention can occur at primary, secondary, and tertiary levels of prevention. While primary prevention avoids the onset of disease, secondary and tertiary levels of prevention encompass activities aimed at preventing disease progression and the development of symptoms, as well as reducing the adverse effects of an already established disease by restoring function and alleviating disease-related complications.

[0072] antigen The present invention provides compositions for inducing an immune response in a subject. In one embodiment, the composition comprises an HCV antigen. In one embodiment, the composition comprises a nucleic acid sequence encoding an HCV antigen, or a fragment or variant thereof. For example, in some embodiments, the composition comprises nucleoside-modified RNA encoding an HCV antigen, or a fragment or variant thereof. In some embodiments, the composition comprises purified nucleoside-modified RNA encoding an HCV antigen, or a fragment or variant thereof. Antigens include, but are not limited to, polypeptides, peptides, proteins, viruses, or cells that induce an immune response in a subject.

[0073] In one embodiment, the antigen comprises an HCV-associated polypeptide or peptide that induces an immune response against the antigen and therefore against HCV, hi one embodiment, the antigen comprises a fragment of an HCV-associated polypeptide or peptide that induces an immune response against HCV.

[0074] In some embodiments, the HCV antigen is at least one of an HCV envelope E1 protein, an HCV envelope E2 protein, an HCV core (C) protein, or a fragment thereof.

[0075] In some embodiments, the core is used to form secreted subviral particles that contain E1 and E2, and in some instances, these secreted particles are in a good format for presentation to B cells.

[0076] In one embodiment, the antigen comprises a protein comprising a signal peptide (SP) from MHC class II. Other signal peptides that can be used include, but are not limited to, signal sequences from IL-2, tPA, mouse and human IgG, and synthetically optimized signal sequences.

[0077] In one embodiment, the composition comprises a nucleoside-modified RNA comprising a nucleic acid sequence encoding E1-E2, wherein the nucleic acid sequence is encoded by a DNA sequence comprising at least one of SEQ ID NOs: 1-9 or 37-39, or a fragment or variant thereof, and the nucleic acid sequence comprises at least one modified nucleoside.

[0078] In one embodiment, the composition comprises a nucleoside-modified RNA comprising a nucleic acid sequence encoding E1-E2 comprising an amino acid sequence comprising at least one of SEQ ID NOs: 10-18, or a fragment or variant thereof, wherein the nucleic acid sequence comprises at least one modified nucleoside.

[0079] In some embodiments, the composition comprises a nucleic acid sequence encoding an HCV core protein. The HCV core protein may be from any HCV isolate.

[0080] In one embodiment, the composition comprises a nucleoside-modified RNA comprising a nucleic acid sequence encoding C-E1-E2, wherein the nucleic acid sequence is encoded by a DNA sequence comprising at least one of SEQ ID NOs: 19-27, or a fragment or variant thereof, and the nucleic acid sequence comprises at least one modified nucleoside.

[0081] In one embodiment, the composition comprises a nucleoside-modified RNA comprising a nucleic acid sequence encoding C-E1-E2 comprising an amino acid sequence comprising at least one of SEQ ID NOs: 28-36, or a fragment or variant thereof, wherein the nucleic acid sequence comprises at least one modified nucleoside.

[0082] In one embodiment, the composition comprises a nucleoside-modified RNA comprising a nucleic acid sequence encoding E1-E2, wherein the nucleic acid sequence is encoded by a DNA sequence provided in Example 2, Example 3, or Example 4, or a fragment or variant thereof.

[0083] In one embodiment, the composition comprises a nucleoside-modified RNA comprising a nucleic acid sequence encoding E1-E2, wherein the amino acid sequences of E1-E2 are encoded by the DNA sequences provided in Example 2, Example 3, or Example 4, or fragments or variants thereof.

[0084] The HCV antigen may be any species or strain of HCV. For example, in one embodiment, the HCV antigen is a protein or fragment thereof of an HCV strain including, but not limited to, 1a, 1b, 1c, 1e, 1g, 1h, 11, 2a, 2b, 2c, 2d, 2e, 2i, 2j, 2k, 2m, 2q, 2r, 3a, 3b, 3g, 3h, 3i, 3k, 4a, 4b, 4c, 4d, 4f, 4g, 4k, 41, 4m, 4n, 4o, 4p, 4q, 4r, 4t, 4v, 4w, 5a, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 61, 6m, 6n, 6o, 6p, 6q, 6r, 6s, 6t, 6u, 6v, 6w, 6xa, and 7a, or a fragment or variant thereof.

[0085] In some embodiments, the HCV antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an HCV antigen described herein and maintains the immunogenic function of the original amino acid sequence. For example, in some embodiments, the amino acid sequence of the HCV antigen has 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% identity to the original amino acid sequence.

[0086] In one embodiment, the HCV antigen is encoded by a nucleic acid sequence of a nucleic acid molecule. In some embodiments, the nucleic acid sequence comprises DNA, RNA, cDNA, viral DNA, a variant thereof, a fragment thereof, or a combination thereof. In one embodiment, the nucleic acid sequence comprises a modified nucleic acid sequence. For example, in one embodiment, the nucleic acid sequence encoding the HCV antigen comprises nucleoside-modified RNA, as described in detail elsewhere herein. In some examples, the nucleic acid sequence includes an additional sequence encoding a linker sequence or tag sequence that is linked to the antigen by a peptide bond.

[0087] strain immunogen The use of computationally derived clonal lineages has recently been proposed to isolate BnAbs against specific mutant viruses, providing an alternative approach to vaccine design. Three general steps in the lineage-based approach are considered. First, monoclonal antibodies are obtained from a set of clonally related, antigen-specific memory B cells using single-cell techniques. This helps identify the native immunoglobulin heavy (VDJ) and light (VJ) gene pairs. Second, computational methods are used to infer the unmutated ancestral BCR (i.e., the putative receptor on naive B cells that binds antigen and initiates a broadly neutralizing antibody response). In addition, intermediate antibodies, which may represent important branching points for the development of clonal lineages, are identified. Finally, intermediate antigens can be designed to first bind and expand the immunogen to the non-mutated BCR, followed by the ancestral BCR to elicit a broadly neutralizing response (reviewed in Nat Biotechnol. 2012 May 7; 30(5): 423-433. doi: 10.1038 / nbt.2197).

[0088] 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 IVT RNA. In one embodiment, the nucleic acid molecule encoding the adjuvant is nucleoside-modified RNA.

[0089] Exemplary adjuvants include, but are not limited to, alpha interferon, gamma interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86. Other genes that may be useful adjuvants include those encoding MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, C D40L, 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, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, 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, anti-LAG3-Ig, anti-TIM3-Ig, and functional fragments thereof.

[0090] In some embodiments, the composition comprises lipid nanoparticles, wherein the lipid nanoparticles function as an adjuvant.

[0091] nucleic acid In one embodiment, the invention encompasses nucleic acid molecules encoding HCV antigens. In one embodiment, the invention encompasses nucleoside-modified nucleic acid molecules. In one embodiment, the nucleoside-modified nucleic acid molecule encodes an HCV antigen. In one embodiment, the nucleoside-modified nucleic acid molecule encodes multiple antigens, including one or more HCV antigens. In some embodiments, the nucleoside-modified nucleic acid molecule encodes an HCV antigen that induces an adaptive immune response against the HCV antigen. In one embodiment, the invention encompasses nucleoside-modified nucleic acid molecules encoding an adjuvant.

[0092] Nucleic acid molecules can be produced using any method in the art, including, but not limited to, in vitro transcription, chemical synthesis, and the like.

[0093] Nucleotide sequences encoding the HCV antigens or adjuvants described herein may alternatively contain sequence variations, e.g., substitutions, insertions, and / or deletions of one or more nucleotides, relative to the original nucleotide sequence, provided that the resulting polynucleotide encodes a polypeptide according to the invention. Thus, the scope of the present invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences recited herein and that encode an HCV antigen or adjuvant of interest.

[0094] As used herein, a nucleotide sequence is "substantially homologous" to a nucleotide sequence described herein if it has 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% identity to the original nucleotide sequence. Nucleotide sequences substantially homologous to an antigen-encoding nucleotide sequence can typically be isolated from an organism that produces the antigen based on the information contained in the nucleotide sequence, for example, by introducing conservative or non-conservative substitutions. Examples of other possible modifications include the insertion of one or more nucleotides into the sequence, the addition of one or more nucleotides to either of the ends of the sequence, or the deletion of one or more nucleotides at the end or within the sequence. Identity between two polynucleotides can be determined using computer algorithms and methods well known to those skilled in the art.

[0095] Furthermore, 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 that retain the immunogenic function of the original amino acid sequences.

[0096] As used herein, an amino acid sequence is "substantially homologous" to an amino acid sequence described herein if it has 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% identity to the original amino acid sequence. The identity between two amino acid sequences can be determined 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)).

[0097] In one embodiment, the present invention relates to a construct comprising a nucleotide sequence encoding an HCV antigen. In one embodiment, the construct comprises multiple nucleotide sequences encoding multiple HCV antigens. For example, in some embodiments, the construct encodes one or more, two or more, three or more, or all of the HCV antigens. In one embodiment, the present invention relates to a construct comprising a nucleotide sequence encoding an adjuvant. In one embodiment, the construct comprises a first nucleotide sequence encoding an HCV antigen and a second nucleotide sequence encoding an adjuvant.

[0098] In one embodiment, the composition comprises multiple constructs, each construct encoding one or more HCV antigens. In some embodiments, the composition comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more constructs. In one embodiment, the composition comprises about 5-11 constructs. In one embodiment, the composition comprises a first construct comprising a nucleotide sequence encoding an HCV antigen and a second construct comprising a nucleotide sequence encoding an adjuvant.

[0099] In another specific embodiment, the construct is operably linked to a translational control element. The construct can form an expression cassette by incorporating operably linked regulatory sequences for expression of the nucleotide sequence of the present invention.

[0100] vector Nucleic acid sequences encoding HCV antigens or adjuvants can be obtained using recombinant methods known in the art, such as by screening libraries from cells expressing the gene, deriving the gene from a vector known to contain it, or isolating it directly from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0101] Nucleic acids can be cloned into many types of 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.

[0102] Chemical means for introducing polynucleotides into host cells include, for example, colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, carbohydrates, peptides, cationic polymers, and liposomes. An exemplary colloidal system for use in vitro and in vivo as a delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0103] When a non-viral delivery system is utilized, liposomes are an exemplary delivery vehicle. The use of lipid formulations is contemplated for introducing 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 lipid-associated nucleic acid may be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule attached to both the liposome and the oligonucleotide, entrapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in a lipid suspension, contained within or complexed with micelles, or otherwise associated with lipids. The lipid / RNA or lipid / expression vector associated with the composition is not limited to a particular structure in solution. For example, it may exist in a bilayer structure such as a micelle, or in a "collapsed" structure. It may also simply be dispersed within the solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic, including, for example, the lipid droplets that occur naturally 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, amino alcohols, and aldehydes.

[0104] Suitable lipids for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components first undergo self-rearrangement and then form a closed structure, trapping water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions whose solution structure differs from the typical vesicular structure are also contemplated. For example, lipids may assume a micellar structure or simply exist as a heterogeneous aggregate of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0105] Regardless of the method by which exogenous nucleic acid is introduced into a host cell or the cell is exposed to a composition of the invention, various assays can be performed to confirm the presence of an mRNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Northern blotting and RT-PCR; and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunogenic means (ELISA and Western blot) or the assays described herein to identify agents within the scope of the invention.

[0106] In vitro transcribed RNA In one embodiment, a composition of the invention comprises an in vitro transcribed (IVT) RNA encoding an HCV antigen. In one embodiment, a composition of the invention comprises an IVT RNA encoding multiple HCV antigens. In one embodiment, a composition of the invention comprises an IVT RNA encoding an adjuvant. In one embodiment, a composition of the invention comprises an IVT RNA encoding one or more HCV antigens and one or more adjuvants.

[0107] In one embodiment, IVT RNA can be introduced into cells in the form of transient transfection. RNA can be produced by in vitro transcription using a synthetically produced plasmid DNA template. 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 source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or other suitable DNA sources. In one embodiment, the desired template for in vitro transcription is an HCV antigen capable of inducing an adaptive immune response. In one embodiment, the desired template for in vitro transcription is an adjuvant capable of enhancing an adaptive immune response.

[0108] In one embodiment, the DNA used for PCR may comprise an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest, a portion of the gene. 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 used for PCR is a human gene. In another embodiment, the DNA used for PCR is a human gene including 5' and 3' UTRs. In another embodiment, the DNA used for PCR is a gene from a pathogenic or symbiotic organism, such as a bacterium, virus, parasite, or fungus. In another embodiment, the DNA used for PCR is derived from a pathogenic or symbiotic organism, including a bacterium, virus, parasite, or fungus, including 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a sequence including portions of genes that are linked together to form an open reading frame encoding a fusion protein. The segments of DNA that are linked together can be from a single organism or from multiple organisms.

[0109] Genes that can be used as DNA sources for PCR include genes encoding polypeptides that induce or enhance adaptive immune responses in organisms. In some instances, the genes are useful for short-term treatment. In some instances, the genes have safety concerns regarding the amount of gene expressed.

[0110] In various embodiments, a plasmid is used to generate a template for in vitro transcription of mRNA used for transfection.

[0111] Chemical structures capable of promoting stability and / or translation efficiency may also be used. In some embodiments, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3,000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied in various ways, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can vary the 5' and 3' UTR lengths necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0112] The 5' and 3' UTRs can be the natural endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating them into the forward and reverse primers or by otherwise modifying the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for altering RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs well known in the art.

[0113] In one embodiment, the 5' UTR can include the Kozak sequence of the endogenous gene. Alternatively, if a non-endogenous 5' UTR is added to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While the Kozak sequence can increase the efficiency of translation of some RNA transcripts, it is not required for all RNAs to enable efficient translation. The requirement for a Kozak sequence for many mRNAs is known in the art. In another embodiment, the 5' UTR can be derived from an RNA virus whose RNA genome is stable within the cell. In another embodiment, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0114] To enable RNA synthesis from a DNA template, a transcription promoter must be attached upstream of the sequence to be transcribed onto the DNA template. 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 is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, as described elsewhere herein, the promoter is a T7 RNA polymerase promoter. 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.

[0115] In one embodiment, the mRNA has both a cap at the 5' end and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and mRNA stability within the cell. With circular DNA templates, such as plasmid DNA, RNA polymerases generate long concatemeric products that are unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in mRNA of normal size, which is effective for eukaryotic transfection when polyadenylated after transcription.

[0116] 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)).

[0117] The traditional method for incorporating poly(A / T) stretches into DNA templates is molecular cloning. However, poly(A / T) sequences incorporated into plasmid DNA can cause plasmid instability, which can be mitigated by using recombination-incompetent bacterial cells for plasmid propagation.

[0118] The poly(A) tail of an RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as Escherichia coli poly(A) polymerase (E-PAP) or yeast poly(A) polymerase. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides approximately doubles the translation efficiency of the RNA. Furthermore, mRNA stability can be improved by attaching different chemical groups to the 3' end. Such attachments include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further enhance RNA stability.

[0119] The 5' cap also provides stability to the mRNA molecule. In one embodiment, the RNA produced by the method comprises a 5' Cap 1 structure. Such Cap 1 structures can be generated using Vaccinia capping enzyme and 2'-O-methyltransferase enzymes (CellScript, Madison, WI). Alternatively, the 5' cap can be 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)).

[0120] RNA can be introduced into target cells using any of a number of different methods, including, but not limited to, electroporation, commercially available methods such as Amaxa Nucleofector-II4 (Amaxa Biosystems, Cologne, Germany), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or bioparticle delivery systems, e.g., "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)). In some embodiments, RNA of the invention is introduced into cells using a method involving the use of the TransIT®-mRNA transfection kit (Mirus, Madison, Wis.), which in some instances results in highly efficient, low-toxicity transfection.

[0121] Nucleoside-modified RNA In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid encoding an HCV antigen described herein. In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid encoding multiple antigens, including one or more HCV antigens. In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid encoding an adjuvant described herein. In one embodiment, a composition of the invention comprises a nucleoside-modified nucleic acid encoding one or more HCV antigens and one or more adjuvants.

[0122] In one embodiment, a composition of the invention comprises a series of nucleoside-modified nucleic acids encoding one or more HCV antigens that are varied with each subsequent injection to follow a linage scheme.

[0123] For example, in one embodiment, the composition comprises nucleoside-modified RNA. In one embodiment, the composition comprises nucleoside-modified mRNA. Nucleoside-modified mRNA offers advantages over unmodified mRNA, such as, for example, improved stability, reduced or eliminated natural immunogenicity, and enhanced translation, among others. Nucleoside-modified mRNA 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.

[0124] In some embodiments, nucleoside-modified mRNAs do not activate any pathological pathways, are translated very efficiently and almost immediately after delivery, and serve as templates for continuous protein production in vivo, lasting for days to weeks (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small, making them applicable to human therapy. For example, as described herein, nucleoside-modified mRNAs encoding HCV antigens have demonstrated the ability to induce antigen-specific antibody production. For example, in some instances, antigens encoded by nucleoside-modified mRNAs induce the production of greater amounts of antigen-specific antibodies compared to antigens encoded by unmodified mRNAs.

[0125] In some instances, delivering coding mRNA to express proteins offers numerous advantages over methods using protein, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence for the protein of interest is the only material delivered to the cell, avoiding all side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- or viral-based vectors, mRNA does not risk integration into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein have been measured within 15–30 minutes of in vivo injection of coding mRNA. In some embodiments, using mRNA rather than protein offers numerous advantages. While mRNA often has a short half-life in circulation or tissues, protein-based therapies require frequent administration, while mRNA provides a template for continuous protein production over days to weeks. Protein purification can be problematic and can contain aggregates and other impurities that can cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0126] In some embodiments, the nucleoside-modified RNA comprises the naturally occurring modified nucleoside pseudouridine. In some embodiments, the inclusion of pseudouridine makes 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).

[0127] The presence of pseudouridine-containing modified nucleosides in RNA has been demonstrated to suppress their natural immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Furthermore, in vitro transcribed protein-encoding RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). The presence of pseudouridine has subsequently been shown to improve RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and reduce both PKR activation and translation inhibition (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892).

[0128] Similar effects to those described for pseudouridine have also been observed with RNA containing 1-methyl-pseudouridine.

[0129] 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 instances, a preparative high-performance liquid chromatography (HPLC) purification procedure is used to obtain pseudouridine-containing RNA with excellent translation potential and lack of 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 increased serum EPO 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 instances, the nucleoside-modified nucleic acid molecule is purified using chromatographic techniques, 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 U.S. Patent Application Publication No. 2016 / 0032316, which is incorporated herein by reference in its entirety.

[0130] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or modified nucleosides. In some embodiments, the composition comprises an isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises pseudouridine or modified nucleosides. In some embodiments, the composition comprises a vector comprising an isolated nucleic acid encoding an antigen, an adjuvant, or a combination thereof, wherein the nucleic acid comprises pseudouridine or modified nucleosides.

[0131] In one embodiment, as described elsewhere herein, the nucleoside-modified RNA of the present invention is IVT RNA. 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 RNA polymerase.

[0132] 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 without further modification. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the pseudouridines listed above. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0133] 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).

[0134] In another embodiment, the modified nucleoside of the present invention is m 5 In another embodiment, the modified nucleoside is m5 U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A(N 6 -methyladenosine). 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).

[0135] In another embodiment, the modified nucleoside is m 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 2 hn 6 A(2-methylthio-N6 -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(lysidine);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(wibutosine);o2yW(peroxywibutosine);OHyW(hydroxywibutosine);OHyW*(undermodified hydroxywibutosine);imG(wibutosine);mimG(methylwibutosine);Q(queosine);oQ(epoxyqueosine);galQ(galactosylqueosine);manQ(mannosylqueosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G + (Archeosin); 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);mo 5 U(5-methoxyuridine); cmo 5 U(uridine 5-oxyacetic acid);mcmo 5 U(uridine 5-hydroxyacetic 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 D(5-methyldihydrouridine);f 5 Cm (5-formyl-2'-O-methylcytidine);m 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-demethylwyosine); imG2(isowyosine); or ac 6 A(N 6 -acetyladenosine).

[0136] In another embodiment, the nucleoside-modified RNA of the 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.

[0137] 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, or other modified nucleoside bases). 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%.

[0138] 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%.

[0139] 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%.

[0140] 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 relative to all other nucleic acid molecules (e.g., DNA, dsRNA).

[0141] In another embodiment, the nucleoside-modified RNA of the invention is translated more efficiently in cells compared to unmodified RNA molecules of the same sequence. In another embodiment, the nucleoside-modified RNA has an enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced by a factor of 2-fold compared to its unmodified counterpart. In another embodiment, translation is enhanced by 3-fold. In another embodiment, translation is enhanced by 4-fold. In another embodiment, translation is enhanced by 5-fold. In another embodiment, translation is enhanced by 6-fold. In another embodiment, translation is enhanced by 7-fold. In another embodiment, translation is enhanced by 8-fold. In another embodiment, translation is enhanced by 9-fold. In another embodiment, translation is enhanced by 10-fold. In another embodiment, translation is enhanced by 15-fold. In another embodiment, translation is enhanced by 20-fold. In another embodiment, translation is enhanced by 50-fold. In another embodiment, translation is enhanced by 100-fold. In another embodiment, translation is enhanced by 200-fold. In another embodiment, translation is enhanced by 500-fold. In another embodiment, translation is enhanced by 1000-fold. In another embodiment, translation is enhanced by 2000-fold. In another embodiment, the enhancement factor is 10-1000-fold. In another embodiment, the enhancement factor is 10-1000-fold. In another embodiment, the enhancement factor is 10-200-fold. In another embodiment, the enhancement factor is 10-300-fold. In another embodiment, the enhancement factor is 10-500-fold. In another embodiment, the enhancement factor is 20-1000-fold. In another embodiment, the enhancement factor is 30-1000-fold. In another embodiment, the enhancement factor is 50-1000-fold. In another embodiment, the enhancement factor is 100-1000-fold. In another embodiment, the enhancement factor is 200-1000-fold. In another embodiment, the enhancement factor is significantly enhanced by other values ​​or ranges of values.

[0142] In another embodiment, RNA encoding a nucleoside-modified antigen of the invention induces a more robust adaptive immune response compared to an unmodified in vitro synthesized RNA molecule having the same sequence. In another embodiment, the modified RNA molecule induces a 2-fold adaptive immune response compared to 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 200-fold. In another embodiment, the adaptive immune response is increased 500-fold. In another embodiment, the adaptive immune response is increased 1000-fold. In another embodiment, the adaptive immune response is increased 2000-fold. In another embodiment, the adaptive immune response is increased by other measures.

[0143] 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 doubling of the adaptive immune response (e.g., one of the "fold" increases described above). In another embodiment, the term refers to an increase such that the nucleoside-modified RNA can be administered at a lower dose or frequency than an unmodified RNA molecule and still induce an equally effective adaptive immune response. In another embodiment, the increase is such that a single administration of the nucleoside-modified RNA can induce an effective adaptive immune response.

[0144] In another embodiment, the nucleoside-modified RNA of the present invention exhibits significantly lower natural immunogenicity than an unmodified in vitro synthesized RNA molecule of the same sequence. In another embodiment, the modified RNA molecule exhibits a 2-fold less natural immune response than its unmodified counterpart. In another embodiment, the natural immunogenicity is reduced by 3-fold. In another embodiment, the natural immunogenicity is reduced by 4-fold. In another embodiment, the natural immunogenicity is reduced by 5-fold. In another embodiment, the natural immunogenicity is reduced by 6-fold. In another embodiment, the natural immunogenicity is reduced by 7-fold. In another embodiment, the natural immunogenicity is reduced by 8-fold. In another embodiment, the natural immunogenicity is reduced by 9-fold. In another embodiment, the natural immunogenicity is reduced by 10-fold. In another embodiment, the natural immunogenicity is reduced by 15-fold. In another embodiment, the natural immunogenicity is reduced by 20-fold. In another embodiment, natural immunogenicity is reduced 50-fold. In another embodiment, natural immunogenicity is reduced 100-fold. In another embodiment, natural immunogenicity is reduced 200-fold. In another embodiment, natural immunogenicity is reduced 500-fold. In another embodiment, natural immunogenicity is reduced 1000-fold. In another embodiment, natural immunogenicity is reduced 2000-fold. In another embodiment, natural immunogenicity is reduced by other measures.

[0145] In another embodiment, "exhibiting significantly less natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In another embodiment, the term refers to a reduction in natural immunogenicity (e.g., one of the "one-fold" reductions described above). In another embodiment, the term refers to a reduction such that administration of an effective amount of nucleoside-modified RNA does not elicit a detectable innate immune response. In another embodiment, the term refers to a reduction such that the nucleoside-modified RNA can be repeatedly administered without eliciting a sufficient innate immune response to reduce the detectable production of the protein encoded by the modified RNA. In another embodiment, the reduction is such that the nucleoside-modified RNA can be repeatedly administered without eliciting a sufficient innate immune response to eliminate the detectable production of the protein encoded by the modified RNA.

[0146] lipid nanoparticles In one embodiment, the delivery of nucleoside-modified RNA comprises any suitable delivery method, including the exemplary RNA transfection method described herein.In some embodiments, the delivery of nucleoside-modified RNA to a subject comprises mixing nucleoside-modified RNA with a transfection reagent before contacting step.In another embodiment, the method of the present invention further comprises administering 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.

[0147] 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 hydrate-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 (登録商標) , Lipofectamine (登録商標) , or TransIT (登録商標) In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0148] In another embodiment, the transfection reagent forms liposomes, which in another embodiment, enhance intracellular stability, increase uptake efficiency, and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a manner similar to that of the lipids that make up cell membranes. In another embodiment, they have an internal aqueous space for entrapment of water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, liposomes can deliver RNA to cells in a biologically active form.

[0149] In one embodiment, a composition comprises a lipid nanoparticle (LNP) described herein and one or more nucleic acid molecules. For example, in one embodiment, a composition comprises a LNP and one or more nucleoside-modified RNA molecules encoding one or more antigens, adjuvants, or a combination thereof.

[0150] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising one or more lipids, e.g., lipids of Formula (I), (II), or (III). In some embodiments, the lipid nanoparticle is included in a formulation comprising a nucleoside-modified RNA described herein. In some embodiments, such lipid nanoparticles comprise one or more excipients selected from cationic lipids (e.g., lipids of Formula (I), (II), or (III)) and neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., pegylated lipids, such as pegylated lipids of structure (IV), such as compound Iva). In some embodiments, the nucleoside-modified RNA is encapsulated within the lipid portion of the lipid nanoparticle or within an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects elicited by mechanisms of the host organism or cells, such as, for example, a harmful immune response.

[0151] 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 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or They 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 nanoparticles, is resistant to degradation by nucleases in aqueous solution.

[0152] LNPs may include any lipid capable of forming particles with one or more nucleic acid molecules attached thereto or encapsulating one or more nucleic acid molecules therein. The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are characterized by being generally insoluble in water but soluble in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids," including fats, oils, and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derivative lipids," such as steroids.

[0153] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and PEGylated lipids.

[0154] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term "cationic lipid" refers to a lipid that becomes cationic or cationic (protonated) when the pH is lower than 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 bind to negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid, which becomes positively charged as the pH decreases.

[0155] In some embodiments, cationic lipids include any of a number of lipid species that carry an overall 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-(2,3-dioleoyloxy)propyl)-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-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercial formulations of cationic lipids are available that can be used in the present invention. Examples include: LIPOFECTIN (登録商標) (Commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE (登録商標) (Commercially available cationic liposomes containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), GIBCO / BRL); and TRANSFECTAM (登録商標)(Commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, Promega Corp., Madison, Wis.) The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0156] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the present invention include those described in WO2012 / 016184, the entire contents of which are incorporated herein by reference. Representative amino lipids include, but are not limited to, the following: 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-T). DLin-MA.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).

[0157] Suitable amino lipids include those having the formula: [ka] wherein R and R are 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 It is acyl; R3 and R4 are the same or different and independently represent an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, or R3 and R4 may join to form an optionally substituted heterocycle having 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R5 is absent or present, and if present is hydrogen or C1-C6 alkyl; m, n, and p are the same or different and independently 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 the same or different and independently O, S, or NH.

[0158] 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.

[0159] Representative useful dilinoleylamino lipids have the formula: [ka] where n is 0, 1, 2, 3, or 4.

[0160] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (n is 2 in the formula of DLin-K-DMA above).

[0161] In one embodiment, the cationic lipid portion of the LNP has the structure of formula (I): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (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 occurrence, either (a) or (b) below: (a) either H or C1-C12 alkyl, or (b) R 1a is H or C1-C 12 alkyl, and R 1b forms a carbon-carbon double bond with adjacent carbon atoms; R 2a and R 2b is, independently at each occurrence, either (a) or (b) below: (a) either H or C1-C12 alkyl, or (b) R 2a or C1-C 12 alkyl, and R 2b forms a carbon-carbon double bond with adjacent carbon atoms; R 3a and R 3b is, independently at each occurrence, either (a) or (b) below: (a) either H or C1-C12 alkyl, or (b) R 3a or C1-C 12 alkyl, and R 3b forms a carbon-carbon double bond with adjacent carbon atoms; R 4a and R 4bis, independently at each occurrence, either (a) or (b) below: (a) either H or C1-C12 alkyl, or (b) R 4a or C1-C 12 alkyl, and R 4b forms a carbon-carbon double bond with adjacent carbon atoms; R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is independently H or C1-C for each occurrence 12 is alkyl; R 8 and R 9 are each independently C1-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 heterocyclic ring 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).

[0162] In some embodiments of Formula (I), R 1a , R 2a , R 3a or R 4a At least one of the following is 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 1b is not isopropyl when a is 6, and is n-butyl when a is 8.

[0163] In a further embodiment of formula (I), R 1a , R 2a , R 3a or R 4a At least one of the following is C1-C 12 alkyl or L 1 or L2 is —O(C═O)— or —(C═O)O—; and R 1a and R 1b is not isopropyl when a is 6, and is n-butyl when a is 8.

[0164] In another embodiment of Formula (I), R 8 and R 9 are each independently an unsubstituted C-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 heterocyclic ring containing one nitrogen atom.

[0165] In some embodiments of Formula (I), L 1 or L 2 Either one of L may 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.

[0166] In some embodiments of Formula (I), L 1 or L 2 In another embodiment, one of L 1 and L 2 All of these are -O(C=O)-.

[0167] In some embodiments of Formula (I), L 1 or L 2 In another embodiment, L 1 and L 2 are both -(C=O)O-.

[0168] In some other embodiments of Formula (I), L 1 or L 2 In another embodiment, one of L 1 and L 2 Both of these are carbon-carbon double bonds.

[0169] 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.

[0170] As used throughout this specification, a "carbon-carbon" double bond is represented by the following structure: [ka] is understood to refer to either (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, for each independently, H, C1-C 12 Alkyl or cycloalkyl, e.g., H or C1-C 12 (It is alkyl).

[0171] In another embodiment, the lipid compound of formula (I) has the following structure (Ia): [ka] It has.

[0172] In another embodiment, the lipid compound of formula (I) has the following structure (Ib): [ka] It has.

[0173] In yet another embodiment, the lipid compound of formula (I) has the following structure (Ic): [ka] It has.

[0174] In some embodiments of the lipid compound of Formula (I), 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 a further embodiment, a is 3. In yet another embodiment, a is 4. In some embodiments, a is 5. In another embodiment, a is 6. In a further embodiment, a is 7. In yet another embodiment, a is 8. In some embodiments, a is 9. In another embodiment, a is 10. In a further embodiment, a is 11. In yet another embodiment, a is 12. In some embodiments, a is 13. In another embodiment, a is 14. In a further embodiment, a is 15. In yet another embodiment, a is 16.

[0175] In some further embodiments of Formula (I), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In yet other embodiments, b is 16.

[0176] In some further embodiments of Formula (I), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In yet other embodiments, c is 16.

[0177] In some further 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 yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In yet other embodiments, d is 16.

[0178] 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 particular embodiments, a and d are the same, and b and c are the same.

[0179] In Formula (I), the sum of a and b and the sum of c and d are factors that can be varied to obtain a lipid of Formula (I) with desired properties. In one embodiment, a and b are selected so that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected so that their sum is an integer in the range of 14 to 24. In yet another embodiment, 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 in the range of 14 to 24. In a further embodiment, a, b, c, and d are selected so that the sum of a and b and the sum of c and d are 12 or greater.

[0180] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0181] R in formula (I) 1a , R 2a , R 3a and R 4a The substituents of R are not particularly limited. 1a , R 2a , R 3a and R 4a is H in each occurrence. In some further embodiments, at least one of R 1a , R 2a , R 3a and R 4a is C1-C 12 In some other embodiments, at least one of R is alkyl. 1a , R 2a , R 3a and R 4a is C1-C8 alkyl. In some other embodiments, at least one of R 1a , R 2a , R 3a and R 4a is C1-C6 alkyl. In some of the foregoing embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0182] In some embodiments of Formula (I), R 1a , R 1b , R 4a and R 4b is C1-C at each occurrence 12 It is alkyl.

[0183] 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 in each occurrence.

[0184] In some embodiments of Formula (I), R 1b forms a carbon-carbon double bond with the adjacent carbon atom. 4b forms a carbon-carbon double bond with the adjacent carbon atom.

[0185] 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 6 In some other embodiments, one or both of the cycloalkyl groups are cycloalkyl, e.g., cyclohexyl. In these embodiments, the cycloalkyl groups may be substituted or unsubstituted. In some other embodiments, the cycloalkyl groups are C-C 12 It is substituted with alkyl, for example tert-butyl.

[0186] R 7 The substituents in are not particularly limited in the foregoing embodiments of Formula (I). In some embodiments, at least one R 7 is H. In some other embodiments, R 7 is H in each occurrence. In some other embodiments, R 7 is C1-C12 It is alkyl.

[0187] In some other of the foregoing embodiments of Formula (I), R 8 or R 9 In another embodiment, one of R 8 and R 9 Both are methyl.

[0188] 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. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, such as a pyrrolidinyl ring.

[0189] In various different embodiments, the lipid of formula (I) has one of the structures shown in Table 1 below.

[0190] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0191] 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, or 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.

[0192] In some alternative embodiments, the cationic lipid component of the LNP has the structure of formula (II): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (In the formula, L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, or -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 -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a or a direct bond; G 3 is C1-C6 alkylene; R a or C1-C 12 is alkyl; R 1a and R 1b is, independently at each occurrence, either (a) or (b) below: (a) H or C-C 12 or (b) R1a or C1-C 12 alkyl, and R 1b forms a carbon-carbon double bond with the adjacent carbon atom; R 2a and R 2b is, independently at each occurrence, either (a) or (b) below: (a) H or C-C 12 or (b) R 2a or C1-C 12 alkyl, and R 2b forms a carbon-carbon double bond with the adjacent carbon atom; R 3a and R 3b is, independently at each occurrence, either (a) or (b) below: (a) H or C-C 12 or (b) R 3a or C1-C 12 alkyl, and R 3b forms a carbon-carbon double bond with the adjacent carbon atom; R 4a and R 4b is, independently at each occurrence, either (a) or (b) below: (a) H or C-C 12 or (b) R 4a or C1-C 12 alkyl, and R 4b forms a carbon-carbon double bond with the adjacent carbon atom; R 5 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-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 heterocyclic ring; a, b, c, and d are each independently an integer from 1 to 24; and where x is 0, 1 or 2).

[0193] 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; and G 1 and G 2 are each independently —(C═O)— or a direct bond.

[0194] In some different embodiments of Formula (II), L 1 and L 2 are each independently -C(=O)-, -O-, or -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -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-, -NR a S(O) x NR a -, -NR a S(O) x -or-S(O) x NR a -It is.

[0195] In another example of the aforementioned embodiment of formula (II), the lipid compound has the following structure (IIA) or (IIB): [ka] The device has one of the following:

[0196] In some embodiments of formula (II), the lipid compound has the structure (IIA): In other embodiments, the lipid compound has the structure (IIB):

[0197] In any of the foregoing 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)—.

[0198] In some different embodiments of Formula (II), L 1 or L 2 One of the groups is —(C═O)O—. For example, in some embodiments, L 1 and L 2 Each of is —(C═O)O—.

[0199] In another 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 ) is absent. For example, in some embodiments, L 1 and L 2 Each of is a direct bond.

[0200] In yet another embodiment of Formula (II), R 1a and R 1b In at least one occurrence of R 1a is H or C1-C12 alkyl, and R 1b forms a carbon-carbon double bond with the adjacent carbon atom.

[0201] In yet another embodiment of Formula (II), R 4a and R 4b In at least one occurrence of R 4a is H or C1-C12 alkyl, and R 4b forms a carbon-carbon double bond with the adjacent carbon atom.

[0202] In a further embodiment of formula (II), R 2a and R 2b In at least one occurrence of R 2a is H or C1-C12 alkyl, and R 2b forms a carbon-carbon double bond with the adjacent carbon atom.

[0203] In yet another embodiment of Formula (II), R 3a and R 3b In at least one occurrence of R 3a is H or C1-C12 alkyl, and R 3b forms a carbon-carbon double bond with the adjacent carbon atom.

[0204] In other various embodiments of formula (II), the lipid compound has the following structure (IIC) or (IID): [ka] have one of (wherein e, f, g and h each independently represent an integer of 1 to 12).

[0205] In some embodiments of formula (II), the lipid compound has the structure (IIC): In other embodiments, the lipid compound has the structure (IID):

[0206] In various embodiments of structure (IIC) or (IID), e, f, g, and h are each independently an integer from 4 to 10.

[0207] 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 a further embodiment, a is 3. In yet another embodiment, a is 4. In some embodiments, a is 5. In another embodiment, a is 6. In a further embodiment, a is 7. In yet another embodiment, a is 8. In some embodiments, a is 9. In another embodiment, a is 10. In a further embodiment, a is 11. In yet another embodiment, a is 12. In some embodiments, a is 13. In another embodiment, a is 14. In a further embodiment, a is 15. In yet another embodiment, a is 16.

[0208] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In a further embodiment, b is 3. In yet another embodiment, b is 4. In some embodiments, b is 5. In another embodiment, b is 6. In a further embodiment, b is 7. In a further embodiment, b is 8. In some embodiments, b is 9. In another embodiment, b is 10. In a further embodiment, b is 11. In a further embodiment, b is 12. In some embodiments, b is 13. In another embodiment, b is 14. In a further embodiment, b is 15. In a further embodiment, b is 16.

[0209] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In a further embodiment, c is 3. In yet another embodiment, c is 4. In some embodiments, c is 5. In another embodiment, c is 6. In a further embodiment, c is 7. In a further embodiment, c is 8. In some embodiments, c is 9. In another embodiment, c is 10. In a further embodiment, c is 11. In a further embodiment, c is 12. In some embodiments, c is 13. In another embodiment, c is 14. In a further embodiment, c is 15. In a further embodiment, c is 16.

[0210] In some embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In a further embodiment, d is 3. In yet another embodiment, d is 4. In some embodiments, d is 5. In another embodiment, d is 6. In a further embodiment, d is 7. In a further embodiment, d is 8. In some embodiments, d is 9. In another embodiment, d is 10. In a further embodiment, d is 11. In a further embodiment, d is 12. In some embodiments, d is 13. In another embodiment, d is 14. In a further embodiment, d is 15. In a further embodiment, d is 16.

[0211] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In yet 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.

[0212] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In yet other embodiments, f is 12.

[0213] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In yet other embodiments, g is 12.

[0214] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In yet other embodiments, h is 12.

[0215] 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 particular embodiments, a and d are the same, and b and c are the same.

[0216] In formula (II), the sum of a and b and the sum of c and d are factors that can be varied to obtain lipids with desired properties. In one embodiment, a and b are selected so that their sum is an integer ranging from 14 to 24. In another embodiment, c and d are selected so that their sum is an integer ranging from 14 to 24. In yet another embodiment, 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 ranging from 14 to 24. In a further embodiment, a, b, c, and d are selected so that the sum of a and b and the sum of c and d are 12 or greater.

[0217] R in formula (II) 1a , R 2a , R 3a and R 4a The substituents in R are not particularly limited. 1a , R 2a , R 3a and R 4a At least one of R is H. In some embodiments, 1a , R 2a , R 3a and R 4a is H in each occurrence. In some other embodiments, R 1a , R 2a , R 3a and R 4a At least one of the following is C1-C 12 In some other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C-C alkyl. 1a , R 2a , R 3a and R 4a At least one of is C1-C6 alkyl. In some of the foregoing embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0218] In some embodiments of Formula (II), R 1a , R 1b , R 4a and R 4b is C1-C at each occurrence 12 It is alkyl.

[0219] 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 in each occurrence.

[0220] In some embodiments of Formula (II), R 1b forms a carbon-carbon double bond with the adjacent carbon atom. 4b forms a carbon-carbon double bond with the adjacent carbon atom.

[0221] R in formula (II) 5 and R 6 The substituents in R are not particularly limited in the above embodiments. 5 or R 6 One of R is methyl. 5 or R 6 Each of is methyl.

[0222] 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 -(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 NR a R b , -NR a S(O) x R b or -S(O) x NR a R b wherein R a is H or C1-C 12 alkyl; R b is C1-C 15 alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 -(C=O)OR b or -O(C=O)R b is replaced by

[0223] In the various embodiments of formula (II) above, R b is branch C1-C 15 For example, in some embodiments, R b has the following structure: [ka] The device has one of the following:

[0224] In some other of the foregoing embodiments of formula (II), R 8 or R 9 In another embodiment, one of R 8 and R 9Both are methyl.

[0225] 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. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, e.g., a pyrrolidinyl ring. In some further embodiments of the foregoing, R 8 and R 9 together with the nitrogen atom to which they are attached form a six-membered heterocyclic ring, such as a piperazinyl ring.

[0226] In yet another embodiment of the aforementioned lipids of formula (II), G 3 is C2-C4 alkylene, for example C3 alkylene.

[0227] In various different embodiments, the lipid compound has any of the structures shown in Table 2 below:

[0228] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0229] 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.

[0230] In some alternative embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof (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 a C(=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(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 -or-NR a C(=O)O- or a direct bond; G 1 and G 2are each independently an unsubstituted C-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-C 24 Alkyl or C6-C 24 is 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 where x is 0, 1 or 2).

[0231] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka] have one of (In the formula, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 each occurrence independently H, OH, or C-C 24 is alkyl; where n is an integer ranging from 1 to 15.

[0232] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0233] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] have one of (wherein y and z are each independently an integer ranging from 1 to 12).

[0234] In any of the foregoing 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 foregoing, L 1 and L 2 are 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—.

[0235] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] It has one of the following.

[0236] In some different embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII), or (IIIJ): [ka] It has one of the following.

[0237] In some of the foregoing 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.

[0238] In some other such 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.

[0239] In some of the foregoing embodiments of formula (III), R 6 is H. In other embodiments of the foregoing, R 6 is C1-C 24 In another embodiment, R 6 is OH.

[0240] In some embodiments of Formula (III), G 3 is unsubstituted. In another embodiment, G is substituted. In various different embodiments, G 3 is a straight chain C1-C 24 Alkylene or straight chain C1-C 24 It is alkenylene.

[0241] In some other aforementioned embodiments of formula (III), R 1 or R 2 Either or both are C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the following structure: [ka] have (In the formula, R 7a and R 7b are, for each occurrence, independently H or C-C 12is alkyl; and a is an integer from 2 to 12, where R 7a , R 7b and a are 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.

[0242] In some of the foregoing embodiments of formula (III), R 7a is H in at least one occurrence. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the foregoing, R 7b is C1-C8 alkyl at least one occurrence. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0243] In another embodiment of the formula, R 1 or R 2 one or both of the following structures (III): [ka] The device has one of the following:

[0244] In some of the foregoing 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.

[0245] In various different embodiments, the cationic lipid of formula (III) has any of the structures shown in Table 3 below:

[0246] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0247] In some embodiments, the LNP comprises a lipid of formula (III), a nucleoside-modified RNA, and, optionally, 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.

[0248] In some embodiments, the cationic lipid is present in the LNP in an amount of about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP is composed solely of cationic lipid.

[0249] In some embodiments, the LNPs include one or more additional lipids that stabilize the particle during its formation.

[0250] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0251] The term "neutral lipid" refers to any of a number of lipid species that exist in uncharged or neutral zwitterionic form at physiological pH. Exemplary neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0252] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0253] 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 about 8:1.

[0254] In various embodiments, the LNP further comprises a steroid or steroid analog. A "steroid" is a compound that contains the following carbon skeleton: [ka]

[0255] In some embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of cationic lipid (e.g., a lipid of Formula (I)) to cholesterol ranges from about 2:1 to about 1:1.

[0256] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH, including phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other neutral lipids with attached anionic modifying groups.

[0257] In some embodiments, the LNPs comprise a glycolipid (e.g., monosialoganglioside GM1). In some embodiments, the LNPs comprise a sterol, such as cholesterol.

[0258] In some embodiments, the LNP comprises a lipid-conjugated polymer. The term "lipid-conjugated polymer" refers to a molecule that contains both a lipid portion and a polymer portion. One example of a lipid-conjugated polymer is a pegylated lipid. The term "pegylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include, for example, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0259] In some embodiments, the LNPs comprise an additional stabilizing lipid that is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. 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-dimyristoylpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In another embodiment, the LNP includes: 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) PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, e.g., 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.

[0260] In some embodiments, the LNP has the following structure (IV): [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. (In the formula, R 10 and R 11 are each independently a linear 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 linkages; and The average value of z is 30-60).

[0261] In some of the foregoing embodiments of PEGylated lipid (IV), when z is 42, R 10 and R 11 In some other embodiments, none of R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 18 carbon atoms. 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 to 16 carbon atoms. 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. 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 straight 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, and R 11 is a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0262] In various embodiments, z is in a range selected to provide an average molecular weight of the PEG portion of (II) of about 400 to about 6000 g / mol. In some embodiments, z has an average of about 45.

[0263] In another embodiment, the PEGylated lipid has the following structure: [ka] have one of where n is an integer selected so that the average molecular weight of the PEGylated lipid is about 2500 g / mol.

[0264] In some embodiments, the additional lipid is present in the LNP in an amount of about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 mole percent or about 1.5 mole percent.

[0265] 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 a different embodiment, the neutral lipid is DSPC. In another embodiment, the steroid is cholesterol. In yet a different embodiment, the PEGylated lipid is compound IVa.

[0266] In some embodiments, the LNPs comprise one or more targeting moieties capable of targeting the LNPs to a cell or cell population, for example, in one embodiment, the targeting moiety is a ligand that directs the LNP to a receptor on the surface of a cell.

[0267] 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 on the cell surface and induce receptor-mediated uptake of the LNP. In some embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can be recognized by a cell surface receptor that induces internalization. For example, in one embodiment, the LNP binds to systemic ApoE, resulting in uptake of the LNP and the bound substance.

[0268] Other exemplary LNPs and their manufacture are described in the art, for example, in US Patent Application Publication No. US20120276209; 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 acid Acids, 1: e37;Jayaraman et 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.

[0269] The following reaction schemes illustrate methods for preparing lipids of formula (I), (II), or (III).

[0270] [ka] Embodiments of lipids of formula (I) (e.g., compound A-5) can be prepared according to general reaction scheme 1 ("Method A"), where R is a saturated or unsaturated C-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 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. A mixture of A-1, A-2, and DMAP is treated with DCC to produce bromide A-3. A mixture of bromide A-3, a base (e.g., N,N-diisopropylethylamine), and N,N-dimethyldiamine A-4 is heated at a temperature and for a time sufficient to produce A-5 after any necessary workup and / or purification steps.

[0271] [ka] Another embodiment of the compound of formula (I) (e.g., compound B-5) can be prepared according to general reaction scheme 2 ("Method B"), wherein R is a saturated or unsaturated C-C 24where m is alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. 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.

[0272] 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.

[0273] [ka] Different embodiments of lipids of formula (I) (e.g., compounds C-7 or C9) 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. With reference to General Reaction Scheme 3, compounds of structure C-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art.

[0274] [ka] 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 , R4a , 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 19 represents alkyl. Referring to General Reaction Scheme 1, compounds of structures D-1 and D-2 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. A solution of D-1 and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to provide D-3 after necessary workup. A solution of D-3 and a base (e.g., trimethylamine, DMAP) is treated with an acyl chloride D-4 (or a carboxylic acid and DCC) to provide D-5 after any necessary workup and / or purification. D-5 can be reduced with LiAlH4 D-6 to provide D-7 after any necessary workup and / or purification.

[0275] [ka] Embodiments of lipids of formula (II) (e.g., compound 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 7 , R 8 , R 9 , L 1 , L 2 , G 3, a, b, c, and d are as described herein. With reference to General Reaction Scheme 2, compounds of structures E-1 and E-2 can 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) provides E-3 after any necessary workup. Treating a solution of E-3 and a base (e.g., trimethylamine, DMAP) with an acyl chloride E-4 (or a carboxylic acid and DCC) provides E-5 after any necessary workup and / or purification.

[0276] [ka] 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 described herein for formula (III), and G 1' is G 1 This refers to a one-carbon shorter homolog of the formula (III). Compounds of structure F-1 can be purchased or prepared according to methods known in the art. Reaction of F-1 with diol F-2 under appropriate condensation conditions (e.g., DCC) produces ester / alcohol F-3, which can be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions provides lipids of formula (III).

[0277] 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, L 1 and L 2 Other lipids of formula (III) other than esters can be prepared according to similar methods using appropriate starting materials. Further, general reaction scheme 6 shows the general reaction scheme of G 1 and G 2 The preparation of lipids of formula (III) in which G is identical is shown, however, this is not a required aspect of the invention, and 1 and G 2The above reaction scheme can be modified to produce different compounds.

[0278] It will be understood 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 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 described herein. The use of protecting groups is detailed in Green, T.W. and P.G.M.Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley, as will be understood by those skilled in the art. The protecting group may be a polymer resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0279] Pharmaceutical Composition The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparative methods include the step of bringing into association 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 multiple dosage units.

[0280] While the description of pharmaceutical compositions provided herein primarily refers to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to animals of all species. Modifications to pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and implement such modifications, with routine experimentation as necessary. Administration of the pharmaceutical compositions of the present invention is contemplated to various mammals, including, but not limited to, humans and other primates, non-human primates, and commercial mammals such as cows, pigs, horses, sheep, cats, and dogs.

[0281] Pharmaceutical compositions useful in the methods of the invention can be prepared, packaged, or sold in formulations suitable for intraocular, oral, enteral, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other possible formulations include projected nanoparticles, liposomal formulations, resealed red blood cells containing the active ingredient, and immunogen-based formulations.

[0282] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk as one single unit dose or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a convenient fraction thereof, such as, for example, one-half or one-third of such a dosage.

[0283] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route of administration of the composition. For example, the composition may contain from 0.1% to 100% (w / w) of the active ingredient.

[0284] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutically active agents.

[0285] Controlled- or sustained-release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques.

[0286] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by the physical disruption of a subject's tissue and the administration of the pharmaceutical composition through tissue disruption. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration is intended to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and kidney dialysis infusion techniques.

[0287] Pharmaceutical compositions suitable for parenteral administration include an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, and sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, and sold in unit dosage forms, such as ampoules, or in multidose 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 ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0288] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using, for example, non-toxic parenterally acceptable diluents or solvents, 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 useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may include pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0289] Pharmaceutical compositions of the invention may be prepared, packaged, and sold in formulations suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising the active ingredient having a diameter in the range of about 0.5 to about 7 nanometers. In some embodiments, the formulations may comprise dry particles comprising the active ingredient having a diameter in the range of about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir capable of directing a stream of propellant to disperse the powder, or a device employing a self-propelling solvent / powder dispensing container, such as a device in which the active ingredient is dissolved or suspended in a low-boiling point 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 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions comprise a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0290] Low boiling point propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant will 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 components such as a liquid nonionic or solid anionic surfactant or a solid diluent (optionally having a particle size on the same order as the particles containing the active ingredient).

[0291] Pharmaceutical compositions suitable for parenteral administration include an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, and sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, and sold in unit dosage forms, such as ampoules, or in multidose 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 ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0292] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions can be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using, for example, non-toxic parenterally acceptable diluents or solvents, 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 useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions may include pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts.

[0293] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; 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, PA), which is incorporated herein by reference.

[0294] Treatment method The present invention provides methods for inducing an adaptive immune response to HCV in a subject, comprising administering an effective amount of a composition of one or more isolated nucleic acids encoding one or more HCV antigens.

[0295] In one embodiment, the method provides immunity to HCV, HCV infection, or an HCV-related disease or disorder in a subject. Accordingly, the present invention provides methods for treating or preventing an HCV-related infection, disease, or disorder.

[0296] In one embodiment, the composition is administered to a subject with an HCV-related infection, disease, or disorder. In one embodiment, the composition is administered to a subject at risk of developing an HCV-related infection, disease, or disorder. For example, the composition may be administered to a subject at risk of contact with HCV. 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 HCV is prevalent. In one embodiment, the composition is administered to a subject who has come into contact with, or is expected to come into contact with, others who live in, have traveled to, or are expected to travel to a geographic area where HCV is prevalent. In one embodiment, the composition is administered to a subject known to have been exposed to HCV through occupational, sexual, or other contact.

[0297] In one embodiment, the method comprises administering a composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more HCV antigens. In one embodiment, the method comprises administering a composition comprising a first nucleoside-modified nucleic acid molecule encoding one or more HCV antigens and a second nucleoside-modified nucleic acid molecule encoding one or more HCV antigens. In one embodiment, the method comprises administering a composition comprising one or more nucleoside-modified nucleic acid molecules encoding multiple lineage HCV antigens described herein.

[0298] In one embodiment, the method comprises administering one or more compositions each comprising one or more nucleoside-modified nucleic acid molecules encoding one or more HCV antigens. In one embodiment, the method comprises administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more HCV antigens and a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more HCV antigens. In one embodiment, the method comprises administering multiple compositions each comprising one or more nucleoside-modified nucleic acid molecules encoding one or more lineage HCV antigens described herein. In some embodiments, the method comprises administering the multiple compositions in a staggered manner.

[0299] In some embodiments, administering to the subject multiple nucleoside-modified nucleic acid molecules encoding multiple HCV antigens, adjuvants, or combinations thereof.

[0300] In some embodiments, the methods of the invention allow for sustained expression of an HCV antigen or adjuvant described herein for at least several days after administration. The methods of the invention allow for sustained expression of an HCV antigen or adjuvant described herein for at least two weeks after administration. The methods of the invention allow for sustained expression of an HCV antigen or adjuvant described herein for at least one month after administration. However, in some embodiments, the nucleic acid is not integrated into the subject's genome, and therefore, in some embodiments, the methods also provide for transient expression.

[0301] In some embodiments, the methods involve administering a nucleoside-modified RNA that provides stable expression of an HCV antigen or adjuvant described herein. In some embodiments, administration of the nucleoside-modified RNA induces an effective adaptive immune response while eliciting little or no innate immune response.

[0302] In some embodiments, the method provides durable protection against HCV. For example, in some embodiments, the method provides durable protection against HCV for more than two weeks. In some embodiments, the method provides durable protection against HCV for one month or more. In some embodiments, the method provides durable protection against HCV for two months or more. In some embodiments, the method provides durable protection against HCV for three months or more. In some embodiments, the method provides durable protection against HCV for four months or more. In some embodiments, the method provides durable protection against HCV for five months or more. In some embodiments, the method provides durable protection against HCV for six months or more. In some embodiments, the method provides durable protection against HCV for one year or more.

[0303] In one embodiment, a single vaccination of the composition induces durable protection against HCV for one or more months, two or more months, three or more months, four or more months, five or more months, six or more months, or one or more years.

[0304] In therapeutic methods, the compositions of the present invention can be administered in many different ways using methods known in the art. In one embodiment, the methods of the present invention involve systemic administration to the subject, for example, enteral or parenteral administration. In some embodiments, the methods involve intradermal delivery of the composition. In another embodiment, the methods involve intravenous delivery of the composition. In some embodiments, the methods involve intramuscular delivery of the composition. In one embodiment, the methods involve subcutaneous delivery of the composition. In one embodiment, the methods involve inhalation of the composition. In one embodiment, the methods involve intranasal delivery of the composition.

[0305] It will be understood that the compositions of the present invention can be administered to a subject alone or in combination with another agent.

[0306] Thus, the therapeutic and prophylactic methods of the present invention encompass the use of pharmaceutical compositions encoding the HCV antigens, adjuvants, or combinations 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 the 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.

[0307] Typically, dosages that can be administered to a mammal, such as a human, using the methods of the present invention range from 0.01 μg to about 50 mg per kilogram of mammalian body weight, although the exact dosage administered will vary depending on a variety of factors, including, but not limited to, the type of mammal, the condition and type of disease 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 to about 10 mg per kilogram of mammalian body weight. In some embodiments, the dosage varies from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0308] The composition may be administered to the mammal several times daily, or less frequently, e.g., once a day, once a week, once every two weeks, once a month, etc., or even less frequently, e.g., every few months, every few years, or even less frequently, e.g., every 10-20 years, every 15-30 years, or even less frequently, e.g., every 50-100 years, etc. The frequency of administration 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.

[0309] In some embodiments, administration of the immunogenic compositions or vaccines of the invention may be by single administration or may be boosted by multiple administrations.

[0310] In one embodiment, the invention encompasses methods comprising administering one or more compositions encoding one or more HCV antigens or adjuvants described herein. In some embodiments, the methods have an additive effect, where the overall effect of administering the combination is approximately equal to the sum of the effects of administering each HCV antigen or adjuvant. In other embodiments, the methods have a synergistic effect, where the overall effect of administering the combination is greater than the sum of the effects of administering each HCV antigen or adjuvant.

[0311] The present invention will be described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.

[0312] Without further description, it is believed that one skilled in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed methods. Accordingly, the following examples are not to be construed as limiting the disclosure in any way. [Example]

[0313] Example 1: Protection against Hepatitis C Virus (HCV) by Nucleoside-Modified mRNA Vaccination We have developed a novel vaccine platform utilizing nucleoside-modified mRNA. This platform specifically induces potent T follicular helper (Tfh) cell responses. Tfh cells induce B cell proliferation, affinity maturation, class switching, high-level antibody production, and long-term memory. This platform was used to develop a vaccine for hepatitis C virus (HCV) using a novel viral envelope strain design approach. The HCV vaccine encodes a strain of envelope proteins expressed as a single protein / mRNA containing core and envelope 1 and 2 proteins (C-E1-E2), which self-assembles and buds from transfected cells as noninfectious virus-like particles. In some embodiments, the vaccine platform encapsulates the modified RNA using lipid nanoparticles (LNPs). LNP encapsulation allows for efficient delivery and expression of mRNA in vivo, and the modification using 1-methylpseudouridine nucleoside instead of uridine is important for the potency of the antibody response induced by the mRNA platform through the induction of Tfh cells, although a variety of other nucleoside-modified or unmodified mRNAs and other lipid, carbohydrate, protein, polymer, and other delivery systems can also be used to create similar vaccines.

[0314] The unmodified sequences of HCV E1 and E2 antigens are identified by analyzing the evolution of HCV sequences of the complete E1E2 envelope genes over a 12-month period in treatment-naive subjects, beginning with acute infection before anti-HCV antibody seroconversion through resolution of plasma viremia and cure, as described by Bailey et al. (2017, Broadly neutralizing antibodies with few somatic mutations and hepatitis C virus clearance, Journal of Clinical Investigation Insight, Volume 2), which is incorporated herein by reference in its entirety.

[0315] This subject developed broadly neutralizing antibodies (bNAbs) that neutralized his own autologous virus, as well as a large and significant proportion of heterologous viruses. Using single-genome sequencing (SGS) techniques, three distinct transmitted / founder (T / F) viral genomes responsible for productive clinical infection in this subject were identified. Using the same SGS method, the evolution of HCV E1E2 sequences was characterized over a one-year period after the subject spontaneously resolved viremia. Analysis of sequential viral E1E2 sequences obtained at seven time points over this 12-month period revealed a series of stringent viral population bottlenecks. This was due to the development of autologous, strain-specific neutralizing antibodies (NAbs) that evolved to ultimately achieve neutralizing breadth (bNAbs). Within the epidemic of evolving viruses, we identified a series of viral NAbs that circumvented E1E2 variants that maintained viremia by continuing NAb evolution until the breadth and potency of NAbs were sufficient to clear viral infection. This resulted in sustained viral control and clinical cure of the infection in this subject. While coevolution of viral antibodies leading to neutralization breadth is an established concept in human immunodeficiency virus type 1 (HIV-1) infection, it has not previously been reported for HCV infection. Here, we describe a series of evolving HCV E1E2 sequences that elicited NAb breadth and potency sufficient to clear HCV infection and neutralize a broad panel of heterologous HCV viruses. These HCV E1E2 sequences are predicted to be sufficient to elicit bNAbs in humans vaccinated with this novel RNA expression platform or other vaccine delivery systems, including viruses, plasmids, proteins, and peptides, and to protect humans from HCV infection. HCV E1E2 sequences used in this novel HCV vaccine include, but are not limited to, those disclosed herein.

[0316] As shown in Figures 1A and 2A, HCV E1E2 antigens, including those designated AL in Example 2 below, are capable of binding to mAbs. Figures 1B and 2B show dendrograms illustrating the phenotypic relationships among different HCV E1E2 antigens. Figures 3A and 3B show a phylogenetic tree and highlighted plots of HCV E1E2 antigens. Figure 4 shows Western blots of 293T cells transfected with HCV A and HCV B, demonstrating the presence of all proteins.

[0317] Example 2: Sequences The immunogens shown below are labeled AI based on the dendrograms in Figures 1, 2, and 3. In some embodiments, the immunogens include E1 and E2 as identified above. In some embodiments, the immunogens further include core protein. The core protein may be from any HCV isolate. The amino acid sequences of E1E2 and the amino acid and nucleotide sequences of the core-E1E2 immunogens of the AIs are provided.

[0318] E1E2 DNA sequences Sequence A: E1E2 DNA (A-CE1E2-11108_20040817_FB08_A-DNA) (SEQ ID NO: 1) Sequence B: E1E2 DNA (B-CE1E2-inferred-11108-DNA) (SEQ ID NO: 2) Sequence C: E1E2 DNA (C-CE1E2-11108_20050210_FD13_C-DNA) (SEQ ID NO: 3) Sequence D: E1E2 DNA (D-CE1E2-11108_20050512_FB05_D-DNA) (SEQ ID NO: 4) Sequence E: E1E2 DNA (E-CE1E2-11108_20050823_5A12_E-DNA) (SEQ ID NO: 5) Sequence F: E1E2 DNA (F-CE1E2-11108_20040817_FA01_F-DNA (SEQ ID NO: 6) Sequence G: E1E2 DNA (G-CE1E2-11108_20050210_FB02_G-DNA) (SEQ ID NO: 7) Sequence H: E1E2 DNA (H-CE1E2-11108_20050512_FA22_H-DNA) (SEQ ID NO: 8) Sequence I: E1E2 DNA (I-CE1E2-11108_20050720_FE05_I-DNA) (SEQ ID NO: 9)

[0319] E1E2 protein sequences Sequence A: E1E2 protein (A-CE1E2-11108_20040817_FB08_A) (SEQ ID NO: 10) Sequence B: E1E2 protein (B-CE1E2-inferred-11108) (SEQ ID NO: 11) Sequence C: E1E2 protein (C-CE1E2-11108_20050210_FD13_C) (SEQ ID NO: 12) Sequence D: E1E2 protein (D-CE1E2-11108_20050512_FB05_D) (SEQ ID NO: 13) Sequence E: E1E2 protein (E-CE1E2-11108_20050823_5A12_E) (SEQ ID NO: 14) Sequence F: E1E2 protein (F-CE1E2-11108_20040817_FA01_F) (SEQ ID NO: 15) Sequence G: E1E2 protein (G-CE1E2-11108_20050210_FB02_G) (SEQ ID NO: 16) Sequence H: E1E2 protein (H-CE1E2-11108_20050512_FA22_H) (SEQ ID NO: 17) Sequence I: E1E2 protein (I-CE1E2-11108_20050720_FE05_I) (SEQ ID NO: 18)

[0320] Core-E1E2 DNA sequence Sequence A: C1E1E2 DNA (A-CE1E2-11108_20040817_FB08_A-DNA) (SEQ ID NO: 19) Sequence B: C1E1E2 DNA (B-CE1E2-inferred-11108-DNA) (SEQ ID NO: 20) Sequence C: C1E1E2 DNA (C-CE1E2-11108_20050210_FD13_C-DNA) (SEQ ID NO: 21) Sequence D: C1E1E2 DNA (D-CE1E2-11108_20050512_FB05_D-DNA) (SEQ ID NO: 22) Sequence E: C1E1E2 DNA (E-CE1E2-11108_20050823_5A12_E-DNA) (SEQ ID NO: 23) Sequence F: C1E1E2 DNA (F-CE1E2-11108_20040817_FA01_F-DNA) (SEQ ID NO: 24) Sequence G: C1E1E2 DNA (G-CE1E2-11108_20050210_FB02_G-DNA) (SEQ ID NO: 25) Sequence H: C1E1E2 DNA (H-CE1E2-11108_20050512_FA22_H-DNA) (SEQ ID NO: 26) Sequence I: C1E1E2 DNA (I-CE1E2-11108_20050720_FE05_I-DNA) (SEQ ID NO: 27)

[0321] Core-E1E2 protein sequence Sequence A: C1E1E2 protein (A-CE1E2-11108_20040817_FB08_A) (SEQ ID NO: 28) Sequence B: C1E1E2 protein (B-CE1E2-inferred-11108) (SEQ ID NO: 29) Sequence C: C1E1E2 protein (C-CE1E2-11108_20050210_FD13_C) (SEQ ID NO: 30) Sequence D: C1E1E2 protein (D-CE1E2-11108_20050512_FB05_D) (SEQ ID NO: 31) Sequence E: C1E1E2 protein (E-CE1E2-11108_20050823_5A12_E) (SEQ ID NO: 32) Sequence F: C1E1E2 protein (F-CE1E2-11108_20040817_FA01_F) (SEQ ID NO: 33) Sequence G: C1E1E2 protein (G-CE1E2-11108_20050210_FB02_G) (SEQ ID NO: 34) Sequence H: C1E1E2 protein (H-CE1E2-11108_20050512_FA22_H) (SEQ ID NO: 35) Sequence I: C1E1E2 protein (I-CE1E2-11108_20050720_FE05_I) (SEQ ID NO: 36)

[0322] Example 3: Additional sequences (J, K, L) Based on the phylogenetic tree in Figure 2, the E1E2 DNA sequences of the immunogens labeled JL are provided. Sequence J: s117_20041118_FB05 (SEQ ID NO: 37) Sequence K: s117_20050210_FB05 (SEQ ID NO: 38) Sequence L: s117_20050210_1a051 (SEQ ID NO: 39)

[0323] Example 4: All sequences The nucleotide sequence of each HCV isolate listed in Figure 3 is provided.

[0324] >s117.D110.1a049_U01 (SEQ ID NO: 40) >s117.D110.A02 (SEQ ID NO: 41) >s117.D110.A03 (SEQ ID NO: 42) >s117.D110.A04 (SEQ ID NO: 43) >s117.D110.A06 (SEQ ID NO: 44) >s117.D110.A07 (SEQ ID NO: 45) >s117.D110.A10 (SEQ ID NO: 46) >s117.D110.A11 (SEQ ID NO: 47) >s117.D110.A16 (SEQ ID NO: 48) >s117.D110.A21 (SEQ ID NO: 49) >s117.D110.A23 (SEQ ID NO: 50) >s117.D110.A24 (SEQ ID NO: 51) >s117.D110.A25 (SEQ ID NO: 52) >s117.D110.A27 (SEQ ID NO: 53) >s117.D110.A28 (SEQ ID NO: 54) >s117.D110.A30 (SEQ ID NO: 55) >s117.D110.A31 (SEQ ID NO: 56) >s117.D110.A32 (SEQ ID NO: 57) >s117.D110.B01 (SEQ ID NO: 58) >s117.D110.B02 (SEQ ID NO: 59) >s117.D110.B03 (SEQ ID NO: 60) >s117.D110.B04 (SEQ ID NO: 61) >s117.D110.B05 (SEQ ID NO: 62) >s117.D110.B06 (SEQ ID NO: 63) >s117.D110.B07 (SEQ ID NO: 64) >s117.D110.B08 (SEQ ID NO: 65) >s117.D110.B09 (SEQ ID NO: 66) >s117.D110.B10 (SEQ ID NO: 67) >s117.D110.B12 (SEQ ID NO: 68) >s117.D110.B14 (SEQ ID NO: 69) >s117.D110.B15 (SEQ ID NO: 70) >s117.D110.B16 (SEQ ID NO: 71) >s117.D110.FA01 (SEQ ID NO: 72) >s117.D110.FA02 (SEQ ID NO: 73) >s117.D110.FA04 (SEQ ID NO: 74) >s117.D110.FA07 (SEQ ID NO: 75) >s117.D110.FA08 (SEQ ID NO: 76) >s117.D110.FB03 (SEQ ID NO: 77) >s117.D110.FB04 (SEQ ID NO: 78) >s117.D110.FB05 (SEQ ID NO: 79) >s117.D110.FB11 (SEQ ID NO: 80) >s117.D110.FB13 (SEQ ID NO: 81) >s117.D110.FB14 (SEQ ID NO: 82) >s117.D110.FT01 (SEQ ID NO: 83) >s117.D110.FT03 (SEQ ID NO: 84) >s117.D110.T03 (SEQ ID NO: 85) >s117.D110.T06 (SEQ ID NO: 86) >s117.D110.T07 (SEQ ID NO: 87) >s117.D17.1a053_v02 (sequence number 88) >s117.D17.FA01 (SEQ ID NO: 89) >s117.D17.FA10 (SEQ ID NO: 90) >s117.D17.FA12 (SEQ ID NO: 91) >s117.D17.FB01 (sequence number 92) >s117.D17.FB03 (SEQ ID NO: 93) >s117.D17.FB06 (SEQ ID NO: 94) >s117.D17.FB08 (SEQ ID NO: 95) >s117.D17.FB12 (SEQ ID NO: 96) >s117.D17.FB18 (SEQ ID NO: 97) >s117.D17.FB19 (SEQ ID NO: 98) >s117.D17.FB23 (SEQ ID NO: 99) >s117.D17.FB24 (sequence number 100) >s117.D17.FC01 (sequence number 101) >s117.D17.FC10 (SEQ ID NO: 102) >s117.D17.FD02 (sequence number 103) >s117.D17.FD06 (sequence number 104) >s117.D17.FD09 (sequence number 105) >s117.D17.FT02 (sequence number 106) >s117.D194.1a050_U02 (sequence number 107) >s117.D194.1a051_U02 (sequence number 108) >s117.D194.A02 (sequence number 109) >s117.D194.A05 (SEQ ID NO: 110) >s117.D194.A06 (SEQ ID NO: 111) >s117.D194.A07 ​​(SEQ ID NO: 112) >s117.D194.A08 (SEQ ID NO: 113) >s117.D194.A10 (SEQ ID NO: 114) >s117.D194.A12 (SEQ ID NO: 115) >s117.D194.A13 (SEQ ID NO: 116) >s117.D194.A14 (SEQ ID NO: 117) >s117.D194.A17 (sequence number 118) >s117.D194.A18 (SEQ ID NO: 119) >s117.D194.A19 (sequence number 120) >s117.D194.B01 (sequence number 121) >s117.D194.B02 (sequence number 122) >s117.D194.B03 (sequence number 123) >s117.D194.B04 (sequence number 124) >s117.D194.B05 (sequence number 125) >s117.D194.B06 (sequence number 126) >s117.D194.B07 (sequence number 127) >s117.D194.B08 (sequence number 128) >s117.D194.B09 (sequence number 129) >s117.D194.B10 (sequence number 130) >s117.D194.FA01 (SEQ ID NO: 131) >s117.D194.FA14 (SEQ ID NO: 132) >s117.D194.FB01 (sequence number 133) >s117.D194.FB02 (sequence number 134) >s117.D194.FB04 (SEQ ID NO: 135) >s117.D194.FB05 (SEQ ID NO: 136) >s117.D194.FB07 (SEQ ID NO: 137) >s117.D194.FB09 (SEQ ID NO: 138) >s117.D194.FB12 (SEQ ID NO: 139) >s117.D194.FB13 (SEQ ID NO: 140) >s117.D194.FB14 (SEQ ID NO: 141) >s117.D194.FC03 (SEQ ID NO: 142) >s117.D194.FD02 (sequence number 143) >s117.D194.FD04 (SEQ ID NO: 144) >s117.D194.FD06 (SEQ ID NO: 145) >s117.D194.FD07 (SEQ ID NO: 146) >s117.D194.FD10 (SEQ ID NO: 147) >s117.D194.FD11 (SEQ ID NO: 148) >s117.D194.FD13 (SEQ ID NO: 149) >s117.D194.FD14 (sequence number 150) >s117.D194.FD16 (SEQ ID NO: 151) >s117.D194.FD18 (SEQ ID NO: 152) >s117.D194.FD19 (sequence number 153) >s117.D194.FD20 (sequence number 154) >s117.D285.1a052_U03 (sequence number 155) >s117.D285.A01 (sequence number 156) >s117.D285.A04 (sequence number 157) >s117.D285.A05 (sequence number 158) >s117.D285.A07 (SEQ ID NO: 159) >s117.D285.A08 (sequence number 160) >s117.D285.A09 (SEQ ID NO: 161) >s117.D285.A10 (SEQ ID NO: 162) >s117.D285.A11 (sequence number 163) >s117.D285.A12 (sequence number 164) >s117.D285.A16 (sequence number 165) >s117.D285.A19 (SEQ ID NO: 166) >s117.D285.A20 (sequence number 167) >s117.D285.B05 (sequence number 168) >s117.D285.B11 (SEQ ID NO: 169) >s117.D285.B13 (SEQ ID NO: 170) >s117.D285.B16 (SEQ ID NO: 171) >s117.D285.B19 (SEQ ID NO: 172) >s117.D285.FA03 (SEQ ID NO: 173) >s117.D285.FA04 (SEQ ID NO: 174) >s117.D285.FA06 (SEQ ID NO: 175) >s117.D285.FA12 (SEQ ID NO: 176) >s117.D285.FA15 (SEQ ID NO: 177) >s117.D285.FA17 (SEQ ID NO: 178) >s117.D285.FA18 (SEQ ID NO: 179) >s117.D285.FA22 (SEQ ID NO: 180) >s117.D285.FA23 (SEQ ID NO: 181) >s117.D285.FA24 (SEQ ID NO: 182) >s117.D285.FB04 (sequence number 183) >s117.D285.FB05 (sequence number 184) >s117.D285.FB08 (sequence number 185) >s117.D285.FB09 (sequence number 186) >s117.D285.FB10 (sequence number 187) >s117.D285.FB12 (sequence number 188) >s117.D285.FB14 (sequence number 189) >s117.D285.FB15 (sequence number 190) >s117.D285.FB17 (sequence number 191) >s117.D285.FB22 (sequence number 192) >s117.D285.FB23 (sequence number 193) >s117.D285.FT06 (sequence number 194) >s117.D285.FT07 (sequence number 195) >s117.D354.FA04 (SEQ ID NO: 196) >s117.D354.FB01 (sequence number 197) >s117.D354.FB04 (sequence number 198) >s117.D354.FB05 (sequence number 199) >s117.D354.FB09 (sequence number 200) >s117.D354.FBO6 (sequence number 201) >s117.D354.FC06 (sequence number 202) >s117.D354.FC07 (sequence number 203) >s117.D354.FC12 (SEQ ID NO: 204) >s117.D354.FC13 (SEQ ID NO: 205) >s117.D354.FC17 (SEQ ID NO: 206) >s117.D354.FC22 (SEQ ID NO: 207) >s117.D354.FD06 (sequence number 208) >s117.D354.FD08 (sequence number 209) >s117.D354.FD11 (sequence number 210) >s117.D354.FD14 (sequence number 211) >s117.D354.FD15 (sequence number 212) >s117.D354.FD16 (sequence number 213) >s117.D354.FD17 (sequence number 214) >s117.D354.FD20 (sequence number 215) >s117.D354.FD24 (sequence number 216) >s117.D354.FD27 (sequence number 217) >s117.D354.FE01 (sequence number 218) >s117.D354.FE05 (sequence number 219) >s117.D354.FE10 (sequence number 220) >s117.D354.FE11 (sequence number 221) >s117.D354.FE14 (sequence number 222) >s117.D354.FF01 (sequence number 223) >s117.D388.5A03 (sequence number 224) >s117.D388.5A04 (sequence number 225) >s117.D388.5A05 (sequence number 226) >s117.D388.5A11 (sequence number 227) >s117.D388.5A12 (sequence number 228) >s117.D388.5A13 (sequence number 229) >s117.D388.5T01 (sequence number 230) >s117.D388.5T03 (sequence number 231) >s117.D46.A02 (sequence number 232) >s117.D46.A08 (sequence number 233) >s117.D46.A10 (sequence number 234) >s117.D46.A12 (sequence number 235) >s117.D46.A14 (sequence number 236) >s117.D46.A17 (sequence number 237) >s117.D46.A18 (sequence number 238) >s117.D46.A20 (sequence number 239) >s117.D46.A22 (sequence number 240) >s117.D46.A25 (sequence number 241) >s117.D46.A27 (sequence number 242) >s117.D46.A35 (sequence number 243) >s117.D46.B02 (sequence number 244) >s117.D46.B10 (sequence number 245) >s117.D46.B13 (sequence number 246) >s117.D46.B26 (sequence number 247) >s117.D46.B30 (sequence number 248) >s117.D46.B35 (SEQ ID NO: 249) >s117.D46.C01 (sequence number 250) >s117.D46.FA01 (sequence number 251) >s117.D46.FA02 (sequence number 252) >s117.D46.FA03 (sequence number 253) >s117.D46.FA07 (sequence number 254) >s117.D46.FA08 (sequence number 255) >s117.D46.FA13 (sequence number 256) >s117.D46.FA17 (sequence number 257) >s117.D46.FA22 (sequence number 258) >s117.D46.FB03 (sequence number 259) >s117.D46.FB04 (sequence number 260) >s117.D46.FB07 (sequence number 261) >s117.D46.FB09 (sequence number 262) >s117.D46.FB10 (sequence number 263) >s117.D46.FB12 (sequence number 264) >s117.D46.FB14 (sequence number 265) >s117.D46.FB16 (sequence number 266) >s117.D46.FB20 (sequence number 267) >s117.D46.FT02 (sequence number 268)

[0325] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While 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 made by others skilled in the art without departing from the true spirit and scope of the invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A composition for inducing an immune response against hepatitis C virus (HCV) in a subject, comprising at least one isolated nucleoside-modified RNA encoding at least one HCV antigen.

2. 2. The composition of claim 1, wherein the at least one isolated nucleoside modified RNA comprises pseudouridine.

3. 2. The composition of claim 1, wherein the at least one isolated nucleoside modified RNA comprises 1-methyl-pseudouridine.

4. 2. The composition of claim 1, wherein the at least one isolated nucleoside-modified RNA is a purified nucleoside-modified RNA.

5. 2. The composition of claim 1, wherein the at least one HCV antigen comprises at least one HCV antigen selected from the group consisting of envelope protein E1 (E1), envelope protein E2 (E2), and core protein (C).

6. 2. The composition of claim 1, wherein the at least one HCV antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:

36.

7. 2. The composition of claim 1, wherein the at least one nucleoside-modified RNA comprises a nucleotide sequence encoded by a DNA sequence comprising at least one nucleotide 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, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:

39.

8. The composition of claim 1 , wherein the composition further comprises an adjuvant.

9. 10. The composition of claim 1, wherein the at least one nucleoside-modified RNA further encodes at least one adjuvant.

10. 10. The composition of claim 1, further comprising a lipid nanoparticle (LNP).

11. 13. The composition of claim 12, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP.

12. The composition of claim 1, wherein the composition is a vaccine.

13. A method for inducing an adaptive immune response against hepatitis C virus (HCV) in a subject, comprising administering to the subject an effective amount of a composition comprising at least one nucleoside-modified RNA encoding at least one HCV antigen.

14. 14. The method of claim 13, wherein the at least one isolated nucleoside modified RNA comprises pseudouridine.

15. 14. The method of claim 13, wherein the at least one isolated nucleoside modified RNA comprises 1-methyl-pseudouridine.

16. 14. The method of claim 13, wherein the at least one isolated nucleoside-modified RNA is a purified nucleoside-modified RNA.

17. 14. The method of claim 13, wherein the at least one HCV antigen comprises at least one HCV antigen selected from the group consisting of envelope protein E1 (E1), envelope protein E2 (E2), and core protein (C).

18. 14. The method of claim 13, wherein the at least one HCV antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:

36.

19. 14. The method of claim 13, wherein the at least one nucleoside-modified RNA comprises a nucleotide sequence encoded by a DNA sequence comprising at least one nucleotide 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, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:

39.

20. 14. The method of claim 13, wherein the method further comprises administering to the subject an effective amount of an adjuvant.

21. 14. The method of claim 13, wherein the at least one nucleoside-modified RNA further encodes at least one adjuvant.

22. 14. The method of claim 13, wherein the composition further comprises a lipid nanoparticle (LNP).

23. 14. The method of claim 13, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP.

24. 14. The method of claim 13, wherein the composition is a vaccine.

25. 14. The method of claim 13, wherein the composition is administered by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

26. 14. The method of claim 13, wherein the method comprises a single administration of the composition.

27. 14. The method of claim 13, wherein the method comprises multiple administrations of the composition.

28. 14. The method of claim 13, wherein the method treats or prevents an HCV-related infection, disease, or disorder in the subject.

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