VLP Enterovirus Vaccine

Abstract

Aspects of the present disclosure relate to messenger RNA vaccine compositions and methods of administration thereof. The compositions provided herein include one or more RNA polynucleotides having an open reading frame encoding a picornavirus capsid polyprotein and protease. The compositions provided herein include one or more RNA polynucleotides having an open reading frame encoding an active product that modulates the expression, structure or function of at least one other RNA or its product.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 157,543, entitled "VLP Enteroviral Vaccines," filed March 5, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] The Picornaviridae family of viruses is one of the largest viral families, constituting the genus Enterovirus, and includes many species of pathogens, such as enterovirus and rhinovirus species, that are responsible for some of the most common infectious diseases in humans. Excluding rhinovirus infections, which are highly prevalent, it is estimated that approximately 10 to 15 million symptomatic enterovirus infections occur annually in the United States alone (van der Linden et al., 2015).

[0003] Vaccination is an effective method of providing preventive protection against infectious diseases, including but not limited to viral, bacterial, and / or parasitic diseases, such as influenza, HIV, hepatitis virus infection, cholera, malaria, and tuberculosis, as well as many other diseases. However, the development of vaccines targeting some enteroviruses has proven difficult, at least in part, because difficult-to-target and highly variable regions are exposed on the surface of enterovirus particles, while conserved regions suitable for targeting are hidden between the enterovirus particles.

[0004] Recent advances in recombinant DNA technology have led to increased interest in the use of virus-like particles (VLPs) for use in vaccine development. VLPs are formed by the self-assembly of viral structural proteins that mimic the morphology of pathogens and have been shown to be both non-infectious and highly immunogenic. However, previous attempts to generate enterovirus VLPs have met with limited success due to difficulties in achieving proper expression and / or folding of the structural proteins required to form VLPs that mimic enterovirus pathogens.

[0005] This disclosure describes how mRNA can be used to code and deliver both a catalytic enzyme protein and a polyprotein encoding multiple substrates for the enzyme, and then allow the enzyme to promote assembly of higher order structures in the form of VLPs to act as immunogens in vivo.Therefore, it is of great interest to develop enterovirus VLPs from mRNA for use as enterovirus vaccines as a new approach to combat infectious diseases and agents. Summary of the Invention [Problem to be solved by the invention]

[0006] Given the lack of effective vaccines against infections caused by enteroviruses, with the exception of polio vaccines, there is a great need for safe and effective vaccines in all patient populations to prevent and / or treat other types of enterovirus infections. Described herein are compositions and methods for nucleic acid vaccines. In particular, described herein are compositions and methods for nucleic acid vaccines (e.g., mRNA vaccines). [Means for solving the problem]

[0007] Aspects of the present disclosure relate to a composition comprising: (i) a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a picornavirus capsid polyprotein, the capsid polyprotein comprising a viral P1 precursor polyprotein; and (ii) a lipid nanoparticle (LNP).

[0008] A further aspect of the present disclosure relates to a composition comprising: (i) a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a protease, wherein the protease is a picornavirus 3C protease; and (ii) a lipid nanoparticle (LNP).

[0009] Further aspects of the present disclosure relate to immunogenic compositions comprising: (i) a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a picornavirus 3C protease; (ii) an mRNA comprising an ORF encoding a picornavirus capsid polyprotein, the capsid polyprotein comprising a viral P1 precursor polyprotein; and (iii) a lipid nanoparticle (LNP).

[0010] In some embodiments, the precursor polyprotein comprises two or more capsid proteins having a viral protease specific cleavage site between the two or more capsid proteins. In some embodiments, the two or more capsid proteins comprise two or more of viral protein 0 (VP0), viral protein 1 (VP1), and viral protein 3 (VP3). In some embodiments, VP0 further comprises viral protein 2 (VP2) and viral protein 4 (VP4), where VP2 and VP4 comprise the cleavage site for capsid maturation.

[0011] In some embodiments, the protease is picornavirus 3C (3CD). In some embodiments, the 3CD is specific to species of the genus Enterovirus in the family Picornaviridae. In some embodiments, the 3CD is specific to species of human rhinovirus (HRV) A, B, or C. In some embodiments, the HRV species is HRV-A16. In some embodiments, the HRV species is HRV-B14. In some embodiments, the 3CD is specific to an enterovirus species. In some embodiments, the enterovirus genotype is EV-D68. In some embodiments, the enterovirus genotype is EV-A71.

[0012] In some embodiments, the capsid proteins form protomers. In some embodiments, the protomers form pentamers. In some embodiments, the pentamers form virus-like particles (VLPs).

[0013] In some embodiments, the mRNA comprising an ORF encoding the viral P1 precursor polyprotein and the mRNA comprising an ORF encoding the picornavirus 3C protease are present in one of the following ratios (P1:3CD): 20:1, 10:1, 7:1, 5:1, 4:1, 3:1, 2:1, 1:1. In some embodiments, the ratio of the mRNA comprising an ORF encoding the viral P1 precursor protein to the mRNA comprising an ORF encoding the picornavirus 3C protease is 10:1. In some embodiments, the ratio of the mRNA comprising an ORF encoding the viral P1 precursor polyprotein to the mRNA comprising an ORF encoding the 3C protease as either 3C or 3CD is 2:1. In some embodiments, the protease comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:14. In some embodiments, the protease comprises the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:14. In some embodiments, the protease comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 26. In some embodiments, the protease comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 26.

[0014] In some embodiments, the capsid polyprotein comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 5, 11, 17, or 23. In some embodiments, the capsid polyprotein comprises the amino acid sequence of any one of SEQ ID NOs: 5, 11, 17, or 23. In some embodiments, the VLP comprises a neutralizing immunogenic (NIm) site.

[0015] In some embodiments, the LNP comprises an ionic amino lipid, a PEG-modified lipid, a structured lipid, and a phospholipid.

[0016] In some embodiments, the mRNA comprising an ORF encoding the viral P1 precursor polyprotein and the mRNA comprising an ORF encoding the picornavirus 3C protease are co-formulated into at least one LNP. In some embodiments, the mRNA comprising an ORF encoding the viral P1 precursor polyprotein and the mRNA comprising an ORF encoding the picornavirus 3C protease are each formulated into separate LNPs. In some embodiments, the LNPs formulated with the mRNA comprising an ORF encoding the viral P1 precursor polyprotein and the mRNA comprising an ORF encoding the picornavirus 3C protease are present in one of the following ratios: 20:1, 10:1, 7:1, 5:1, 4:1, 3:1, 2:1, 1:1. In some embodiments, the LNPs comprise an ionic amino lipid, a sterol, a neutral lipid, and a PEG-modified lipid. In some embodiments, the lipid nanoparticles comprise 40-55 mol% ionic amino lipid, 30-45 mol% sterol, 5-15 mol% neutral lipid, and 1-5 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticles comprise 40-50 mol% ionic amino lipid, 35-45 mol% sterol, 10-15 mol% neutral lipid, and 2-4 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticles comprise 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol% ionic amino lipid. In some embodiments, the ionic amino lipid has the structure of Compound 2: [ka] In some embodiments, the sterol is cholesterol or a variant thereof. In some embodiments, the neutral lipid is 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0017] A further aspect of the present disclosure relates to a method comprising administering to a subject an immunogenic composition comprising (i) a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding a picornavirus protease, and (ii) a messenger ribonucleic acid (mRNA) comprising an open reading frame encoding a capsid polyprotein comprising precursor proteins, the precursor proteins comprising two or more capsid proteins having a cleavage site specific for the protease between the two or more capsid proteins, in an amount effective to induce an immune response in the subject against a viral infection from a member of the Enterovirus genus.

[0018] In some embodiments, the immune response includes binding antibody titers against human rhinovirus species of the Enterovirus genus. In some embodiments, the immune response includes neutralizing antibody titers against human rhinovirus species of the Enterovirus genus. In some embodiments, the immune response includes a T cell response against human rhinovirus species of the Enterovirus genus. In some embodiments, the human rhinovirus species is selected from the group consisting of genotypes A2, A16, B14, and C15. In some embodiments, the human rhinovirus genotype is human rhinovirus 16 (HRV16).

[0019] In some embodiments, the immune response includes binding antibody titers against a Human Enterovirus species of the Enterovirus genus. In some embodiments, the immune response includes neutralizing antibody titers against a Human Enterovirus species of the Enterovirus genus. In some embodiments, the immune response includes a T cell response against a Human Enterovirus species of the Enterovirus genus. In some embodiments, the Human Enterovirus species virus is selected from the group consisting of RV-A, RV-B, RV-C, EV-A, and EV-D. In some embodiments, the Human Enterovirus species virus is selected from the group consisting of genotypes RV-A16, RV-B14, RV-C15, EV-A71, and EV-D68. In some embodiments, the Human Enterovirus species virus is Enterovirus D68 (EV-D68). In some embodiments, the Human Enterovirus species virus is Enterovirus A71 (EV-A71).

[0020] In some embodiments, the (i) mRNA is formulated in a composition comprising at least one lipid nanoparticle. In some embodiments, the (ii) mRNA is formulated in a composition comprising at least one lipid nanoparticle. In some embodiments, the (i) mRNA is administered to the subject simultaneously with the (ii) mRNA. In some embodiments, the (i) and (ii) mRNAs are formulated in a composition comprising at least one lipid nanoparticle. In some embodiments, the (i) and (ii) mRNAs are formulated in a composition comprising two lipid nanoparticles.

[0021] Further aspects of the disclosure relate to a composition comprising: (i) a first messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a first product; (ii) a second mRNA comprising an ORF encoding a second product; and (iii) a lipid nanoparticle (LNP), wherein the first product is an active protein, and the active protein can regulate the expression, structure, or activity of the second mRNA and / or the second product. In some embodiments, the composition is an immunogenic composition. In some embodiments, the first product is a catalytic protein. In some embodiments, the first product is an enzymatic protein. In some embodiments, the first product is a binding protein. In some embodiments, the first product is a polyprotein. In some embodiments, the second product is a substrate. In some embodiments, the first product is a polyprotein.

[0022] Each limitation of the present disclosure may encompass various embodiments of the present disclosure. Thus, each limitation of the present disclosure involving any one element or combination of elements is anticipated to be included in each aspect of the present disclosure. The present disclosure is not limited in its application to the details of construction or to the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0023] The accompanying drawings are not drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For clarity, not every component may be labeled in every figure. [Brief description of the drawings]

[0024] [Figure 1A]Schematic diagram of the enterovirus genome. Schematic showing the relative sizes of the capsid proteins (VP1, VP2, VP3, and VP4) and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). Protease cleavage sites of the polyprotein (P1, P2, and P3) are indicated by inverted triangles. [Figure 1B] A schematic diagram of the resulting icosahedral virion is shown. The Enterovirus icosahedral virion obtained after polyprotein processing and assembly is shown. The capsid proteins VP1, VP2, and VP3 are shown on the outside of the icosahedral virion. [Figure 2A] Figure 1 shows an mRNA vaccine composed of HRV-A16 P1+3CD mRNA demonstrating proper P1 processing in vitro and generating neutralizing antibody titers in mice. Western blot showing cell lysates from 293T cells transfected with 2 μg HRV-A16 P1 mRNA and increasing amounts of HRV-A16 3CD mRNA to give the indicated P1:3CD molar ratios. Results show that P1 is proteolytically processed into individual VP proteins. Cleavage is evidenced by the disappearance of the high molecular weight P1 band and the appearance of a lower molecular weight band corresponding to VP0. Wild-type HRV-A16 virion lysate is included for molecular weight comparison, and arrows indicate the P1 cleavage products. Cell lysates blotted for GAPDH are shown as a loading control. [Figure 2B] Figure 1 shows an mRNA vaccine composed of HRV-A16 P1+3CD mRNA demonstrating proper P1 processing in vitro and generating neutralizing antibody titers in mice. Virus-like particles (VLPs) produced by co-expression of P1 and 3CD are shown. Expi293 cells were transfected with plasmids encoding P1 and 3CD. VLPs were purified from the supernatant by PEG precipitation followed by anion exchange chromatography. VLPs were eluted and imaged using negative staining transmission electron microscopy. A scale bar of 200 nm is included for reference. [Figure 2C]Figure 1 shows an mRNA vaccine consisting of HRV-A16 P1+3CD mRNA demonstrating proper P1 processing in vitro and generating neutralizing antibody titers in mice. Graph showing that vaccination with HRV-A16 P1:3CD mRNA generates detectable neutralizing antibody titers in mice at levels comparable to those induced by injection of replicating virus. Mice were vaccinated with 10 μg of P1 mRNA and variable amounts of 3CD mRNA constituting molar ratios of P1:3CD of 10:1 and 2:1. Mice were vaccinated on days 1 and 22, and sera were collected on days 21 (after dose 1, grey) and 35 (after dose 2, black) and tested in the HRV-A16 neutralization assay. Sera from cotton rats injected with replicating HRV-A16 on days 1 and 22 were tested in parallel (cotton rat HRV-A16A / S). Horizontal bars represent geometric mean titers (GMT) and vertical bars represent standard deviations. The dotted line represents the assay limit of detection (LOD) = 25. Animals below the LOD were assigned a titer of 12.5. [Figure 2D] Figure 1 shows an mRNA vaccine consisting of HRV-A16 P1+3CD mRNA demonstrating proper P1 processing in vitro and generating neutralizing antibody titers in mice. Figure 2 shows that vaccination with EV-D68 P1:3CD mRNA at various ratios generates neutralizing antibody titers against two heterologous virus isolates. Mice were vaccinated with 10 μg P1 mRNA and variable amounts of 3CD mRNA constituting final molar ratios of 10:1, 5:1, 2:1, and 1:1 P1 mRNA:3CD mRNA. Mice were vaccinated on days 1 and 22 and sera collected on day 35 were pooled by group. Sera from pooled groups were tested for neutralization of EV-D68 clade B1 isolate US / MO / 14-18947 and clade A2 isolate US / KY / 14-18953. The dotted line represents the assay limit of quantification (LOQ=100). [Figure 3A]FIG. 1 is a graph showing neutralizing antibody responses to vaccination with P1+3CD mRNA at a molar ratio of 2:1 for different enterovirus species. FIG. 2 is a graph showing that vaccination with HRV-A16 P1+3CD mRNA results in dose-dependent production of neutralizing antibodies. The X-axis shows the μg amount of P1 and 3CD mRNA given to each dose group. Bars represent GMTs for groups of 8 mice, with individual animals represented by dots. Error bars represent standard deviation. Commercially available HRV-A16 antiserum from hyperimmune guinea pigs was included as a benchmark control (GP A / S). The dotted line on the Y-axis represents the assay LOD=25. [Figure 3B] FIG. 1 shows neutralizing antibody responses to vaccination with P1+3CD mRNA at a molar ratio of 2:1 for different enterovirus species. FIG. 2 shows that vaccination with HRV-B14 P1+3CD mRNA at doses of 5 μg, 1 μg, and 0.2 μg P1 mRNA resulted in similar levels of neutralizing antibodies and exceeded the neutralizing antibody titers of a commercial hyperimmune antiserum. The X-axis shows the μg amount of P1 and 3CD mRNA given to each dose group. Bars represent GMTs for groups of 8 mice, with individual animals represented by dots. Error bars represent standard deviation. A commercial HRV-B14 antiserum from a hyperimmune guinea pig was included as a benchmark control (GP A / S). The dotted line on the Y-axis represents the assay LOD=25. [Figure 3C]Graph showing neutralizing antibody responses to vaccination with P1+3CD mRNA at a molar ratio of 2:1 for different enterovirus species. Graph showing that vaccination with EV-D68 P1+3CD mRNA at doses of 5, 1, and 0.2 μg P1 mRNA elicits strong neutralizing antibody responses even at low doses. Sera were tested for neutralization of EV-D68 clade B1 isolate US / MO / 14-18947 and clade A2 isolate US / KY / 14-18953. X-axis shows μg amount of P1 and 3CD mRNA given to each group. Bars represent GMTs for groups of 8 mice, with individual animals represented by dots. Error bars show standard deviation. Dotted line on Y-axis represents assay limit of detection (LOD)=25. Animals with titers below the LOD were assigned a value of 12.5. Bar shading corresponds to the legend and represents the virus isolates tested. [Figure 3D] Graph showing neutralizing antibody responses to vaccination with P1+3CD mRNA at a molar ratio of 2:1 for different enterovirus species. Graph showing that vaccination with EV-A71 P1+3CD mRNA results in a dose-dependent production of antibodies capable of neutralizing clinical isolates recovered from acute flaccid myelitis patients. X-axis shows μg amount of P1 and 3CD mRNA given to each group. Bars represent GMT per dose group. Error bars show standard deviation. Dotted line on Y-axis represents assay LOD=20. Animals with titers below the LOD were assigned a titer of 10. [Figure 4]AB show that the combination of P1+3CD from two different species demonstrates P1 processing in vitro and is immunogenic in vivo. A is a Western blot showing cell lysates from 293T cells transfected with 2 μg P1 mRNA±0.75 μg 3CD mRNA blotted for P1 processing. Lane labels at the bottom of the blot indicate the HRV species of P1 and 3CD mRNA transfected. HRV-B14 P1 is cleaved when co-transfected with 3CD mRNA from either HRV-B14 or HRV-A16. Cleavage is indicated by the disappearance of the high molecular weight band corresponding to P1 and the appearance of a low molecular weight band corresponding to VP1. Cell lysates blotted for GAPDH are shown as a loading control. B is a graph showing that vaccination with the combination of HRV-B14 P1+HRV-A16 3CD mRNA produces anti-HRV-B14 neutralizing antibodies. Mice were vaccinated on days 1 and 22 with 2.5 μg of the indicated P1 mRNA + 0.94 μg of HRV-A16 3CD. Serum collected on day 35 was examined for neutralization of either HRV-A16 or HRV-B14 viruses. Commercially available hyperimmune antisera against each virus were included as benchmark controls. Solid bars represent GMTs for groups of 8 mice, with individual animals represented by dots. Error bars represent standard deviation. Hashed bars represent neutralization titers of hyperimmune antisera. Dotted line on Y-axis represents assay LOD=25. [Figure 5A]Figure 1 shows that vaccination with the P1 / 3CD mRNA combination confers dose-dependent protection from HRV16 challenge in cotton rats. Figure 2 shows that animals vaccinated with the P1 / 3CD prime-boost regimen show dose-dependent production of nAbs. Four out of five animals in each dose group that received high doses of P1 / 3CD (25, 5, and 1 μg) and five out of five animals vaccinated with live HRV16 showed detectable HRV16 nAbs at the time of challenge (2 weeks post-boost). Animals vaccinated with 0.2 μg P1 / 3CD or those that received PBS as a negative control did not have detectable nAb titers. [Figure 5B] Figure 1 shows that vaccination with the P1 / 3CD mRNA combination provides dose-dependent protection from HRV16 challenge in cotton rats. Figure 2 shows that vaccination with P1 / 3CD provides dose-dependent protection of lung tissue. Viral load in lung tissue was measured 8 hours after HRV16 challenge. Four of five animals receiving 25 μg P1 / 3CD mRNA and three of five animals receiving 5 μg were fully protected, and all animals vaccinated with 1 μg showed reduced viral load compared to PBS-vaccinated controls. No lung tissue protection was seen in animals receiving the lowest dose (0.2 μg). Animals vaccinated with a live HRV16 prime-boost regimen were included as positive and comparator controls for protection. [Figure 5C] Figure 1 shows that vaccination with the P1 / 3CD mRNA combination provides dose-dependent protection from HRV16 challenge in cotton rats. Figure 2 shows that vaccination with P1 / 3CD provides complete protection of nasal tissues at the highest dose and some protection at lower doses. Five of five animals receiving 25 μg P1 / 3CD had no detectable viral load in nasal tissues at 8 hours post-HRV16 challenge. Two of five animals vaccinated with 5 μg or 1 μg showed complete protection. No nasal protection was seen in animals receiving 0.2 μg. [Figure 5D] Figure 1 shows that vaccination with the P1 / 3CD mRNA combination confers dose-dependent protection against HRV16 challenge in cotton rats. Figure 2 shows that vaccination with P1 / 3CD confers a reduction in lung viral load as measured by qPCR for HRV16(-)vRNA. Significant reductions in lung viral titers were seen in five of five animals receiving 25 μg P1 / 3CD and in some animals receiving 5 μg or 1 μg. No reductions were seen in animals vaccinated with 0.2 μg. For each panel, bars represent GMTs for groups of five cotton rats, with individual animals represented by dots. Error bars represent geometric standard deviation. The dotted line on the Y-axis represents assay LOD=200. [Figure 6] 1 is a table showing individual cotton rat endpoint assay results from the HRV-A16 vaccination+inoculation study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In the field of therapeutics, diagnostics, reagents, and biological assays, it is of great interest to be able to design, synthesize, and deliver nucleic acids, e.g., ribonucleic acid (RNA), into a cell, whether in vitro, in vivo, in situ, or ex vivo, e.g., to effect a beneficial physiological outcome for the cell, tissue, or organ, and ultimately for the organism. One beneficial outcome is to cause intracellular translation of the nucleic acid and production of at least one encoded peptide or polypeptide of interest.

[0026] Virus-like particles (VLPs) are spherical particles that have structural and antigenic characteristics similar to live viruses. However, VLPs are distinct from live viruses in that they do not contain viral genetic material and are therefore non-infectious. Due to the antigenicity of VLPs as well as their non-infectious nature, there has been growing interest in exploring the application of VLPs in vaccination.

[0027] Currently, most VLPs are produced using recombinant or cloning strategies.VLPs can be self-assembled particles.Non-limiting examples of self-assembled VLPs and methods for producing self-assembled VLPs are described in International Patent Publication No. WO2013122262, the contents of which are incorporated herein by reference in their entirety.

[0028] VLPs are formed from the assembly of structural viral proteins (e.g., envelope and / or capsid proteins). The size and shape of a VLP depends, at least in part, on the particular structural viral proteins that are incorporated into the particle upon assembly. VLPs assembled from structural viral proteins of enveloped viruses can, for example, contain one or more envelope proteins and one or more capsid proteins. VLPs assembled from structural viral proteins of non-enveloped viruses can, for example, contain one or more capsid proteins.

[0029] Multiple capsid proteins can be organized by co-expression of capsid proteins from bicistronic or multicistronic vectors in the same cell. Attempts have been made to produce VLPs that mimic Enterovirus viruses, including expressing viral structural proteins on different vectors in the same cell and / or designing fusion proteins of viral structural proteins and chaperone proteins, but the resulting VLPs do not form properly and they fail to mimic the morphology of enterovirus VLPs.

[0030] Very surprisingly, the inventors have found that, according to an embodiment of the present invention, multiple RNAs can be delivered, one of which produces a protein that acts on the other RNA or the protein produced by the RNA to achieve complex physiological processes in cells. For example, in some cases, complex structures such as VLPs can be properly organized from one or more capsid precursor polyproteins that are expressed and then processed in cells from messenger ribonucleic acid (mRNA) delivered in lipid nanoparticles (LNPs). For example, the inventors have confirmed that a composition comprising an mRNA that includes an open reading frame (ORF) that encodes a picornavirus capsid polyprotein and an mRNA that includes an ORF that encodes a picornavirus 3C protease is sufficient for the formation of virus-like particles. In some embodiments, the composition is formulated into one or more lipid nanoparticles.

[0031] The inventors have also discovered, according to an aspect of the present invention, a method comprising administering an immunogenic composition comprising an mRNA comprising an ORF encoding a picornavirus capsid polyprotein and, optionally, an mRNA comprising an ORF encoding a picornavirus 3C protease, in an amount effective to induce an immune response in a subject against viral infection from a member of the Enterovirus genus.

[0032] Further aspects of the present disclosure relate to compositions in which two or more mRNAs are delivered to a subject, the mRNAs encoding at least a first and a second product that can interact in the body to achieve an end result. In the above example, one product is a precursor protein or polyprotein (substrate) and the other is an enzyme. In other words, the first product is an active protein that can regulate the expression, structure, or activity of the second mRNA and / or the second product. These two products can be a substrate and enzyme pair. Alternatively, the first product can be a binding protein that affects the second mRNA translation process or the function of the second product.

[0033] Described herein are compositions that include one or more polynucleotides that encode picornavirus capsid polyproteins. Thus, the present invention is directed, in part, to polynucleotides, specifically messenger ribonucleic acid (mRNA), that include an open reading frame encoding one or more picornavirus capsid polyproteins and / or components thereof. In some embodiments, the picornavirus capsid polyprotein comprises two or more capsid proteins with a cleavage site specific for a viral protease between the two or more capsid proteins.

[0034] Further described herein are compositions that include one or more polynucleotides that encode a picornavirus 3C protease. Accordingly, the present invention is directed, in part, to polynucleotides, particularly mRNAs, that include an open reading frame that encodes a picornavirus 3C protease.

[0035] Further described herein are compositions that include one or more polynucleotides encoding a picornaviral capsid polyprotein and one or more polynucleotides encoding a picornaviral 3C protease. Accordingly, the present invention is directed, in part, to polynucleotides, specifically, mRNAs that include an open reading frame encoding a picornaviral capsid polyprotein and mRNAs that include an open reading frame encoding a picornaviral 3C protease.

[0036] In some embodiments of the present invention, the precursor polyprotein is a capsid polyprotein. In some embodiments, the capsid polyprotein is a picornaviral capsid polyprotein. In some embodiments, the picornaviral capsid polyprotein is a viral P1 precursor polyprotein.

[0037] Picornaviral capsid polyproteins may be encoded by a single ribonucleic acid (RNA) molecule, which may be a bicistronic molecule encoding two separate polypeptide chains or a polycistronic molecule encoding three separate polypeptide chains (FIG. 1A). Such an RNA molecule may contain a signal sequence between the two or three coding sequences, so that two or three separate polypeptides are produced during the translation process. Alternatively, the RNA molecule may contain a sequence encoding a cleavage site (e.g., a protease cleavage site) between the capsid proteins, so that a single capsid protein is produced, which can be processed via cleavage at the cleavage site to produce two or more separate capsid proteins. Alternatively, the capsid protein may be encoded by two or three separate RNA molecules.

[0038] In some embodiments, the capsid polyprotein comprises two or more capsid proteins. In some embodiments, the two or more capsid proteins comprise two or more of viral protein 0 (VP0), viral protein 1 (VP1), and viral protein 3 (VP3). In some embodiments, the viral protein 0 (VP0) further comprises viral protein 2 (VP2) and viral protein 4 (VP4).

[0039] A capsid polyprotein comprising two or more capsid proteins may further comprise one or more cleavage sites specific for a viral protease. For example, in some embodiments of the present invention, a precursor polyprotein comprises two or more capsid proteins, with a cleavage site specific for a viral protease between the two or more capsid proteins. In some embodiments, a cleavage site specific for a viral protease exists between one or more of VP0, VP1, and VP3. A capsid polyprotein may further comprise a cleavage site for capsid maturation. In some embodiments, a capsid protein comprising VP0 further comprises VP2 and VP4, with VP2 and VP4 comprising a cleavage site for capsid maturation (FIG. 1A).

[0040] In some embodiments, the mRNA encoding the picornavirus capsid polyprotein is specific to a human rhinovirus (HRV) species. In some embodiments, the HRV species is HRV-A16. In some embodiments, the mRNA encoding the picornavirus capsid polyprotein comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO:3. In some embodiments, the nucleotide sequence of the mRNA encoding the picornavirus capsid polyprotein comprises SEQ ID NO:3. In some embodiments, the HRV species is HRV-B14. In some embodiments, an mRNA encoding a picornavirus capsid polyprotein comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity to SEQ ID NO: 10. In some embodiments, the nucleotide sequence of an mRNA encoding a picornavirus capsid polyprotein comprises SEQ ID NO:10.

[0041] In some embodiments, the picornavirus capsid polyprotein is specific to a human rhinovirus (HRV) species. In some embodiments, the HRV species is HRV-A16. In some embodiments, the picornavirus capsid polyprotein comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO:5. In some embodiments, the amino acid sequence of the picornavirus capsid polyprotein comprises SEQ ID NO:5. In some embodiments, the HRV species is HRV-B14. In some embodiments, the picornavirus capsid polyprotein comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity to SEQ ID NO: 11. In some embodiments, the amino acid sequence of the picornavirus capsid polyprotein comprises SEQ ID NO:11.

[0042] In some embodiments, the mRNA encoding the picornavirus capsid polyprotein is specific to an enterovirus species. In some embodiments, the enterovirus species is EV-D68. In some embodiments, the mRNA encoding the picornavirus capsid polyprotein comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO: 16. In some embodiments, the nucleotide sequence of the mRNA encoding the picornavirus capsid polyprotein comprises SEQ ID NO: 16. In some embodiments, the enterovirus species is EV-A71. In some embodiments, an mRNA encoding a picornavirus capsid polyprotein comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity to SEQ ID NO: 22. In some embodiments, the nucleotide sequence of an mRNA encoding a picornavirus capsid polyprotein comprises SEQ ID NO:22.

[0043] In some embodiments, the picornavirus capsid polyprotein is specific to an enterovirus species. In some embodiments, the enterovirus species is EV-D68. In some embodiments, the picornavirus capsid polyprotein comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO: 17. In some embodiments, the amino acid sequence of the picornavirus capsid polyprotein comprises SEQ ID NO: 17. In some embodiments, the enterovirus species is EV-A71. In some embodiments, the picornavirus capsid polyprotein comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the amino acid sequence of the picornavirus capsid polyprotein comprises SEQ ID NO:23.

[0044] Prior to assembly of VLPs, the capsid polyprotein may be processed or cleaved by nonstructural viral proteins (e.g., viral proteases). As used herein, viral proteases refer to proteases that can recognize viral protease-specific cleavage sites between one or more capsid proteins in the capsid polyprotein. In some embodiments, the viral protease is picornavirus 3C (3CD) protease. The viral protease 3CD is part of the nonstructural polyprotein P3 (Figure 1A). The viral protease 3CD may be cleaved into 3C and 3D. The picornavirus 3C protease may be provided by 3C or 3CD. In some embodiments, the picornavirus 3C protease is provided by 3C. In some embodiments, the 3C protease is provided by 3CD. In some embodiments, the 3CD protease is specific to Enterovirus species.

[0045] In some embodiments, the mRNA encoding picornavirus 3C protease (3CD) is specific to a human rhinovirus (HRV) species. In some embodiments, the HRV species is HRV-A16. In some embodiments, the mRNA encoding 3CD comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO:7. In some embodiments, the nucleotide sequence of the mRNA encoding 3CD comprises SEQ ID NO:7. In some embodiments, the HRV species is HRV-B14. In some embodiments, the mRNA encoding 3CD comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO: 13. In some embodiments, the nucleotide sequence of the mRNA encoding 3CD comprises SEQ ID NO:13.

[0046] In some embodiments, the 3CD protease is specific for a human rhinovirus (HRV) species. In some embodiments, the HRV species is HRV-A16. In some embodiments, the 3CD protease comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO:8. In some embodiments, the amino acid sequence of the 3CD protease comprises SEQ ID NO:8. In some embodiments, the HRV species is HRV-B14. In some embodiments, the 3CD protease comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the 3CD protease comprises SEQ ID NO:14.

[0047] In some embodiments, the mRNA encoding picornavirus 3C protease (3CD) is specific to an enterovirus species. In some embodiments, the enterovirus species is EV-D68. In some embodiments, the mRNA encoding 3CD comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO: 19. In some embodiments, the nucleotide sequence of the mRNA encoding 3CD comprises SEQ ID NO: 19. In some embodiments, the enterovirus species is EV-A71. In some embodiments, the mRNA encoding 3CD comprises a nucleotide sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO: 25. In some embodiments, the nucleotide sequence of the mRNA encoding 3CD comprises SEQ ID NO:25.

[0048] In some embodiments, the 3CD protease is specific to an enterovirus species. In some embodiments, the enterovirus genotype is EV-D68. In some embodiments, the 3CD protease comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity with SEQ ID NO:20. In some embodiments, the amino acid sequence of the 3CD protease comprises SEQ ID NO:20. In some embodiments, the enterovirus genotype is EV-A71. In some embodiments, the 3CD protease comprises an amino acid sequence that shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% identity to SEQ ID NO: 26. In some embodiments, the amino acid sequence of the 3CD protease comprises SEQ ID NO:26.

[0049] According to the present invention, the polynucleotides are described in compositions, in which the polynucleotides encoding one or more picornavirus capsid polyproteins and / or the picornavirus 3C protease are included in the composition in a specific ratio. As used herein, "ratio" refers to the ratio of picornavirus 3C protease and picornavirus capsid polyproteins capable of producing VLPs. In some embodiments, the ratio refers to a molar ratio. In some embodiments, the ratio refers to a mass ratio.

[0050] In some embodiments, the ratio of an mRNA comprising an open reading frame encoding a picornavirus 3C protease to an mRNA comprising an open reading frame encoding a picornavirus capsid polyprotein is at least 20:1, at least 10:1, at least 7:1, at least 5:1, at least 4:1, at least 3:1, at least 2:1, or at least 1:1. In some embodiments, the ratio of an mRNA comprising an open reading frame encoding a picornavirus 3C protease to an mRNA comprising an open reading frame encoding a picornavirus capsid polyprotein is 10:1. In some embodiments, the ratio of an mRNA comprising an open reading frame encoding a picornavirus 3C protease to an mRNA comprising an open reading frame encoding a picornavirus capsid polyprotein is 2:1.

[0051] In some embodiments, the polynucleotides encoding one or more picornavirus capsid polyproteins and / or picornavirus 3C proteases are included in the composition in a ratio such that the capsid proteins form protomers. In some embodiments, the protomers form pentamers. In some embodiments, the pentamers form VLPs.

[0052] Neutralizing antibodies are produced against the surface-exposed regions of virus particles. For example, in human rhinovirus (HRV), viral protein VP1 is the most surface-exposed of the viral proteins, and therefore the most immunogenic viral protein. Neutralizing antibodies are often directed against neutralizing immunogenic (NIm) sites on virus particles. The amino acid sequences of NIm sites vary widely between virus serotypes. Although virus particles contain highly conserved regions, these highly conserved regions are usually buried inside the virus particle and are inaccessible to neutralizing antibodies. In some embodiments of the present invention, the VLP comprises neutralizing immunogenic (NIm) sites.

[0053] In some embodiments, the compositions of the present invention are formulated into at least one lipid nanoparticle. In some embodiments, the compositions of the present invention are formulated into two lipid nanoparticles.

[0054] The polynucleotides and / or compositions of the invention are useful for assembling VLPs that mimic a virus or viral particle and elicit an immunogenic response when administered to a subject, in some embodiments, the virus is a member of the Picornaviridae family of viruses.

[0055] Picornaviridae viruses are classified into 29 genera, one of which is the Enterovirus genus. The Enterovirus genus is further divided into 12 species, including Enteroviruses A-D, Rhinoviruses A-C, and five Enterovirus species that infect only animal species (van der Linden et al., 2015). Enterovirus species that infect humans (e.g., Enteroviruses A-D and Rhinoviruses A-C) cause infections that range in severity from a simple cold to life-threatening illnesses.

[0056] Rhinovirus species are responsible for symptoms associated with the common cold. In some embodiments of the invention, the rhinovirus is human rhinovirus type 16 (HRV-A16). In some embodiments, the rhinovirus is human rhinovirus type 14 (HRV-B14).

[0057] Enterovirus species that infect humans cause more severe symptoms and disease. For example, Enterovirus A71 (EV-A71), a genotype of virus from the Enterovirus A species, is associated with polio-like symptoms affecting the central nervous system, such as acute flaccid myelitis causing quadriplegia. As another example, viruses from the Enterovirus B (e.g., Coxsackievirus B3) species are associated with hand, foot and mouth disease. As yet another example, viruses from the Enterovirus C species are associated with poliovirus infection (polio disease). As yet another example, Enterovirus D68 (EV-D68), a genotype of virus from the Enterovirus D species, is associated with severe respiratory disease and / or acute flaccid myelitis. In some embodiments of the invention, the enterovirus is EV-A71. In some embodiments, the enterovirus is EV-D68.

[0058] Examples of viruses that may be immunized against using the compositions or constructs of the invention include members of the Enterovirus A species, formerly known as Human Enterovirus A (e.g., serotypes Coxsackievirus A2 (CVA2), CVA3, CVA4, CVA5, CVA6, CVA7, CVA8, CVA10, CVA12, CVA14, CVA16, Enterovirus A71 (EV-A71), EV-A76, EV-A89, EV-A90, EV-A91, EV-A92, EV-A93, EV-A94, EV-A95, EV-A96, EV-A97, EV-A99, EV-A98, EV-A9 ...9, EV-A100, EV-A101, EV-A102, EV-A103, EV-A104, EV-A105, EV-A106, EV-A107, EV-A109, EV-A102, EV-A105, EV-A106, EV-A107, EV-A108, EV-A109, EV-A109, EV-A109, EV-A110, EV-A111, EV-A112, EV-A113, EV-A114, EV-A115, EV-A116, EV-A117, EV-A118, EV-A119, , EV-A91, EV-A92, EV-A114, EV-A119, EV-A120, EV-A121, EV-A122 (formerly SV19), EV-A123 (formerly SV43), EV-A124 (formerly SV46), and EV-125 (formerly BA13)), members of the enterovirus B species formerly called human enterovirus B (e.g., serotypes Coxsackievirus B1 (CVB1), CVB2, CVB3, and CVB4 (swine vesicular disease virus 2 SVDV-2), CVB5 (including SVDV-1), CVB6, CVA9, echovirus 1 (including E1, E8), E2, E3, E4, E5, E6, E7, E9 (including CVA23), E11, E12, E13, E14, E15, E16, E17, E18, E19, E20, E21, E24, E25, E26, E27, E29, E30, E31, E32, E33, enterovirus B69 (EV-B69), EV-B73, EV-B74, EV-B75, EV-B 77, EV-B78, EV-B79, EV-B80, EV-B81, EV-B82, EV-B83, EV-B84, EV-B85, EV-B86, EV-B87, EV-B88, EV-B93, EV-B97, EV-B98, EV-B100, EV-B101, EV-B106, EV-B107, EV-B110 (chimpanzee-derived), EV-B111, EV-B112 (chimpanzee-derived), EV-B113 (mandrill-derived), and EV-B114 (formerly known as simian agent5 (SA5)), members of the enterovirus C species, formerly known as human enterovirus C (e.g., poliovirus (PV) 1, PV2, PV3, coxsackievirus A1 (CVA1), CVA11, CVA13, CVA17, CVA19, CVA20, CVA21, CVA22, CVA24, EV-C95, EV-C96, EV-C99, EV-C102, EV-C104, EV-C105,EV-C109, EV-C113, EV-C116, EV-C117, and EV-C118), members of the Enterovirus D species formerly known as Human Enterovirus D (e.g., serotypes EV-D68, EV-D70, EV-D94, EV-D111 (from both humans and chimpanzees), and EV-D120 (from gorillas)), members of the Rhinovirus A species formerly known as Human Rhinovirus A (e.g., serotypes rhinovirus (RV) A1, A2, A7, A8, A9, A10, A11, A12, A13, A15, A16, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A47, A48, A49, A50, A51, A52, A53, A54, A55, A56, A57, A58, A59, A60, A61, A62, A63, A64, A65, A66, A67, A68, A69, A70, A71, A72, A73, A74, A75, A76, A77, A78, A79, A80, A81, A82, A83, A84, A85, A86, A87, A88, A89, A90, A91, A92, A93, A94, A95, A9 , A19, A20, A21, A22, A23, A24, A25, A28, A29, A30, A31, A32, A33, A34, A36, A38, A39, A40, A41, A43, A45, A46, A47, A49, A50, A51, A53, A54, A55, A56, A 57, A58, A59, A60, A61, A62, A63, A64, A65, A66, A67, A68, A71, A73, A74, A7 5, A76, A77, A78, A80, A81, A82, A85, A88, A89, A90, A94, A96, A100, A101, A A102, A103, A104, A105, A106, A107, A108, A109), members of the rhinovirus B species formerly known as human rhinovirus B (e.g., serotypes rhinovirus (RV) B3, B4, B5, B6, B14, B17, B26, B27, B35, B37, B42, B48, B52, B69, B70, B72, B79, B83, B84, B86, B91, B92, B93, B97, B99, B100, B101, B102, B103, B104, B105, and B106), and members of the rhinovirus B species formerly known as human rhinovirus C ... These include, but are not limited to, members of the rhinovirus C species, formerly known as serotypes C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, and C51.

[0059] The compositions of the present disclosure may be designed as a single prophylactic therapeutic agent to immunize a subject against various pathogenic strains of enterovirus.

[0060] In some aspects, the methods of the disclosure include administering to a subject an immunogenic composition as described herein. As used herein, "immunogenic composition" refers to a composition comprising an mRNA comprising an open reading frame encoding a protease and an mRNA comprising an open reading frame encoding a capsid polyprotein comprising a precursor protein, the precursor protein comprising two or more capsid proteins having a cleavage site specific for the protease between the two or more capsids, in an amount effective to induce an immune response in the subject against a viral infection from a member of the Enterovirus genus.

[0061] The immune response includes binding antibody titers against the virus species. In some embodiments, the immune response includes binding antibody titers against a virus of the human rhinovirus species (e.g., HRV-A16 or HRV-B14). In some embodiments, the immune response includes binding antibody titers against a virus of the human enterovirus species (e.g., EV-A71 or EV-D68).

[0062] The immune response further includes neutralizing antibody titers against the virus species. In some embodiments, the immune response includes neutralizing antibody titers against a virus of the human rhinovirus species (e.g., HRV-A16 or HRV-B14). In some embodiments, the immune response includes neutralizing antibody titers against a virus of the human enterovirus species (e.g., EV-A71 or EV-D68).

[0063] The immune response further includes a T cell response to the virus species. In some embodiments, the immune response includes a T cell response to a virus of the human rhinovirus species (e.g., HRV-A16 or HRV-B14). In some embodiments, the immune response includes a T cell response to a virus of the human enterovirus species (e.g., EV-A71 or EV-D68).

[0064] According to the present invention, polynucleotides or constructs and compositions related thereto may be designed to produce commercially viable vaccines, variants thereof or parts thereof in vivo.

[0065] The polynucleotides of the present invention may be used to protect infants from infections or diseases, including but not limited to diseases caused by Picornaviridae viruses. Examples of diseases caused by the Picornaviridae viruses of the present invention include, but are not limited to, aseptic meningitis (the most common acute viral disease of the CNS), encephalitis, upper respiratory tract disease, lower respiratory tract disease, common cold, febrile rash disease (hand, foot and mouth disease), conjunctivitis, herpangina, myositis and myocarditis, hepatitis, poliovirus infection, polio-like viral disease, acute flaccid myelitis (AFM), gastrointestinal disorders and conjunctivitis.

[0066] In some embodiments, the polynucleotides of the invention may encode at least one picornavirus capsid polyprotein and / or picornavirus 3C protease that, when administered to a subject, forms a VLP and immunizes a subpopulation for the prevention, management, or treatment of an enterovirus infection.

[0067] In one embodiment, a polynucleotide of the invention is or functions as a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded peptide polypeptide of interest.

[0068] The major components of an mRNA molecule typically include at least a coding region, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a polyA tail. The polynucleotides of the present disclosure may function as mRNAs, but may be distinguished from wild-type mRNAs in functional and / or structural design features that help overcome existing problems of effective polypeptide expression using nucleic acid-based therapeutics.

[0069] The polynucleotides of the present disclosure are codon-optimized in some embodiments. Codon optimization methods are known in the art and can be used as described herein. Codon optimization can be used in some embodiments to match codon frequencies in target and host organisms to ensure proper folding, to bias GC content to increase mRNA stability or reduce secondary structures, to minimize runs of tandem repeat codons or bases that may impair gene organization or expression, to customize transcriptional and translational regulatory regions, to insert or remove protein trafficking sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) of the encoded protein, to add, remove, or swap protein domains, to insert or delete restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to adjust translation rates and correctly fold various domains of a protein, or to reduce or remove problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0070] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof).

[0071] In some embodiments, the codon-optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof). In some embodiments, the codon-optimized sequence shares 65% to 75 or about 80% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest, e.g., a picornaviral capsid polyprotein and / or a picornaviral 3C protease thereof).

[0072] In some embodiments, the codon-optimized RNA may, for example, have an increased G / C level. The G / C content of a nucleic acid molecule may affect the stability of the RNA. RNA with increased amounts of guanine (G) and / or cytosine (C) residues may be more functionally stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses pharmaceutical compositions comprising mRNAs stabilized by modification of sequences in the translation region. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with those that further increase the stability of the RNA without changing the resulting amino acid. The approach is limited to the coding region of the RNA.

[0073] Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. Polypeptides may be single molecules or multi-molecular complexes, such as dimers, trimers, or tetramers. Polypeptides may also include single chain or multi-chain polypeptides, such as antibodies, and may be bound or linked. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analog of the corresponding naturally occurring amino acid.

[0074] The term "polypeptide variant" refers to a molecule that differs in amino acid sequence from a native or reference sequence. The amino acid sequence variant may have substitutions, deletions, and / or insertions at certain positions in the amino acid sequence compared to the native or reference sequence. Typically, the variant has at least 50% identity to the native or reference sequence. In some embodiments, the variant shares at least 80% or at least 90% identity with the native or reference sequence.

[0075] In some embodiments, a "variant mimic" is provided. As used herein, the term "variant mimic" includes at least one amino acid that mimics an activation sequence. For example, glutamic acid can function as a mimic of phosphorothreonine and / or phosphoroserine. Alternatively, the variant mimic may be inactivated or may produce an inactivated product that includes the mimic, for example, phenylalanine may function as an inactivating replacement for tyrosine, and alanine may function as an inactivating replacement for serine.

[0076] "Ortholog" refers to genes in different species that have evolved from a common ancestral gene by speciation. Orthologs usually retain the same function during evolution. Identification of orthologs is important for reliable prediction of gene function in newly sequenced genomes.

[0077] "Analog" is meant to include polypeptide variants that differ by one or more amino acid alterations, e.g., substitution, addition or deletion of an amino acid residue, that still retain one or more properties of a parent or starting polypeptide.

[0078] The present disclosure provides several types of compositions based on polynucleotides or polypeptides, including variants and derivatives. These include, for example, substitutions, insertions, deletions, and covalent variants and derivatives. The term "derivative" is used interchangeably with the term "variant", but generally refers to a molecule that is modified and / or altered in some way relative to a reference or starting molecule.

[0079] Thus, polynucleotides encoding peptides or polypeptides that contain substitutions, insertions, and / or additions, deletions, and covalent modifications with respect to a reference sequence, particularly a polypeptide sequence disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequence (e.g., at its N-terminus or C-terminus). Sequence tags can be used for detection, purification, or localization of the peptide. Lysines can be used to increase the solubility of the peptide or to allow biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted, resulting in a truncated sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) can alternatively be deleted depending on the use of the sequence, e.g., expression of the sequence as part of a larger sequence that is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., fold-on regions), and the like, can be replaced with alternative sequences that achieve the same or similar functions. In some embodiments, cavities within the core of the protein may be filled, for example by introducing larger amino acids, to improve stability. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic residues to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art.

[0080] "Substitutional variants" when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule is substituted, or multiple, where two or more amino acids are substituted in the same molecule.

[0081] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the replacement of one polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Furthermore, the replacement of a basic residue, such as lysine, arginine, or histidine, with another, or the replacement of an acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, are also further examples of conservative substitutions. Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, methionine, with a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, or replacing a polar residue with a non-polar residue.

[0082] A "feature" when referring to a polypeptide or polynucleotide is defined as a component of a distinct amino acid sequence-based or nucleotide-based molecule, respectively. Features of a polypeptide encoded by a polynucleotide include surface expression, local conformational shape, fold, loop, half-loop, domain, half-domain, site, terminus, or any combination thereof.

[0083] As used herein, the term "domain" when referring to a polypeptide refers to a motif in a polypeptide having one or more distinguishable structural or functional features or characteristics (e.g., a binding capacity to serve as a site of protein-protein interaction).

[0084] As used herein, with respect to amino acid-based embodiments in reference to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain." As used herein, with respect to nucleotide-based embodiments in reference to polynucleotides, the term "site" is used synonymously with "nucleotide." A site represents a position within a peptide, or polypeptide, or polynucleotide that may be modified, manipulated, altered, derivatized, or changed within that polypeptide or polynucleotide-based molecule.

[0085] As used herein, the term "terminus" or "terminus" when referring to a polypeptide or polynucleotide refers to the end of the polypeptide or polynucleotide, respectively. Such ends are not limited to only the first or last site of the polypeptide or polynucleotide, but may also include additional amino acids or nucleotides in the terminal region. A polypeptide-based molecule may be characterized as having both an N-terminus (terminated with an amino acid with a free amino group (NH2)) and a C-terminus (terminated with an amino acid with a free carboxyl group (COOH)). Proteins are composed of multiple polypeptide chains (e.g., multimers, oligomers) that are held together, in some cases, by disulfide bonds or by non-covalent forces. These proteins have multiple N- and C-termini. Alternatively, the ends of polypeptides may be modified so that they optionally begin or end with non-polypeptide-based moieties, e.g., organic conjugates.

[0086] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptide of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence but otherwise identical) of a reference protein that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that is 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be used in accordance with the present disclosure. In some embodiments, the polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. In another example, any protein containing a stretch of 20, 30, 40, 50, or 100 amino acids that is more than 80%, 90%, 95% or 100% identical to any of the sequences described herein, and having a stretch of 5, 10, 15, 20, 25, or 30 amino acids that is less than 80%, 75%, 70%, 65%, or 60% identical to any of the sequences described herein can be used in accordance with the present disclosure.

[0087] The polypeptide or polynucleotide molecule of the present disclosure may share a certain degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or polynucleotide) and, for example, with a molecule described in the art (e.g., an artificially created or designed molecule or a wild-type molecule). The term "identity" as known in the art refers to the relationship between two or more polypeptide or polynucleotide sequences and is determined by comparing the sequences. In the art, identity also means the degree of sequence correlation between them and is determined by the number of matches between two or more strings of amino acid or nucleic acid residues. Identity measures the percentage of perfect matches between the smaller of two or more sequences with gap alignment (if any) that is addressed by a certain mathematical model or computer program (e.g., "algorithm"). The identity of related peptides can be easily calculated by known methods. "% identity" as applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for such alignment are well known in the art. It is understood that the identity depends on the calculation of percent identity, but the value may vary depending on the gaps and penalties introduced in the calculation. In general, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters known to those skilled in the art and described herein.Such alignment tools include those in the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). Another well-known local alignment technique is based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences", J. Mol. Biol. 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol. Biol. 48: 443-453.). More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is intended to generate global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0088] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymer molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold of similarity or identity determined by alignment of matching residues are called homologous. Homology is a qualitative term that indicates the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that determines the degree of sequence match between two compared sequences. In some embodiments, polymer molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical for at least one stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Two protein sequences are considered to be homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.

[0089] Homology implies that the compared sequences have branched off during evolution from a common origin. The term "homolog" refers to a first amino acid or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) that is related by descent from a common ancestral sequence to a second amino acid or nucleic acid sequence. The term "homolog" can apply to the relationship between genes and / or proteins that have been separated by a speciation event or to the relationship between genes and / or proteins that have been separated by a gene duplication event. An "ortholog" is a gene (or protein) in a different species that has evolved from a common ancestral gene (or protein) by speciation. Orthologs usually retain the same function during evolution. A "paralog" is a gene (or protein) that is related by duplication within a genome. An ortholog retains the same function during evolution, whereas a paralog evolves new functions, even if related to the original function.

[0090] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the compared sequences. The nucleotides at corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) as implemented in the ALIGN program (version 2.0) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleic acid sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs.Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0091] The RNA (e.g., mRNA) therapeutics of the present disclosure may include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a picornavirus capsid polyprotein that includes at least one chemical modification.The RNA (e.g., mRNA) therapeutics of the present disclosure may include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a picornavirus 3C protease that includes at least one chemical modification.

[0092] The terms "chemical modification" and "chemically modified" refer to modifications to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides in at least one of their positions, patterns, percentages or populations. In general, these terms do not refer to ribonucleotide modifications in the naturally occurring 5'-terminal mRNA cap portion. In the context of polypeptides, the term "modification" refers to modifications to the standard set of 20 amino acids. The polypeptides provided herein are also considered "modified" if they contain amino acid substitutions, insertions, or a combination of substitutions and insertions.

[0093] A polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide), in some embodiments, comprises a variety (multiple) of different modifications. In some embodiments, a particular region of a polynucleotide comprises one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or organism exhibits reduced degradation in the cell or organism, respectively, compared to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified mRNA polynucleotide) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively.

[0094] Modifications of polynucleotides include, but are not limited to, those described herein. Polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides) may contain naturally occurring modifications, non-naturally occurring modifications, or the polynucleotides may contain a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may contain, for example, any useful modifications of sugars, nucleobases, or internucleoside linkages (e.g., to phosphate linkages, to phosphodiester linkages, or to phosphodiester backbones).

[0095] A polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide), in some embodiments, comprises non-naturally occurring modified nucleotides that are introduced during or after synthesis of the polynucleotide to achieve a desired function or property. The modifications can be in the internucleotide linkage, the purine or pyrimidine base, or the sugar. The modifications can be introduced by chemical synthesis or can be introduced at the end of the chain or anywhere in the chain using a polymerase enzyme. Any region of the polynucleotide can be chemically modified.

[0096] The present disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides). A "nucleoside" refers to a compound that includes a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes one or more phosphate groups. Modified nucleotides can be synthesized in any useful manner, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A polynucleotide can include a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide includes a region of nucleotides.

[0097] Base pairing of modified nucleotides encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases, or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers may be incorporated into the polynucleotides of the present disclosure.

[0098] Those of skill in the art will understand that unless otherwise indicated, the polynucleotide sequences set forth in this application recite a "T" in a representative DNA sequence, but when the sequence represents RNA, the "T" is replaced with a "U."

[0099] Modifications of polynucleotides (e.g., RNA polynucleotides, e.g., mRNA polynucleotides) useful in the compositions, methods, and synthetic processes of the present disclosure include, but are not limited to, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, 1,2'-O-dimethyladenosine, 1-methyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), 2-methyladenosine, 2-methylthio O-N6-isopentenyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, 2'-O-methyladenosine, 2'-O-ribosyladenosine (phosphate), isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, N6,N6-dimethyladenosine, N6-acetyladenosine, N6-hydroxynorvalylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, 2-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 7-deazaadenosine, N1-methyl-adenosine, N6,N6-(dimethyl)adenine, N6-cis-hydroxy-isopentenyl-adenosine, α-thio-adenosine, 2-(amino)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 2-(alkyl)adenines, 2-(aminoalkyl)adenines, 2-(aminopropyl)adenine, 2-(halo)adenine, 2-(halo)adenine, 2-(propyl)adenine, 2'-amino-2'-deoxy-ATP, 2'-azido-2'-deoxy-ATP, 2'-deoxy-2'-a-aminoadenosine TP, 2'-deoxy-2'-a-azidoadenosine TP, 6-(alkyl)adenine, 6-(methyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(thioalkyl)adenine, 8-(alkenyl)adenine, 8-(alkyl) Adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, 8-azido-adenosine, azaadenine, deazaadenine, N6-(methyl)adenine, N6-(isopentyl)adenine, 7-deaza-8-aza-adenosine, 7-methyladenine, 1-deazaadenosine TP, 2'-fluoro-N6-Bz-deoxyadenosine TP , 2'-OMe-2-amino-ATP, 2'-O-methyl-N6-Bz-deoxyadenosine TP, 2'-a-ethynyl adenosine TP, 2-aminoadenine, 2-aminoadenosine TP, 2-amino-ATP, 2'-a-trifluoromethyl adenosine TP, 2-azidoadenosine TP, 2'-b-ethynyl adenosine TP, 2-bromoadenosine TP, 2'-b-trifluoromethyl adenosine TP, 2-chloroadenosine TP, 2'-deoxy-2',2'-Difluoroadenosine TP, 2'-deoxy-2'-a-mercaptoadenosine TP, 2'-deoxy-2'-a-thiomethoxyadenosine TP, 2'-deoxy-2'-b-aminoadenosine TP, 2'-deoxy-2'-b-azidoadenosine TP, 2'-deoxy-2'-b-bromoadenosine TP, 2'-deoxy-2'-b-chloroadenosine TP, 2'-deoxy-2'-b-fluoroadenosine TP, 2'-deoxy-2'-b-iodoadenosine TP, 2 '-Deoxy-2'-b-mercaptoadenosine TP, 2'-deoxy-2'-b-thiomethoxyadenosine TP, 2-fluoroadenosine TP, 2-iodoadenosine TP, 2-mercaptoadenosine TP, 2-methoxy-adenine, 2-methylthio-adenine, 2-trifluoromethyladenosine TP, 3-deaza-3-bromoadenosine TP, 3-deaza-3-chloroadenosine TP, 3-deaza-3-fluoroadenosine TP, 3-deaza-3-iodoadenosine TP, 3-Deazaadenosine TP, 4'-Azidoadenosine TP, 4'-Carbocyclic Adenosine TP, 4'-Ethynyladenosine TP, 5'-Homo-Adenosine TP, 8-Aza-ATP, 8-Bromo-Adenosine TP, 8-Trifluoromethyl-Adenosine TP, 9-Deazaadenosine TP, 2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-Deaza-8-Aza-2,6-Diaminopurine, 7-Deaza-8-Aza-2-Aminopurine, 2,6-Diaminopurine, 7- Deaza-8-aza-adenine, 7-deaza-2-aminopurine, 2-thiocytidine, 3-methylcytidine, 5-formylcytidine, 5-hydroxymethylcytidine, 5-methylcytidine, N4-acetylcytidine, 2'-O-methylcytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, lysine, N4,2'-O-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N4-methylcytidine, N4,N4-Dimethyl-2'-OMe-cytidine TP, 4-Methylcytidine, 5-Aza-cytidine, Pseudo-iso-cytidine, Pyrrolo-cytidine, α-Thio-cytidine, 2-(Thio)cytosine, 2'-Amino-2'-deoxy-CTP, 2'-Azido-2'-deoxy-CTP, 2'-Deoxy-2'-α-aminocytidine TP, 2'-Deoxy-2'-α-azidocytidine TP, 3-(Deaza)-5-(Aza)cytosine, 3-(Methyl)cytosine, 3-(Alkyl)cytosine, 3-(Deaza)-5-(Aza)cytosine , 3-(methyl)cytidine, 4,2'-O-dimethylcytidine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 5-bromo-cytidine, 5-iodo-cytidine, 5-propynylcytosine, 6-(azo)cytosine, 6-aza-cytidine, azacytosine, deazacytosine, N4-(acetyl)cytosine, N4-(acetyl)cytosine, N5-(acetyl)cytosine, N6-(acetyl)cytosine, N7-(acetyl)cytosine, N8-(acetyl)cytosine, N9-(acetyl)cytosine, N1-(acetyl)cytosine, N2-(acetyl)cytosine, N3-(acetyl)cytosine, N4-(acetyl)cytosine, N5-(acetyl)cytosine, N6-(acetyl)cytosine, N7-(acetyl)cytosine, N8-(acetyl)cytosine, N9-(acetyl)cytosine, N1-(acetyl)cytosine, N1-(acetyl)cytosine, N2-(acetyl)cytosine, N2-(acetyl)cytosine, N3-(acetyl)cytosine, N4-(acetyl)cytosine, N5-(acetyl)cytosine, N6-(acetyl)cytosine, N1-(acetyl)cytosine, N2-(acetyl)cytosine, N3-(acetyl)cytosine, N4-(acetyl)cytosine, ) Cytosine, 1-methyl-1-deaza-pseudoisocytidine, 1-methyl-pseudoisocytidine, 2-methoxy-5-methyl-cytidine, 2-methoxy-cytidine, 2-thio-5-methyl-cytidine, 4-methoxy-1-methyl-pseudoisocytidine, 4-methoxy-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-pseudoisocytidine, 5-aza-zebularine, 5-methyl-zebularine, pyrrolo-pseudoisocytidine Socytidine, Zebularine, (E)-5-(2-bromo-vinyl)cytidine TP, 2,2'-anhydro-cytidine TP hydrochloride, 2'-fluoro-N4-Bz-cytidine TP, 2'-fluoro-N4-acetyl-cytidine TP, 2'-O-methyl-N4-acetyl-cytidine TP, 2'-O-methyl-N4-Bz-cytidine TP, 2'-a-ethynylcytidine TP, 2'-a-trifluoromethylcytidine TP, 2'-b-ethynylcytidine TP, 2'-b-trifluoromethylcytidine TP, 2'-deoxy-2',2'-difluorocytidine TP, 2'-deoxy-2'-a-mercaptocytidine TP, 2'-deoxy-2'-a-thiomethoxycytidine TP, 2'-deoxy-2'-b-aminocytidine TP, 2'-deoxy-2'-b-azidocytidine TP, 2'-deoxy-2'-b-bromocytidine TP, 2'-deoxy-2'-b-chlorocytidine TP, 2'-deoxy-2'-b-fluorocytidine TP, 2'-deoxy-2'-b-iodocytidine TP, 2' -Deoxy-2'-b-mercaptocytidine TP, 2'-deoxy-2'-b-thiomethoxycytidine TP, 2'-O-methyl-5-(1-propynyl)cytidine TP, 3'-ethynylcytidine TP, 4'-azidocytidine TP, 4'-carbocyclic cytidine TP, 4'-ethynylcytidine TP, 5-(1-propynyl)ara-cytidine TP, 5-(2-chloro-phenyl)-2-thiocytidine TP, 5-(4-amino-phenyl)-2-thiocytidine T P, 5-aminoallyl-CTP, 5-cyanocytidine TP, 5-ethynyl-cytidine TP, 5-ethynyl-cytidine TP, 5'-homo-cytidine TP, 5-methoxycytidine TP, 5-trifluoromethyl-cytidine TP, N4-amino-cytidine TP, N4-benzoyl-cytidine TP, pseudoisocytidine, 7-methylguanosine, N2,2'-O-dimethylguanosine, N2-methylguanosine, 1,2'-O-dimethyl ... -Methylguanosine, 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 2'-O-methylguanosine, 2'-O-ribosylguanosine (phosphate), 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, archaeosine, methylwyosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, 6-thio-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, N1-methyl-guanosine, α-thio-guanosine, 2-(propyl)guanine, 2-(alkyl)guanine, 2'-amino-2'-deoxy-GTP, 2'-azido-2'-deoxy-GTP, 2'-deoxy-2'-α-aminoguanosine TP, 2'-deoxy-2'-α-azidoguanosine TP, 6-(methyl)guanine, 6-(alky)guanine 6-(alkyl)guanine, 6-methylguanine, 6-methyl-guanosine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 7-(alkyl)guanine, 7-(deaza)guanine, 7-(methyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(halo)guanine, 8-(thioalkyl)guanine, 8-(alkenyl)guanine, 8-(alkyl)guanine, 8-(alkynyl)guanine, 8-(amino ... N)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, azaguanine, deazaguanine, N-(methyl)guanine, N-(methyl)guanine, 1-methyl-6-thio-guanosine, 6-methoxy-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7- Methyl-8-oxo-guanosine, N2,N2-dimethyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, 1-Me-GTP, 2'-fluoro-N2-isobutyl-guanosine TP, 2'-O-methyl-N2-isobutyl-guanosine TP, 2'-a-ethynylguanosine TP, 2'-a-trifluoromethylguanosine TP, 2'-b-ethynylguanosine TP, 2'-b-trifluoromethylguanosine TP, 2'-deoxy-2',2'-difluoroguanosine TP, 2'-deoxy-2'-a-mercaptoguanosine TP, 2'-deoxy-2'-a-thiomethoxyguanosine TP, 2'-deoxy-2'-b-aminoguanosine TP, 2'-deoxy-2'-b-azidoguanosine TP, 2'-deoxy-2'-b-bromoguanosine TP, 2'-deoxy-2'-b-chloroguanosine TP, 2'-deoxy-2'-b-fluoroguanosine TP, 2'-deoxy-2'-b-iodoguanosine, Inosine TP, 2'-deoxy-2'-b-mercaptoguanosine TP, 2'-deoxy-2'-b-thiomethoxyguanosine TP, 4'-azidoguanosine TP, 4'-carbocyclic guanosine TP, 4'-ethynylguanosine TP, 5'-homo-guanosine TP, 8-bromo-guanosine TP, 9-deazaguanosine TP, N2-isobutyl-guanosine TP, 1-methylinosine, inosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 7-methylinosine, 2'-O-methylinosine, epoxyqueuosine, galactosyl-queuosine, mannosylqueuosine, queuosine, allylamino-thymidine, azathymidine, deazathymidine, deoxy-thymidine, 2'-O-methyluridine, 2-thiouridine, 3-methyluridine, 5-carboxymethyluridine, 5 -Hydroxyuridine, 5-methyluridine, 5-taurinomethyl-2-thiouridine, 5-taurinomethyluridine, dihydrouridine, pseudouridine, (3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine, 1-methylpseudouridine, 1-methyl-pseudouridine, 2'-O-methyluridine, 2'-O-methylpseudouridine, 2'-O-methyluridine, 2-thio-2'-O-methyluridine, 3-(3-amino-3-carboxypropyl)uridine, 3,2'-O-dimethyluridine, 3-methyl-pseudouridine TP, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(carboxyhydroxymethyl)uridine methyl ester, 5,2'-O-dimethyluridine, 5,6-Dihydro-uridine, 5-aminomethyl-2-thiouridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyluridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyluridine, 5-carbamoyl uridine TP, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methyluridine), 5-methoxyuridine, 5-methyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methyldihydrouridine, 5-oxyacetic acid-uridine TP, 5-oxyacetic acid-methylester-uridine TP, N1-methyl-pseudo-uridine, N1 -ethylpseudouridine, uridine-5-oxyacetic acid, uridine-5-oxyacetic acid methyl ester, 3-(3-amino-3-carboxypropyl)-uridine TP, 5-(iso-pentenylaminomethyl)-2-thiouridine TP, 5-(iso-pentenylaminomethyl)-2'-O-methyluridine TP, 5-(iso-pentenylaminomethyl)uridine TP, 5-propynyluracil, α-thio-uridine, 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl) 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2-(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-substituted 2-(thio)-pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP, 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP, 1-methyl-pseudo-UTP, 2-(thio)pseudouracil, 2'-deoxyuridine, 2'-fluorouridine, 2-(thio )uracil, 2,4-(dithio)pseudouracil, 2'-methyl,2'-amino,2'-azido,2'-fluoro-guanosine, 2'-amino-2'-deoxy-UTP, 2'-azido-2'-deoxy-UTP, 2'-azido-deoxyuridine TP, 2'-O-methylpseudouridine, 2'-deoxyuridine, 2'-fluorouridine, 2'-deoxy-2'-a-aminouridine TP, 2'-deoxy-2'-a-azidouridine TP, 2-methylpseudouridine, 3-(3- 4-(amino-3-carboxypropyl)uracil, 4-(thio)pseudouracil, 4-(thio)uracil, 4-thiouracil, 5-(1,3-diazolyl-1-alkyl)uracil, 5-(2-aminopropyl)uracil, 5-(aminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidinium alkyl)uracil, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(methylamino ... -(methyl)-2-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(alkyl)-2,4(dithio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(alkyl)pseudouracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(halo)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(methoxy)uracil, 5-(methoxy carbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(methyl)-2-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methyl)-2-(thio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(methyl)pseudouracil, 5-(methylaminomethyl)-2-(thio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 5-aminoallyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-uracil, 6-(azo)uracil, 6-(azo)uracil, 6-aza-uridine, allylamino-uracil, azauracil, deazauracil, N3-(methyl)uracil, pseudo-UTP-1-2-ethanoic acid, pseudouracil, 4-thio-pseudo-UTP, 1-caproic acid 1-Propynyl-uridine, 1-Taurinomethyl-1-methyl-uridine, 1-Taurinomethyl-4-thio-uridine, 1-Taurinomethyl-pseudouridine, 2-Methoxy-4-thio-pseudouridine, 2-Thio-1-methyl-1-deaza-pseudouridine, 2-Thio-1-methyl-pseudouridine, 2-Thio-5-aza-uridine, 2-Thio-dihydropseudouridine, 2-Thio-dihydrouridine, 2-Thio opseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, (±)1-(2-hydroxypropyl)pseudouridine TP, (2R)-1-(2-hydroxypropyl)pseudouridine TP, (2S)-1-(2-hydroxypropyl)pseudouridine TP, (E)-5-(2-bromo-vinyl)ara-uridine TP, (E)-5 -(2-Bromo-vinyl)uridine TP, (Z)-5-(2-Bromo-vinyl)ara-uridine TP, (Z)-5-(2-Bromo-vinyl)uridine TP, 1-(2,2,2-trifluoroethyl)-pseudo-UTP, 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP, 1-(2,2-diethoxyethyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudouridine TP, 1-(2,4,6-trimethylbenzyl)pseudouridine TP, 1-(2,4,6-trimethyl-phenyl)pseudo-UTP, 1-(2-amino-2-carboxyethyl)pseudo-UTP, 1-(2-amino-ethyl)pseudo-UTP, 1-(2-hydroxyethyl)pseudouridine TP, 1-(2-methoxyethyl)pseudouridine TP, 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine TP, 1-(3,4-dimethoxybenzyl)pseudouridine TP, 1-(3-amino-3-carboxypropyl)pseudouridine TP, -UTP, 1-(3-amino-propyl)pseudo-UTP, 1-(3-cyclopropyl-prop-2-ynyl)pseudo-UTP, 1-(4-amino-4-carboxybutyl)pseudo-UTP, 1-(4-amino-benzyl)pseudo-UTP, 1-(4-amino-butyl)pseudo-UTP, 1-(4-amino-phenyl)pseudo-UTP, 1-(4-azidobenzyl)pseudo-UTP, 1-(4-bromobenzyl)pseudo-UTP, 1-(4- chlorobenzyl)pseudouridine TP, 1-(4-fluorobenzyl)pseudouridine TP, 1-(4-iodobenzyl)pseudouridine TP, 1-(4-methanesulfonylbenzyl)pseudouridine TP, 1-(4-methoxybenzyl)pseudouridine TP, 1-(4-methoxy-benzyl)pseudouridine TP, 1-(4-methoxy-phenyl)pseudouridine TP, 1-(4-methylbenzyl)pseudouridine TP, 1-(4-methyl-benzyl)pseudouridine TP do-UTP, 1-(4-nitrobenzyl)pseudo-UTP, 1-(4-nitro-benzyl)pseudo-UTP, 1-(4-nitro-phenyl)pseudo-UTP, 1-(4-thiomethoxybenzyl)pseudo-UTP, 1-(4-trifluoromethoxybenzyl)pseudo-UTP, 1-(4-trifluoromethylbenzyl)pseudo-UTP, 1-(5-amino-pentyl)pseudo-UTP, 1-(6-amino-hexyl)pseudo-UTP, 1,6-Dimethyl-pseudo-UTP, 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP, 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine TP, 1-acetylpseudouridine TP, 1-alkyl-6-(1-propynyl)-pseudo-UTP, 1-alkyl-6-(2-propynyl)-pseudo-UTP, 1-alkyl-6-allyl-pseudo-UTP, 1-alkyl-6-ethynyl-pseudo-UTP, 1-alkyl-6-homoallyl-pseudo-UTP, 1-alkyl-6-vinyl-pseudo-UTP, 1-allylpseudo, pseudouridine TP, 1-aminomethyl-pseudo-UTP, 1-benzoylpseudouridine TP, 1-benzyloxymethylpseudouridine TP, 1-benzyl-pseudo-UTP, 1-biotinyl-PEG2-pseudouridine TP, 1-biotinyl-pseudo-UTP, 1-butyl-pseudo-UTP, 1-cyanomethylpseudouridine TP, 1-cyclobutylmethyl-pseudo-UTP, 1-cyclobutyl-pseudo-UTP, 1-cycloheptylmethyl-pseudo-UTP, 1-cycloheptyl-pseudo-UTP, 1-cyclo 1-cyclohexylmethyl-pseudo-UTP, 1-cyclohexyl-pseudo-UTP, 1-cyclooctylmethyl-pseudo-UTP, 1-cyclooctyl-pseudo-UTP, 1-cyclopentylmethyl-pseudo-UTP, 1-cyclopentyl-pseudo-UTP, 1-cyclopropylmethyl-pseudo-UTP, 1-cyclopropyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 1-hexyl-pseudo-UTP, 1-homoallylpseudouridine TP, 1-hydroxymethylpseudouridine TP, 1-isopropyl-pseudo-UTP, UTP, 1-Me-2-thio-pseudo-UTP, 1-Me-4-thio-pseudo-UTP, 1-Me-alpha-thio-pseudo-UTP, 1-methanesulfonylmethylpseudouridine TP, 1-methoxymethylpseudouridine TP, 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP, 1-methyl-6-(4-morpholino)-pseudo-UTP, 1-methyl-6-(4-thiomorpholino)-pseudo-UTP, 1-methyl-6-(substituted phenyl)pseudo-UTP, 1-methyl-6-amino-pseudo-UTP, 1 -Methyl-6-azido-pseudo-UTP, 1-methyl-6-bromo-pseudo-UTP, 1-methyl-6-butyl-pseudo-UTP, 1-methyl-6-chloro-pseudo-UTP, 1-methyl-6-cyano-pseudo-UTP, 1-methyl-6-dimethylamino-pseudo-UTP, 1-methyl-6-ethoxy-pseudo-UTP, 1-methyl-6-ethylcarboxylate-pseudo-UTP, 1-methyl-6-ethyl-pseudo-UTP, 1-methyl-6-fluoro-pseudo-UTP, 1-methyl-6-formyl-pseudo-UTP,1-methyl-6-hydroxyamino-pseudo-UTP, 1-methyl-6-hydroxy-pseudo-UTP, 1-methyl-6-iodo-pseudo-UTP, 1-methyl-6-iso-propyl-pseudo-UTP, 1-methyl-6-methoxy-pseudo-UTP, 1-methyl-6-methylamino-pseudo-UTP, 1-methyl-6-phenyl-pseudo-UTP, 1-methyl-6-propyl-pseudo-UTP, 1-methyl-6-tert-butyl-pseudo-UTP, 1-methyl-6-trifluoromethoxy-pseudo-UTP, 1-methyl-6- Trifluoromethyl-pseudo-UTP, 1-morpholinomethylpseudouridine TP, 1-pentyl-pseudouridine TP, 1-phenyl-pseudouridine TP, 1-pivaloyl-pseudouridine TP, 1-propargyl-pseudouridine TP, 1-propyl-pseudouridine TP, 1-propynyl-pseudouridine, 1-p-tolyl-pseudouridine TP, 1-tert-butyl-pseudouridine TP, 1-thiomethoxymethyl-pseudouridine TP, 1-thiomorpholinomethyl-pseudouridine TP, 1-trifluoroacetyl-pseudouridine TP, 1 -Trifluoromethyl-pseudo-UTP, 1-vinylpseudouridine TP, 2,2'-anhydro-uridine TP, 2'-bromo-deoxyuridine TP, 2'-F-5-methyl-2'-deoxy-UTP, 2'-OMe-5-Me-UTP, 2'-OMe-pseudo-UTP, 2'-a-ethynyluridine TP, 2'-a-trifluoromethyluridine TP, 2'-b-ethynyluridine TP, 2'-b-trifluoromethyluridine TP, 2'-deoxy-2',2'-difluorouridine TP, 2'-deoxy-2'-a-mercaptouridine uridine TP, 2'-deoxy-2'-a-thiomethoxyuridine TP, 2'-deoxy-2'-b-aminouridine TP, 2'-deoxy-2'-b-azidouridine TP, 2'-deoxy-2'-b-bromouridine TP, 2'-deoxy-2'-b-chlorouridine TP, 2'-deoxy-2'-b-fluorouridine TP, 2'-deoxy-2'-b-iodouridine TP, 2'-deoxy-2'-b-mercaptouridine TP, 2'-deoxy-2'-b-thiomethoxyuridine TP, 2-methoxy-4-thio-uridine, 2-methoxyuridine,2'-O-methyl-5-(1-propynyl)uridine TP, 3-alkyl-pseudo-UTP, 4'-azidouridine TP, 4'-carbocyclic uridine TP, 4'-ethynyluridine TP, 5-(1-propynyl)ara-uridine TP, 5-(2-furanyl)uridine TP, 5-cyanouridine TP, 5-dimethylaminouridine TP, 5'-homo-uridine TP, 5-iodo-2'-fluoro-deoxyuridine TP, 5-phenylethynyluridine TP, 5-trideuteromethyl-6-deuterouridine TP, 5 -Trifluoromethyl-uridine TP, 5-vinyl auridine TP, 6-(2,2,2-trifluoroethyl)-pseudo-UTP, 6-(4-morpholino)-pseudo-UTP, 6-(4-thiomorpholino)-pseudo-UTP, 6-(substituted-phenyl)-pseudo-UTP, 6-amino-pseudo-UTP, 6-azido-pseudo-UTP, 6-bromo-pseudo-UTP, 6-butyl-pseudo-UTP, 6-chloro-pseudo-UTP, 6-cyano-pseudo-UTP, 6-dimethylamino-pseudo-UTP, 6-etyl-pseudo-UTP, Toxic-pseudo-UTP, 6-ethylcarboxylate-pseudo-UTP, 6-ethyl-pseudo-UTP, 6-fluoro-pseudo-UTP, 6-formyl-pseudo-UTP, 6-hydroxyamino-pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP, 6-isopropyl-pseudo-UTP, 6-methoxy-pseudo-UTP, 6-methylamino-pseudo-UTP, 6-methyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-protoxin-pseudo-UTP, 6-ethylcarboxylate-pseudo-UTP, 6-ethyl-pseudo-UTP, 6-fluoro-pseudo-UTP, 6-formyl-pseudo-UTP, 6-hydroxyamino-pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP, 6-isopropyl-pseudo-UTP, 6-methoxy-pseudo-UTP, 6-methylamino-pseudo-UTP, 6-methyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-phenyl-pseudo-UTP, Propyl-pseudo-UTP, 6-tert-butyl-pseudo-UTP, 6-trifluoromethoxy-pseudo-UTP, 6-trifluoromethyl-pseudo-UTP, alpha-thio-pseudo-UTP, pseudouridine-1-(4-methylbenzenesulfonic acid) TP, pseudouridine-1-(4-methylbenzoic acid) TP, pseudouridine TP-1-[3-(2-ethoxy)]propionic acid, pseudouridine TP-1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid,Pseudouridine TP-1-[3-{2-(2-[2-{2-(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP-1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouridine TP-1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid, pseudouridine TP-1-methylphosphonic acid, pseudouridine TP-1-methylphosphonic acid diethyl ester, pseudo-UTP-N1-3-propionic acid, pseudo-UTP-N1-4-butanoic acid , pseudo-UTP-N1-5-pentanoic acid, pseudo-UTP-N1-6-hexanoic acid, pseudo-UTP-N1-7-heptanoic acid, pseudo-UTP-N1-methyl-p-benzoic acid, pseudo-UTP-N1-p-benzoic acid, wybutosine, hydroxywybutosine, isowyosine, peroxywybutosine, undermodified hydroxywybutosine, 4-demethylwybutosine, 2,6-(diamino)purine, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)wybutosine, so)-phenothiazin-1-yl, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, 2-(amino)purine, 2,4,5-(trimethyl)phenyl, 2'-methyl,2'-amino,2'-azido,2'-fluoro-cytidine, 2'-methyl,2'-amino,2'-azido,2'-fluoro-adenine, 2'-methyl,2'-amino,2'-azido,2'-fluoro-uridine, 2'-amino-2'-deoxyribose, 2-amino-6-chloro-purine, 2-azaino cinyl, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-fluoro-modified base, 2'-O-methyl-ribose, 2-oxo-7-aminopyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, 2-pyridinone, 3-nitropyrrole, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 3-(methyl)isocarbostyrilyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,5-Nitroindole, 5-substituted pyrimidines, 5-(methyl)isocarbostyrylyl, 5-nitroindole, 6-(aza)pyrimidine, 6-(azo)thymine, 6-(methyl)-7-(aza)indolyl, 6-chloro-purine, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1, 3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aza)indolyl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-( Guanidinium alkyl hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkyl hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(propynyl)isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, ropinyl-7-(aza)indolyl, 7-deaza-inosinyl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 9-(methyl)-imidizopyridinyl, aminoindolyl, anthracenyl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, difluorotolyl, hypoxanthine, imidizopyridinyl,Inosinyl, Isocarbostyrilyl, Isoguanisine, N2-Substituted Purines, N6-Methyl-2-amino-purine, N6-Substituted Purines, N-Alkylated Derivatives, Naphthalenyl, Nitrobenzimidazolyl, Nitroimidazolyl, Nitroindazolyl, Nitropyrazolyl, Nubularine, O6-Substituted Purines, O-Alkylated Derivatives, Ortho-(aminoalkylhydroxy)-6-phenyl-, Pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, oxoformycin TP, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pentacenyl, phenanthracenyl, phenyl, propynyl-7-(aza)indolyl, pyrenyl, pyridopyrimidin-3-yl, pyridopyrimidin-3-yl yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, pyrrolo-pyrimidin-2-one-3-yl, pyrrolopyrimidinyl, pyrrolopyridinyl, stilbenzyl, substituted 1,2,4-triazoles, tetracenyl, tubercidin, xanthine, xanthosine-5'-TP, 2-thio-zebularine, 5-aza-2-thio-zebularine, 7-deaza-2-amino-purine, pyridin-4-one ribonucleoside, 2-amino-riboside-TP, formycin ATP, formycin B TP, pyrrolosine TP, 2'-OH-ara-adenosine TP, 2'-OH-ara-cytidine TP, 2'-OH-ara-uridine TP, 2'-OH-ara-guanosine TP, 5-(2-carbomethoxyvinyl)uridine TP, and N6-(19-amino-pentaoxanonadecyl)adenosine TP.

[0100] In some embodiments, a polynucleotide (eg, an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises a combination of at least two (eg, two, three, four or more) of the foregoing modified nucleobases.

[0101] In some embodiments, the modified nucleobase in a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) is pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-methyl-cytosine, 2-thio-1-methyl-1-deaza-pseudouridine ... In some embodiments, the modified nucleobase is selected from the group consisting of 2'-O-methyluridine, ...

[0102] In some embodiments, modified nucleobases in a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) are selected from the group consisting of 1-methyl-pseudouridine (m1Ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (Ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, a polynucleotide comprises a combination of at least two (e.g., two, three, four or more) of the foregoing modified nucleobases.

[0103] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises pseudouridine (Ψ) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1Ψ). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1Ψ) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises methoxy-uridine (mo5U). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2'-O-methyluridine. In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises 2'-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0104] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, e.g., an mRNA polynucleotide) is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), i.e., all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified with respect to any type of nucleoside residue present in the sequence by replacing it with a modified residue such as those described above.

[0105] Exemplary nucleobases and nucleosides having modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0106] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having modified uridine include 5-cyanouridine and 4'-thiouridine.

[0107] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0108] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0109] The polynucleotides of the present disclosure may be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified within the polynucleotides of the present disclosure or within a given sequence region thereof (e.g., within an mRNA, including or excluding a polyA tail). In some embodiments, all nucleotides X within the polynucleotides of the present disclosure (or within a given sequence region thereof) are modified nucleotides, where X can be any one of the nucleotides A, G, U, C, or any one of the following combinations: A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0110] The polynucleotides may contain from about 1% to about 100% modified nucleotides (either with respect to the total nucleotide content, or with respect to one or more of the types of nucleotides, i.e., with respect to any one or more of A, G, U, or C), or any percentage therebetween (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10 ...95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10 % to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It will be understood that the remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0111] The polynucleotide may comprise at least 1% and up to 100% modified nucleotides, or any percentage therebetween, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotide may comprise modified pyrimidines, such as modified uracils or cytosines. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils of the polynucleotide are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracils may be replaced with a compound having a single unique structure, or may be replaced with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the polynucleotide are replaced with modified cytosines (e.g., 5-substituted cytosines), either with a compound having a single unique structure or with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0112] Thus, in some embodiments, the RNA therapeutic comprises a 5'UTR element, optionally a codon-optimized open reading frame, and a 3'UTR element, a poly(A) sequence and / or a polyadenylation signal, and the RNA is not chemically modified.

[0113] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (Ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-Methoxy-uridine (mo5U), Uridine 5-oxyacetic acid (cmo5U), Uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2- Thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1Ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine,1-Methyl-1-deaza-pseudouridine, 2-Thio-1-methyl-1-deaza-pseudouridine, Dihydrouridine (D), Dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyl-dihydrouridine (m5D), 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2 -thio-pseudouridine, N1-methyl-pseudouridine, N1-ethyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio -uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0114] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydro-cytidine (5-hydro-cytidine), 5-amino-cytidine (5-amino ... 5-methyl-2-thiocytidine (s2C), 2-thio-5-methyl-2-thiocytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl- These include tyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0115] In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -Methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarba N6-amino-N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenine adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0116] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OhyW), undermodified hydroxywyosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (o Q), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl ... Chil-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O 2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0117] The antibodies and antigen-binding fragments thereof of the present disclosure comprise at least one RNA polynucleotide, e.g., an mRNA (e.g., a modified mRNA). The mRNA is transcribed in vitro, e.g., from a template DNA, referred to as an "in vitro transcription template." In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a polyA tail. The particular nucleic acid sequence composition and length of the in vitro transcription template depends on the mRNA encoded by the template.

[0118] Array Optimization In some embodiments, the ORFs encoding the antigens of the present disclosure are codon-optimized. Methods of codon optimization are known in the art. For example, any one or more of the ORFs of the sequences provided herein may be codon-optimized. Codon optimization may be used in some embodiments to match codon frequencies in target and host organisms to ensure proper folding, to bias GC content to enhance mRNA stability or reduce secondary structure, to minimize runs of tandem repeat codons or bases that may impair gene organization or expression, to customize transcriptional and translational regulatory regions, to insert or remove protein trafficking sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) of the encoded protein, to add, remove, or swap protein domains, to insert or delete restriction enzyme recognition sites, to modify ribosome binding sites and mRNA degradation sites, to adjust translation rates and correctly fold various domains of the protein, or to reduce or remove problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0119] In some embodiments, the codon-optimized sequence shares less than 95% sequence identity to a naturally occurring or wild-type sequence ORF (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease). In some embodiments, the codon-optimized sequence shares less than 90% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease). In some embodiments, the codon-optimized sequence shares less than 85% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease). In some embodiments, the codon-optimized sequence shares less than 80% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease). In some embodiments, the codon-optimized sequence shares less than 75% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease).

[0120] In some embodiments, the codon optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease). In some embodiments, the codon optimized sequence shares 65% to 75% or about 80% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease).

[0121] In some embodiments, the codon optimized sequence encodes a picornavirus capsid polyprotein and / or picornavirus 3C protease that is the same as or more immunogenic (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% or more) than a picornavirus capsid polyprotein and / or picornavirus 3C protease encoded by a non-codon optimized sequence.

[0122] The modified mRNA, when transfected into a mammalian host cell, has a stability of 12 to 18 hours or more than 18 hours, for example, 24, 36, 48, 60, 72 hours, or more than 72 hours, and is capable of being expressed by the mammalian host cell.

[0123] In some embodiments, a codon-optimized RNA may have an increased G / C level. The G / C content of a nucleic acid molecule (e.g., an mRNA) may affect the stability of the RNA. An RNA with an increased amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than an RNA that contains a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02 / 098443 discloses a pharmaceutical composition comprising an mRNA stabilized by modification of a sequence in the translation region. Due to the degeneracy of the genetic code, the modification works by replacing existing codons with those that further increase the stability of the RNA without changing the resulting amino acid. The approach is limited to the coding region of the RNA.

[0124] Unmodified nucleotides In some embodiments, the RNA (e.g., mRNA) is not chemically modified and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues, e.g., residues present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides, e.g., deoxyribonucleosides present in DNA (e.g., dA, dG, dC, or dT).

[0125] chemical modification The compositions of the present disclosure, in some embodiments, include RNA having an open reading frame encoding a coronavirus antigen, the nucleic acid comprising nucleotides and / or nucleosides that may be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications may include art-recognized modifications at the sugar, backbone, or nucleobase moieties of the nucleotides and / or nucleosides.

[0126] In some embodiments, the naturally occurring modified nucleotides or nucleotides of the present disclosure are those commonly known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0127] In some embodiments, the non-naturally occurring modified nucleotides or nucleosides of the present disclosure are those that are commonly known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published U.S. application Nos. PCT / US2012 / 058519, PCT / US2013 / 075177, PCT / US2014 / 058897, PCT / US2014 / 058891, PCT / US2014 / 070413, PCT / US2015 / 36773, PCT / US2015 / 36759, PCT / US2015 / 36771, or PCT / IB2017 / 051367, all of which are incorporated herein by reference.

[0128] Thus, the nucleic acids of the present disclosure (eg, DNA nucleic acids and RNA nucleic acids, e.g., mRNA nucleic acids) can include standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0129] Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, e.g., mRNA nucleic acids), in some embodiments, contain a variety (two or more) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0130] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into a cell or organism exhibit reduced degradation in the cell or organism, respectively, compared to unmodified nucleic acids comprising standard nucleotides and nucleosides.

[0131] In some embodiments, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively, compared to unmodified nucleic acids comprising standard nucleotides and nucleosides.

[0132] In some embodiments, a nucleic acid (e.g., an RNA nucleic acid, e.g., an mRNA nucleic acid) comprises non-natural modified nucleotides that are introduced during or after the synthesis of the nucleic acid to achieve a desired function or property. The modifications can be in the internucleotide bond, the purine or pyrimidine base, or the sugar. The modifications can be introduced by chemical synthesis or can be introduced at the end of the chain or anywhere in the chain using a polymerase enzyme. Any region of the nucleic acid can be chemically modified.

[0133] The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids, e.g., mRNA nucleic acids). A "nucleoside" refers to a compound that includes a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized in any useful manner, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. A nucleic acid can include a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acid includes a region of nucleotides.

[0134] Base pairing of modified nucleotides encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases, or between two complementary non-standard base structures, for example, in nucleic acids with at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of bases / sugars or linkers may be incorporated into the nucleic acids of the present disclosure.

[0135] In some embodiments, the modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid, e.g., an mRNA nucleic acid) include 1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1Ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (Ψ). In some embodiments, the modified nucleobases in a nucleic acid (e.g., an RNA nucleic acid, e.g., an mRNA nucleic acid) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., two, three, four, or more) of any of the foregoing modified nucleobases (including, but not limited to, chemical modifications).

[0136] In some embodiments, an mRNA of the disclosure comprises 1-methyl-pseudouridine (m1Ψ) substitutions at one or more or all uridine positions of the nucleic acid.

[0137] In some embodiments, an mRNA of the disclosure comprises 1-methyl-pseudouridine (m1Ψ) substitutions at one or more or all of the uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all of the cytidine positions of the nucleic acid.

[0138] In some embodiments, an mRNA of the disclosure contains pseudouridine (Ψ) substitutions at one or more or all uridine positions of the nucleic acid.

[0139] In some embodiments, an mRNA of the disclosure comprises pseudouridine (Ψ) substitutions at one or more or all of the uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all of the cytidine positions of the nucleic acid.

[0140] In some embodiments, an mRNA of the disclosure comprises a uridine at one or more or all uridine positions of the nucleic acid.

[0141] In some embodiments, the mRNA is uniformly modified (e.g., completely modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, i.e., all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified with respect to any type of nucleoside residue present in the sequence by replacing it with a modified residue such as those described above.

[0142] The nucleic acids of the present disclosure may be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified within the nucleic acids of the present disclosure or within a given sequence region thereof (e.g., within an mRNA, including or excluding a poly(A) tail). In some embodiments, all nucleotides X in the nucleic acids of the present disclosure (or in a sequence region thereof) are modified nucleotides, where X is any one of the nucleotides A, G, U, C, or any one of the following combinations: A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0143] Nucleic acids may contain from about 1% to about 100% modified nucleotides (either with respect to the total nucleotide content, or with respect to one or more of the types of nucleotides, i.e., with respect to any one or more of A, G, U, or C), or any percentage therebetween (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, %, 10%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%). It will be understood that the remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0144] The mRNA may comprise a minimum of 1% to a maximum of 100% modified nucleotides, or any range of percentages therebetween, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.For example, the nucleic acid may comprise modified pyrimidines, such as modified uracil or cytosine.In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in the nucleic acid are replaced with modified uracils (e.g., 5-substituted uracils).The modified uracils may be replaced with a compound having a single unique structure, or may be replaced with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the nucleic acid are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines may be replaced with a compound having a single unique structure or may be replaced with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0145] Untranslated Regions (UTRs) The mRNA of the present disclosure may include one or more regions or portions that act or function as untranslated regions. When the mRNA is designed to code for at least one antigen of interest, the nucleic acid may include one or more of these untranslated regions (UTRs). The wild-type untranslated region of a nucleic acid is transcribed but not translated. In an mRNA, the 5'UTR starts at the transcription start site and continues to the start codon but does not include the start codon. On the other hand, the 3'UTR starts immediately after the stop codon and continues to the transcription termination signal. There is a growing body of evidence on the regulatory role that UTRs play in the stability and translation of the nucleic acid molecule. The regulatory function of UTRs can be incorporated into the polynucleotide of the present disclosure, particularly to increase the stability of the molecule. Also, specific functions can be incorporated to ensure that the downregulation of the transcript is controlled in case the transcript is misdirected to an undesired organ site. A variety of 5'UTR and 3'UTR sequences are known and available in the art.

[0146] 5'UTR is the region of an mRNA immediately upstream (5') from the start codon (the first codon of an mRNA transcript that is translated by ribosomes). 5'UTRs do not code for proteins (they are non-coding). Natural 5'UTRs have functions that play a role in translation initiation. They have signatures such as the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate the translation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG (SEQ ID NO:27), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) followed by another "G". 5'UTRs are also known to form secondary structures involved in the binding of elongation factors.

[0147] In some embodiments of the present disclosure, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature associated with a different ORF. In another embodiment, the 5'UTR is a synthetic UTR, i.e., does not exist in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., to increase gene expression, and completely synthetic UTRs. Exemplary 5'UTRs include a-globin or b-globin from Xenopus or human (8278063, 9012219), human cytochrome b-245a polypeptide, and hydroxysteroid (17b) dehydrogenase, as well as tobacco etch virus (US8278063, 9012219). The CMV immediate early 1 (IE1) gene (US20140206753, WO2013 / 185069), sequence GGGAUCCUACC (SEQ ID NO:28) (WO2014144196) can also be used. In another embodiment, the 5'UTR of the TOP gene is a 5'UTR of a TOP gene lacking a 5'TOP motif (oligopyrimidine tract) (e.g., WO / 2015101414, WO2015101415, WO / 2015 / 062738, WO2015024667, WO2015024667, a 5'UTR element from the ribosomal protein large 32 (L32) gene (WO / 2015101414, WO2015024667, 5'UTR elements derived from the 5'UTR of the hydroxysteroid (17-beta) dehydrogenase 4 gene (WO2015024667), or the 5'UTR elements derived from the 5'UTR of the ATP5A1 gene (WO2015024667) may be used. In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0148] In some embodiments, the 5'UTR of the disclosure comprises SEQ ID NO:2.

[0149] The 3'UTR is the region of an mRNA immediately downstream (3') of the stop codon (the codon of the mRNA transcript that signals the end of translation). The 3'UTR does not code for a protein (it is non-coding). Native or wild-type 3'UTRs are known to be embedded with stretches of adenosines and uridines. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al, 1995), where class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III AREs are less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins that bind to AREs are known to destabilize messengers, but members of the ELAV family, particularly HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. Incorporation of a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding, which stabilizes the message in vivo.

[0150] Introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR may be used to modulate the stability of the nucleic acids (e.g., RNA) of the present disclosure. When engineering a particular nucleic acid, one or more copies of an ARE can be introduced to reduce the stability of the nucleic acid of the present disclosure, thereby suppressing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase stability in cells, increasing the translation and production of the resulting protein. Transfection experiments can be performed in relevant cell lines using the nucleic acids of the present disclosure, and protein production can be assayed at various time points after transfection. For example, cells can be transfected with various ARE engineered molecules and ELISA kits can be used for the relevant proteins to assay the proteins produced 6 hours, 12 hours, 24 hours, 48 ​​hours, and 7 days after transfection.

[0151] The 3'UTR may be heterologous or synthetic. For 3'UTR, globin UTRs (including Xenopus β-globin UTR and human β-globin UTR) are known in the art (8278063, 9012219, US20110086907). Modified β-globin constructs with enhanced stability in some cell types have been developed by cloning two consecutive human β-globin 3'UTRs head-to-tail and are known in the art (US2012 / 0195936, WO2014 / 071963). In addition, a2-globin, a1-globin, UTR and their variants are also known in the art (WO2015101415, WO2015024667). Other 3'UTRs described in mRNA constructs in non-patent literature include CYBA (Ferizi et al., 2015) and albumin (Thess et al., 2015). Other exemplary 3'UTRs include those of bovine or human growth hormone (wild type or modified) (WO2013 / 185069, US20140206753, WO2014152774), and rabbit β-globin and Hepatitis B virus (HBV), α-globin 3'UTR and viral VEEV 3'UTR sequences are also known in the art. In some embodiments, the sequence UUUGAAUU (WO2014144196) is used. In some embodiments, the 3'UTRs of human and mouse ribosomal proteins are used. Other examples include rps9 3'UTR (WO2015101414), FIG4 (WO2015101415), and human albumin 7 (WO2015101415).

[0152] In some embodiments, the 3'UTR of the disclosure comprises SEQ ID NO:4.

[0153] One of skill in the art will appreciate that heterologous or synthetic 5'UTRs may be used with any desired 3'UTR sequence. For example, heterologous 5'UTRs may be used with synthetic 3'UTRs and heterologous 3"UTRs.

[0154] Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, an intron or a portion of an intron sequence may be incorporated into a region of the nucleic acid of the present disclosure. The incorporation of an intron sequence may increase protein production and the level of the nucleic acid.

[0155] A combination of features may be included in the flanking regions, among other features. For example, the ORF may be flanked by a 5'UTR that may include a strong Kozak translation initiation signal and / or a 3'UTR that may include an oligo(dT) sequence for templated addition of a polyA tail. The 5'UTR may include a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes (e.g., the 5'UTRs described in U.S. Patent Application Publication No. 20100293625 and PCT / US2014 / 069155, which are incorporated herein by reference in their entireties).

[0156] It should be understood that any UTR from any gene may be incorporated into the region of the nucleic acid. In addition, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs that are not variants of the wild-type region. These UTRs or parts thereof may be placed in the same orientation as the transcript from which they were selected, or may be altered in orientation or position. Thus, the 5' or 3' UTR may be inverted, shortened, extended, or combined with one or more other 5' or 3' UTRs. As used herein, the term "altered" with respect to a UTR sequence means that the UTR is altered in some way relative to the reference sequence. For example, the 3' or 5' UTR may be altered relative to the wild-type or natural UTR by the change in orientation or position as taught above, or by the inclusion of additional nucleotides, deletion of nucleotides, exchange or transposition of nucleotides. Any of these alterations that produce an "altered" UTR (either 3' or 5') include variant UTRs.

[0157] In some embodiments, double, triple, or quadruple UTRs can be used, such as 5'UTR or 3'UTR.As used herein, a "double" UTR is one in which two copies of the same UTR are encoded in tandem or substantially in tandem.For example, double beta-globin 3'UTRs can be used as described in US Patent Publication No. 20100129877, the contents of which are incorporated herein by reference in their entirety.

[0158] It is also within the scope of the present disclosure to have patterned UTR.As used herein, "patterned UTR" refers to UTR that reflects repeating or alternating patterns, such as ABABAB or AABBABBAABB or ABCABCABC, or their variants that are repeated once, twice, or more than three times.In these patterns, each letter A, B, or C represents a UTR that is different at the nucleotide level.

[0159] In some embodiments, the flanking region is selected from a family of transcripts whose proteins share a common function, structure, feature, or characteristic. For example, a polypeptide of interest may belong to a family of proteins that are expressed in a particular cell, tissue, or at a certain time during development. Any UTR from these genes may be exchanged with any other UTR from the same or different protein family to create a new polynucleotide. As used herein, "family of proteins" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, feature, location, origin, or expression pattern.

[0160] The untranslated region may also include a translational enhancer element (TEE). By way of non-limiting example, the TEE may include those described in U.S. Application No. 20090226470, which is incorporated herein by reference in its entirety, and those known in the art.

[0161] In vitro transcription of RNA The cDNA encoding the polynucleotides described herein can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and described in International Publication No. WO / 2014 / 152027 (incorporated herein by reference in its entirety). In some embodiments, the RNA of the present disclosure is prepared according to any one or more of the methods described in WO2018 / 053209 and WO2019 / 036682 (each of which is incorporated herein by reference).

[0162] In some embodiments, the RNA transcript is generated using an unamplified linearized DNA template in an in vitro transcription reaction to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA polynucleotide, such as, but not limited to, a coronavirus mRNA. In some embodiments, cells, such as bacterial cells, such as E. coli, e.g., DH-1 cells, are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template comprises an RNA polymerase promoter, such as a T7 promoter, located 5' to a gene of interest and operably linked to the gene of interest.

[0163] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a poly(A) tail. The particular nucleic acid sequence composition and length of the in vitro transcription template depends on the mRNA encoded by the template.

[0164] "5' untranslated region" (UTR) refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of the mRNA transcript that is translated by the ribosome) that does not code for a polypeptide. When an RNA transcript is being produced, the 5'UTR may contain a promoter sequence. Such promoter sequences are known in the art. It is understood that no such promoter sequences are present in the vaccines of the present disclosure.

[0165] "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (i.e., 3') from a stop codon (i.e., a codon in an mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0166] An "open reading frame" is a contiguous stretch of DNA beginning with a start codon (eg, methionine (ATG)) and ending with a stop codon (eg, TAA, TAG, or TGA) that encodes a polypeptide.

[0167] A "poly(A) tail" is a region of an mRNA downstream, e.g., immediately downstream (i.e., 3'), of a 3'UTR that contains multiple consecutive adenosine monophosphates. A poly(A) tail can contain 10-300 adenosine monophosphates. For example, a poly(A) tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50-250 adenosine monophosphates. In relevant biological environments (e.g., within a cell, in vivo), the poly(A) tail functions to protect the mRNA from enzymatic degradation (e.g., in the cytoplasm) and to assist in transcription termination and / or transport of the mRNA from the nucleus and translation.

[0168] In some embodiments, the nucleic acid comprises 200 to 3,000 nucleotides. For example, the nucleic acid may comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0169] An in vitro transcription system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase.

[0170] The NTPs may be produced in-house, selected from a commercial supplier, or synthesized as described herein. The NTPs may be selected from those described herein, including, but not limited to, natural and unnatural (modified) NTPs.

[0171] Various RNA polymerases or variants can be used in the methods of the present disclosure. The polymerase can be selected from, but is not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or mutant polymerases, such as polymerases that can incorporate modified nucleic acids and / or modified nucleotides, including but not limited to chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNase.

[0172] In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA comprises a 5' end cap, e.g., 7mG(5')ppp(5')NlmpNp.

[0173] chemical synthesis Solid-phase chemical synthesis. The nucleic acids of the present disclosure may be produced in whole or in part using solid-phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a reaction solution. Solid-phase synthesis is useful for site-specific introduction of chemical modifications into nucleic acid sequences.

[0174] Solution Phase Chemical Synthesis. Synthesis of the nucleic acids of the present disclosure by sequential addition of monomer building blocks can be carried out in solution phase.

[0175] Combination of synthetic methods. Each of the above synthetic methods has its own advantages and limitations. Attempts have been made to combine these methods to overcome the limitations. Such combinations of methods are within the scope of this disclosure. The combination of solid-phase or liquid-phase chemical synthesis and enzymatic ligation provides an efficient method for producing long-chain nucleic acids that cannot be obtained by chemical synthesis alone.

[0176] Ligation of nucleic acid regions or subregions Assembly of nucleic acids by ligase can also be used. DNA or RNA ligase promotes intermolecular ligation of the 5' and 3' ends of a polynucleotide chain through the formation of a phosphodiester bond. Nucleic acids, e.g., chimeric polynucleotides and / or circular nucleic acids, can be prepared by ligation of one or more regions or subregions. By joining DNA fragments by a ligase-catalyzed reaction, recombinant DNAs with different functions can be created. Two oligodeoxynucleotides, one containing a 5' phosphoryl group and the other containing a free 3' hydroxyl group, serve as substrates for DNA ligase.

[0177] purification Purification of nucleic acids as described herein may include, but is not limited to, nucleic acid cleanup, quality assurance and quality control. Cleanup may be performed by methods known in the art, such as, but not limited to, AGENCORT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA™ Oligo-T Capture Probes (EXIQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term "purified" when used in reference to nucleic acids, e.g., "purified nucleic acid," refers to something that has been separated from at least one contaminant. A "contaminant" is any substance that renders another unsuitable, impure, or adulterated. Thus, purified nucleic acids (e.g., DNA and RNA) are present in a form or setting that is different from that in which it is found in nature or that is present prior to being subjected to a processing or purification method.

[0178] Quality assurance and / or quality control checks may be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0179] In some embodiments, the nucleic acid may be sequenced by methods including, but not limited to, reverse transcriptase PCR.

[0180] Quantification In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or derived from one or more bodily fluids, including peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's or pre-ejaculate fluid, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyle, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool, pancreatic juice, sinus lavage, bronchopulmonary aspirate, blastodermal cavity fluid, and umbilical cord blood. Alternatively, exosomes may be harvested from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0181] The assay may be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, and exosomes may be isolated using immunohistochemistry such as enzyme-linked immunosorbent assay (ELISA). Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or a combination thereof.

[0182] These methods allow researchers to monitor residual or delivered levels of nucleic acid in real time, which is possible because the nucleic acids of the present disclosure, in some embodiments, differ from endogenous forms by structural or chemical modifications.

[0183] In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed to determine whether the nucleic acid may be of the appropriate size and to confirm that degradation of the nucleic acid has not occurred. Degradation of the nucleic acid may be confirmed by methods such as, but not limited to, agarose gel electrophoresis, HPLC-based purification methods, including, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE).

[0184] Lipid nanoparticle formulations In some embodiments, the RNA (e.g., mRNA) of the present disclosure is formulated into lipid nanoparticles (LNPs), referred to herein as LNP-formulated mRNA vaccines (fLNPs). As used herein, a "lipid nanoparticle" typically comprises an ionizable cationic lipid, typically an ionizable amino lipid, a non-cationic lipid, a sterol and a PEG lipid component, and a nucleic acid cargo of interest. The lipid nanoparticles of the present invention can be produced using components, compositions, and methods generally known in the art. For example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2016 / 014280, PCT / US2017 / 038426, PC See PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, PCT / US2016 / 069491, PCT / US2016 / 069493, and PCT / US2014 / 066242, all of which are incorporated by reference in their entireties.

[0185] The vaccines of the present disclosure are typically formulated into lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise at least one ionic amino lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0186] In some embodiments, the lipid nanoparticles contain 20-60% amino lipid by molar ratio relative to other lipid components. For example, the lipid nanoparticles may contain 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% amino lipid by molar ratio. In some embodiments, the lipid nanoparticles contain 20%, 30%, 40%, 50, or 60% amino lipid by molar ratio.

[0187] In some embodiments, the lipid nanoparticles contain 5-25% molar phospholipids relative to other lipid components. For example, the lipid nanoparticles may contain 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% molar phospholipids. In some embodiments, the lipid nanoparticles contain 5%, 10%, 15%, 20%, 25%, or 30% molar non-cationic lipids.

[0188] In some embodiments, the lipid nanoparticles contain 25-55% molar structured lipid relative to other lipid components. For example, the lipid nanoparticles may contain 10-55%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% structured lipid. In some embodiments, the lipid nanoparticles contain 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% structured lipid.

[0189] In some embodiments, the lipid nanoparticles contain PEG lipids at a molar ratio of 0.5-15% relative to other lipid components. For example, the lipid nanoparticles may contain PEG lipids at a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticles contain PEG lipids at a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0190] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% amino lipid, 5-25% phospholipid, 25-55% structural lipid, and 0.5-15% PEG lipid.

[0191] In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% amino lipid, 5-30% phospholipid, 10-55% structural lipid, and 0.5-15% PEG lipid.

[0192] Amino lipids In some embodiments, the amino lipids of the present disclosure comprise a compound of formula (I): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R1 is C 5-30 Alkyl, C 5-20 Alkenyl, -R * -YR", -YR", and -R"M'R'; R2 and R3 are independently H, C 1-14 Alkyl, C 2-14 Alkenyl, -R * YR”, -YR”, and -R * OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or a carbocycle; R4 is hydrogen, C 3-6 Carbocyclic ring, -(CH2)n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1-6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R , -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2) n n is independently selected from 1, 2, 3, 4, and 5; Each R5 is independently 1-3 Alkyl, C 2-3 alkenyl, and H; Each R6 is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group, where M" is a bond, 1-13 Alkyl or C 2-13 alkenyl, R7 is C 1-3 Alkyl, C 2-3 alkenyl, and H; R8 is C 3-6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 selected from the group consisting of carbocycles and heterocycles; Each R is independently C 1-3 Alkyl, C 2-3 alkenyl, and H; Each R' is independently C 1-18 Alkyl, C 2-18 Alkenyl, -R * is selected from the group consisting of YR″, -YR″, and H; Each R" is independently 3-15 Alkyl and C 3-15 alkenyl, Each R * is independently 1-12 Alkyl and C 2-12 alkenyl, Each Y is independently C 3-6 is a carbocyclic ring, each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, where R4 is -(CH2) n Q, -(CH2) n In the case of CHQR, -CHQR, or -CQ(R), (i) Q is not -N(R) when n is 1, 2, 3, 4, or 5, or (ii) Q is not a 5-, 6-, or 7-membered heterocycloalkyl when n is 1 or 2.

[0193] In certain embodiments, a subset of compounds of formula (I) is represented by formula (IA): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M'; R4 is hydrogen, unsubstituted C 1-3 Alkyl, or -(CH2) n Q, where Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from H, C, 1-14 Alkyl, and C 2-14 For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.

[0194] In certain embodiments, a subset of compounds of formula (I) has formula (IB): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; R4 is hydrogen, unsubstituted C 1-3 Alkyl, or -(CH2) nQ, where Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from H, C, 1-14 Alkyl, and C 2-14 For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.

[0195] In certain embodiments, a subset of compounds of formula (I) are of formula (II): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; R4 is hydrogen, unsubstituted C 1-3 Alkyl, or -(CH2) n Q, where n is 2, 3, or 4; Q is OH, -NHC(S)N(R), -NHC(O)N(R), -N(R)C(O)R, -N(R)S(O)R, -N(R)R, -NHC(=NR)N(R), -NHC(=CHR)N(R), -OC(O)N(R), -N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R and R are independently selected from H, C, 1-14 Alkyl, and C 2-14alkenyl.

[0196] In one embodiment, the compound of formula (I) has the formula (IIa): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R4 is as defined herein.

[0197] In another embodiment, the compound of formula (I) has the formula (IIb): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R4 is as defined herein.

[0198] In another embodiment, the compound of formula (I) has formula (IIc) or (IIe): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein R4 is as defined herein.

[0199] In another embodiment, the compound of formula (I) has the formula (IIf): [ka] or an N-oxide thereof, or a salt or isomer thereof, In the formula, M is -C(O)O- or -OC(O)-, and M" is C 1-6 Alkyl or C 2-6 alkenyl, and R and R are independently 5-14 Alkyl and C 5-14 alkenyl, where n is selected from 2, 3, and 4.

[0200] In a further embodiment, the compound of formula (I) has the formula (IId): [ka] or an N-oxide thereof, or a salt or isomer thereof, where n is 2, 3, or 4, and m, R', R", and R2-R6 are as described herein. For example, each of R2 and R3 can be independently selected from C 5-14 Alkyl and C 5-14 alkenyl.

[0201] In a further embodiment, the compound of formula (I) has the formula (IIg): [ka] or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M'; M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, an aryl group, and a heteroaryl group; R2 and R3 are independently selected from H, C, 1-14 Alkyl, and C 2-14 For example, M" is selected from the group consisting of C 1-6 Alkyl (e.g., C 1-4 Alkyl) or C 2-6 Alkenyl (e.g., C 2-4 alkenyl). For example, R2 and R3 are independently 5-14 Alkyl and C 5-14 alkenyl.

[0202] In some embodiments, the amino lipid is one or more of the compounds described in U.S. Application Nos. 62 / 220,091, 62 / 252,316, 62 / 253,433, 62 / 266,460, 62 / 333,557, 62 / 382,740, 62 / 393,940, 62 / 471,937, 62 / 471,949, 62 / 475,140, ​​and 62 / 475,166, and PCT Application No. PCT / US2016 / 052352.

[0203] In some embodiments, the amino lipid is Compound 1: [ka] or a salt thereof.

[0204] In some embodiments, the amino lipid is Compound 2: [ka] or a salt thereof.

[0205] The central amine moiety of the lipid of formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), or (IIg) can be protonated at physiological pH. Thus, the lipid can have a positive or partial positive charge at physiological pH. Such amino lipids can be called cationic lipids, ionic lipids, cationic amino lipids, or ionic amino lipids. Amino lipids can also be zwitterionic, i.e., neutral molecules that have both positive and negative charges.

[0206] In some embodiments, the amino lipids of the present disclosure comprise a compound of formula (III): [ka] or one or more of its salts or isomers, wherein W is [ka] and Ring A is [ka] and t is 1 or 2; A1 and A2 are each independently selected from CH or N; Z is CH2 or absent, where when Z is CH2, the dashed lines (1) and (2) each represent a single bond, and when Z is absent, the dashed lines (1) and (2) are both absent; R1, R2, R3, R4, and R5 are independently C 5-20 Alkyl, C 5-20 Alkenyl, -R"MR', -R * YR”, -YR”, and -R * OR" R X1 and R X2 are each independently H or C 1-3 is alkyl, each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group; M * is C1-C6 alkyl, W 1 and W 2 are each independently selected from the group consisting of -O- and -N(R6)-; Each R6 is independently H or C 1-5 is selected from the group consisting of alkyl, X 1 , X 2 , and X 3 are independently a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH2) n -C(O)-, -C(O)-(CH2)n -, -(CH2) n -C(O)O-, -OC(O)-(CH2) n -, -(CH2) n -OC(O)-, -C(O)O-(CH2) n is selected from the group consisting of -, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C 3-6 is a carbocyclic ring, Each R * is independently 1-12 Alkyl and C 2-12 alkenyl, Each R is independently C 1-3 Alkyl and C 3-6 is selected from the group consisting of carbocycles; Each R' is independently C 1-12 Alkyl, C 2-12 alkenyl, and H; Each R" is independently 3-12 Alkyl, C 3-12 Alkenyl and -R * MR′, n is an integer from 1 to 6, Here, ring A is [ka] in the case of, i)X 1 , X 2 , and X 3 at least one of is not -CH2-; and / or ii) At least one of R1, R2, R3, R4, and R5 is -R"MR'.

[0207] In some embodiments, the compound is of any of formulas (IIIa1)-(IIIa8): [ka] [ka]

[0208] In some embodiments, the amino lipid is [ka] or a salt thereof.

[0209] The central amine moiety of the lipid of formula (III), (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), or (IIIa8) can be protonated at physiological pH. Thus, the lipid can have a positive or partial positive charge at physiological pH.

[0210] Phospholipids The lipid composition of the lipid nanoparticle compositions disclosed herein may include one or more phospholipids, such as one or more saturated or (poly)unsaturated phospholipids or combinations thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0211] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0212] The fatty acid moiety may be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0213] Certain phospholipids can facilitate fusion with membrane.For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membrane (e.g., cell membrane or intracellular membrane).Fusion of phospholipid with membrane may allow one or more elements (e.g., therapeutic agent) of lipid-containing composition (e.g., LNP) to pass through the membrane, for example, allowing one or more elements to be delivered to target tissue.

[0214] Non-natural phospholipid species, including natural species, with modifications and substitutions, including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cell recognition, or for conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes).

[0215] Phospholipids include, but are not limited to, glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. Phospholipids also include phosphosphingolipids, such as sphingomyelin.

[0216] In some embodiments, the phospholipids of the present invention are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). , 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine -phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0217] In certain embodiments, phospholipids that are useful or potentially useful in the present invention are analogs or variants of DSPC. In certain embodiments, phospholipids that are useful or potentially useful in the present invention are compounds of formula (IV): [ka] or a salt thereof, wherein Each R 1 are independently optionally substituted alkyl, or optionally, two R 1 together with the intervening atoms form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 together with the intervening atoms form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene, wherein the optionally substituted C 1-6 One methylene unit of the alkylene is optionally O, N(R N ), S, C(O), C(O)N(R N ), N.R. N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), N.R. N C(O)O, or NR N C(O)N(R N ), R 2 Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30alkenyl, or optionally substituted C 1-30 alkynyl, optionally where R 2 one or more methylene units are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), N.R. N C(O), NR N C(O)N(R N ), C(O)O, OC(O), -OC(O)O, OC(O)N(R N ), N.R. N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), N.R. N C(=NR N ), N.R. N C(=NR N )N(R N ), C(S), C(S)N(R N ), N.R. N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, -OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or -N(R N )S(O)2O, R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2, However, the compound is not of the formula: [ka] In the formula, R 2 Each instance of is independently an unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0218] In some embodiments, the phospholipid can be one or more of the phospholipids described in PCT Application No. PCT / US2018 / 037922.

[0219] structured lipids The lipid composition of the pharmaceutical compositions disclosed herein may include one or more structured lipids. As used herein, the term "structured lipid" refers to a sterol and also to lipids that contain a sterol moiety.

[0220] The incorporation of structured lipids into the lipid nanoparticles may help reduce the aggregation of other lipids within the particle. The structured lipid may be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is alpha-tocopherol.

[0221] In some embodiments, the structured lipid can be one or more of the structured lipids described in US Application No. 16 / 493,814.

[0222] Polyethylene glycol (PEG)-lipids The lipid composition of the pharmaceutical compositions disclosed herein may include one or more polyethylene glycol (PEG) lipids.

[0223] As used herein, the term "PEG lipid" refers to lipid modified with polyethylene glycol (PEG).Non-limiting examples of PEG lipid include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropan-3-amine.Such lipids are also called PEGylated lipids.For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0224] In some embodiments, the PEG lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0225] In one embodiment, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also called PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0226] In some embodiments, the lipid portion of the PEG-lipid has a molecular weight of about C 14 ~About C 22 , preferably about C 14 ~About C 16 In some embodiments, the PEG moiety, e.g., mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. In one embodiment, the PEG-lipid is a PEG 2k -DMG.

[0227] In one embodiment, the lipid nanoparticles described herein can include a PEG lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEG include PEG-DSG and PEG-DSPE.

[0228] PEG-lipids, such as those described in U.S. Pat. No. 8,158,601 and International Publication No. WO2015 / 130584A2, which are incorporated by reference in their entireties, are known in the art.

[0229] Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein can be synthesized as described in International Patent Application No. PCT / US2016 / 000129, entitled "Compositions and Methods for Delivery of Therapeutic Agents," filed December 10, 2016, which is incorporated by reference in its entirety.

[0230] The lipid component of the lipid nanoparticle composition may include one or more molecules including polyethylene glycol, e.g., PEG or PEG-modified lipid. Such species may alternatively be referred to as PEGylated lipid. PEG lipid is a lipid modified with polyethylene glycol. PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0231] In some embodiments, the PEG-modified lipid is a modified form of PEG DMG. PEG-DMG has the following structure: [ka]

[0232] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety. Any of these exemplary PEG lipids described herein may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0233] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (II). Provided herein is a compound of formula (V): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer from 1 to 100, inclusive; L 1 is an arbitrarily substituted C 1-10 alkylene, wherein the optionally substituted C 1-10 At least one methylene of the alkylene is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, O, N(R N ), S, C(O), C(O)N(R N ), N.R. N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), N.R. N C(O)O, or NR N C(O)N(R N ), D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group represented by the formula: [ka] The L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene, wherein the optionally substituted C 1-6 One methylene unit of the alkylene is optionally O, N(R N ), S, C(O), C(O)N(RN ), N.R. N C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), N.R. N C(O)O, or NR N C(O)N(R N ), R 2 Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl, optionally where R 2 one or more methylene units are independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), N.R. N C(O), NR N C(O)N(R N ), C(O)O, OC(O), -OC(O)O, OC(O)N(R N ), N.R. N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), N.R. N C(=NR N ), N.R. N C(=NR N )N(R N ), C(S), C(S)N(R N ), N.R. N C(S), NR N C(S)N(R N ), S(O), OS(O), S(O)O, -OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(RN ), N(R N )S(O)2N(R N ), OS(O)2N(R N ), or -N(R N )S(O)2O, R N each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; p is 1 or 2.

[0234] In certain embodiments, the compound of formula (V) is a PEG-OH lipid (i.e., R 3 -OR O and R O is hydrogen). In certain embodiments, the compound of formula (V) has the formula (V-OH): [ka] or a salt thereof.

[0235] In certain embodiments, the PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (VI). Provided herein is a compound of formula (VI): [ka] or a salt thereof, wherein R 3 -OR O and R O is hydrogen, an optionally substituted alkyl or an oxygen protecting group; r is an integer from 1 to 100, inclusive; R 5 is an arbitrarily substituted C 10-40 Alkyl, optionally substituted C 10-40 alkenyl, or optionally substituted C 10-40alkynyl, and optionally R 5 One or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R N ), O, S, C(O), C(O)N(R N ), -NR N C(O), NR N C(O)N(R N ), C(O)O, OC(O), OC(O)O, OC(O)N(R N ), N.R. N C(O)O, C(O)S, SC(O), C(=NR N ), C(=NR N )N(R N ), N.R. N C(=NR N ), N.R. N C(=NR N )N(R N ), C(S), C(S)N(R N ), N.R. N C(S), -NR N C(S)N(R N ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R N )S(O), -S(O)N(R N ), N(R N )S(O)N(R N ), OS(O)N(R N ), N(R N )S(O)O, S(O)2, N(R N )S(O)2, S(O)2N(R N ), -N(R N )S(O)2N(R N ), OS(O)2N(R N ), or N(R N )S(O)2O, R N Each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group.

[0236] In certain embodiments, the compound of formula (VI) has the formula (VI-OH): [ka] or a salt thereof. In some embodiments, r is 40-50.

[0237] In yet another embodiment, the compound of formula (VI) is: [ka] or a salt thereof.

[0238] In one embodiment, the compound of formula (VI) [ka] It is.

[0239] In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0240] In some embodiments, the PEG-lipid can be one or more of the PEG-lipids described in US Application No. US15 / 674,872.

[0241] In some embodiments, an LNP of the invention comprises an amino lipid of any of Formula I, II, or III, a phospholipid comprising DSPC, a structured lipid, and a PEG lipid comprising PEG-DMG.

[0242] In some embodiments, the LNPs of the invention comprise an amino lipid of any of Formula I, II, or III, a phospholipid comprising DSPC, a structured lipid, and a PEG lipid comprising a compound having Formula VI.

[0243] In some embodiments, the LNPs of the invention comprise an amino lipid of formula I, II, or III, a phospholipid comprising a compound having formula IV, a structured lipid, and a PEG lipid comprising a compound having formula V or VI.

[0244] In some embodiments, the LNPs of the invention comprise an amino lipid of formula I, II, or III, a phospholipid comprising a compound having formula IV, a structured lipid, and a PEG lipid comprising a compound having formula V or VI.

[0245] In some embodiments, the LNPs of the invention comprise an amino lipid of formula I, II, or III, a phospholipid having formula IV, a structured lipid, and a PEG lipid comprising a compound having formula VI.

[0246] In some embodiments, the LNPs of the invention comprise an N:P ratio of about 2:1 to about 30:1.

[0247] In some embodiments, the LNPs of the invention comprise an N:P ratio of about 6:1.

[0248] In some embodiments, the LNPs of the invention comprise an N:P ratio of about 3:1, 4:1, or 5:1.

[0249] In some embodiments, the LNPs of the invention comprise a weight / weight ratio of the amino lipid component to the RNA of about 10:1 to about 100:1.

[0250] In some embodiments, the LNPs of the invention comprise a weight / weight ratio of said amino lipid component to said RNA of about 20:1.

[0251] In some embodiments, the LNPs of the invention comprise a weight / weight ratio of said amino lipid component to said RNA of about 10:1.

[0252] In some embodiments, the LNPs of the invention have a mean diameter of about 30 nm to about 150 nm.

[0253] In some embodiments, the LNPs of the invention have a mean diameter of about 60 nm to about 120 nm.

[0254] As disclosed herein, lipid nanoparticles (LNPs) refer to nanoscale constructs (e.g., nanoparticles, typically less than 100 nm in diameter) that comprise lipid molecules arranged in a substantially spherical (i.e., spheroidal) geometric shape, and optionally encapsulating one or more additional molecular species. LNPs may comprise one or more types of lipids, including, but not limited to, amino lipids (e.g., ionic amino lipids), neutral lipids, non-cationic lipids, charged lipids, PEG-modified lipids, phospholipids, structured lipids, and sterols. In some embodiments, LNPs may further comprise one or more cargo molecules, including, but not limited to, nucleic acids (e.g., mRNA, plasmid DNA, DNA or RNA oligonucleotides, siRNA, shRNA, snRNA, snoRNA, lncRNA, etc.), small molecules, proteins, and peptides. LNPs may have a unilamellar structure (i.e., have a single lipid layer or lipid bilayer surrounding a central region) or a multilamellar structure (i.e., have multiple lipid layers or lipid bilayers surrounding a central region). In some embodiments, the lipid nanoparticles may be liposomes. Liposomes are nanoparticles that contain lipids arranged in one or more concentric lipid bilayers around a central region that can comprise an aqueous solution, suspension, or other aqueous composition.

[0255] In some embodiments, the lipid nanoparticles may comprise two or more components (e.g., amino lipids and nucleic acids, PEG-lipids, phospholipids, structured lipids). For example, the lipid nanoparticles may comprise amino lipids and nucleic acids. The lipid nanoparticles, such as those described herein, may be used in a wide variety of applications, including stealth delivery of therapeutic payloads with minimal adverse innate immune responses.

[0256] Effective in vivo delivery of nucleic acids represents a continuing medical challenge. Exogenous nucleic acids (i.e., originating from outside the cell or organism) are easily degraded in the body, for example, by the immune system. Thus, effective delivery of nucleic acids to cells often requires the use of particulate carriers (e.g., lipid nanoparticles). The particulate carriers should be formulated to minimize particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids into cells, and induce no or minimal immune response. To achieve minimal particle aggregation and stability prior to delivery, many conventional particulate carriers rely on the presence and / or concentration of certain components (e.g., PEG-lipids). However, it has been discovered that certain components can reduce the stability of encapsulated nucleic acids (e.g., mRNA molecules). The reduced stability can limit the broad applicability of the particulate carriers. Thus, there remains a need for methods to improve the stability of nucleic acids (e.g., mRNA) encapsulated within lipid nanoparticles.

[0257] In some embodiments, the lipid nanoparticles comprise one or more ionic molecules, polynucleotides, and optional components such as structured lipids, sterols, neutral lipids, phospholipids, and molecules that can reduce particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipids), such as those described above.

[0258] In some embodiments, the LNPs described herein may include one or more ionic molecules (e.g., amino lipids or ionic lipids). The ionic molecules may include charged groups and may have a certain pKa. In certain embodiments, the pKa of the ionic molecule may be about 6 or more, about 6.2 or more, about 6.5 or more, about 6.8 or more, about 7 or more, about 7.2 or more, about 7.5 or more, about 7.8 or more, about 8 or more. In some embodiments, the pKa of the ionic molecule may be about 10 or less, about 9.8 or less, about 9.5 or less, about 9.2 or less, about 9.0 or less, about 8.8 or less, or about 8.5 or less. Combinations of the above-referenced ranges are also possible (e.g., 6 or more and about 8.5 or less). Other ranges are also possible. In embodiments in which more than one type of ionic molecule is present in the particle, each type of ionic molecule may independently have one or more pKas in the above ranges.

[0259] In general, an ionic molecule comprises one or more charged groups. In some embodiments, an ionic molecule may have a positive or negative charge. For example, an ionic molecule may have a positive charge. For example, an ionic molecule may comprise an amine group. As used herein, the term "ionic molecule" has its usual meaning in the art and may refer to a molecule or matrix that comprises one or more charged moieties. As used herein, a "charged moiety" is a chemical moiety that has a formal electronic charge, for example, monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. The charged moiety may be anionic (i.e., has a negative charge) or cationic (i.e., has a positive charge). Examples of moieties that have a positive charge include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidizolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate, sulfonate, sulfate, phosphonate, phosphate, hydroxyl, etc. The charge of a charged moiety may in some cases be changed by environmental conditions, e.g., a change in pH may change the charge of the moiety and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule and / or matrix may be selected as desired.

[0260] In some cases, ionic molecules (e.g., amino lipids or ionic lipids) may contain one or more precursor moieties that can be converted into charged moieties. For example, the ionic molecules may contain neutral moieties, such as those described above, that can be hydrolyzed to form charged moieties. As a non-limiting example, the molecule or matrix may contain amides, respectively, that can be hydrolyzed to form amines. Those skilled in the art can determine whether a given chemical moiety has a formal electronic charge (e.g., by inspection, pH titration, ionic conductivity measurement, etc.) and / or whether a given chemical moiety can react (e.g., hydrolyzed) to form a chemical moiety with a formal electronic charge.

[0261] Ionic molecule (for example, amino lipid or ionic lipid) can have any suitable molecular weight.In certain embodiments, the molecular weight of ionic molecule is about 2,500g / mol or less, about 2,000g / mol or less, about 1,500g / mol or less, about 1,250g / mol or less, about 1,000g / mol or less, about 900g / mol or less, about 800g / mol or less, about 700g / mol or less, about 600g / mol or less, about 500g / mol or less, about 400g / mol or less, about 300g / mol or less, about 200g / mol or less, or about 100g / mol or less. In some cases, the molecular weight of the ionic molecule is about 100 g / mol or more, about 200 g / mol or more, about 300 g / mol or more, about 400 g / mol or more, about 500 g / mol or more, about 600 g / mol or more, about 700 g / mol or more, about 1000 g / mol or more, about 1,250 g / mol or more, about 1,500 g / mol or more, about 1,750 g / mol or more, about 2,000 g / mol or more, or about 2,250 g / mol or more. Combinations of the above ranges (e.g., at least about 200 g / mol and 2,500 g / mol or less) are also possible. In embodiments in which more than one type of ionic molecule is present in the particle, each type of ionic molecule can independently have one or more molecular weights in the above ranges.

[0262] In some embodiments, the percentage (e.g., by weight or by molar basis) of a single type of ionic molecule (e.g., amino lipid or ionic lipid) and / or all of the ionic molecules within the particle can be about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 42% or more, about 45% or more, about 48% or more, about 50% or more, about 52% or more, about 55% or more, about 58% or more, about 60% or more, about 62% or more, about 65% or more, or about 68% or more. In some cases, the percentage (e.g., by weight or by molar basis) can be about 70% or less, about 68% or less, about 65% or less, about 62% or less, about 60% or less, about 58% or less, about 55% or less, about 52% or less, about 50% or less, or about 48% or less. Combinations of the above-referenced ranges are also possible (e.g., 20% or more and about 60% or less, 40% or more and about 55% or less, etc.). In embodiments in which more than one type of ionic molecule is present in the particle, each type of ionic molecule can independently have one or more percentages (e.g., by weight or by molar basis) in the above ranges. The percentage (e.g., by weight or by molar basis) can be determined by extracting the ionic molecule(s) from the dried particle, for example, using an organic solvent, and measuring the amount of the agent using high pressure liquid chromatography (i.e., HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS). Those skilled in the art will be familiar with techniques for determining the amount of a component using the techniques referenced above. For example, HPLC can be used to quantify the amount of a component, for example, by comparing the area under the curve of the HPLC chromatogram to a standard curve.

[0263] It should be understood that the term "charged" or "charged moiety" does not refer to a "partial negative charge" or "partial positive charge" of a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art. A "partial negative charge" can arise when a functional group contains a bond that is polarized such that electron density is attracted toward one atom of the bond, forming a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polarized in this way.

[0264] As described herein, in some embodiments, the mRNA is formulated with an LNP such that the mRNA is at least partially contained within the LNP. LNP-formulated mRNA vaccines (fLNPs) contain structures that protect the RNA from environmental components that may cause degradation of the mRNA.

[0265] Insertion and Substitution The present disclosure also includes polynucleotides of the present disclosure that further comprise insertions and / or substitutions.

[0266] In some embodiments, the 5'UTR of the polynucleotide may be replaced by the insertion of at least one region and / or string of nucleosides of the same base. The region and / or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides, and the nucleotides may be natural and / or non-natural. As a non-limiting example, the group of nucleotides may include a string of 5-8 adenines, cytosines, thymines, any of the other nucleotides disclosed herein, and / or combinations thereof.

[0267] In some embodiments, the 5'UTR of the polynucleotide can be replaced by the insertion of at least two regions and / or strings of nucleotides of two different bases, including but not limited to adenine, cytosine, thymine, any of the other nucleotides disclosed herein and / or combinations thereof. For example, the 5'UTR can be replaced by inserting 5-8 adenine bases followed by 5-8 cytosine bases. In another example, the 5'UTR can be replaced by inserting 5-8 cytosine bases followed by 5-8 adenine bases.

[0268] In some embodiments, the polynucleotide may include at least one substitution and / or insertion downstream of the transcription start site that can be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and / or insertion may be made downstream of the transcription start site by substituting at least one nucleic acid in the region immediately downstream of the transcription start site (e.g., but not limited to, +1 to +6). Alterations in the nucleotide region immediately downstream of the transcription start site may affect the initiation rate, increase the apparent nucleotide triphosphate (NTP) reaction constant value, and increase the dissociation of short transcripts from the transcription complex that corrects the initial transcription (Brieba et al, Biochemistry (2002) 41:5144-5149, incorporated herein by reference in its entirety). Modification, substitution and / or insertion of at least one nucleoside may cause silent mutation of the sequence or may cause mutation of the amino acid sequence.

[0269] In some embodiments, the polynucleotide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 substitutions of guanine bases downstream of the transcription start site.

[0270] In some embodiments, the polynucleotide may contain at least one, at least two, at least three, at least four, at least five, or at least six guanine base substitutions in the region immediately downstream of the transcription start site. As a non-limiting example, if the nucleotide in the region is GGGAGA, the guanine base may be replaced by at least one, at least two, at least three, or at least four adenine nucleotides. As another non-limiting example, if the nucleotide in the region is GGGAGA, the guanine base may be replaced by at least one, at least two, at least three, or at least four cytosine bases. As another non-limiting example, if the nucleotide in the region is GGGAGA, the guanine base may be replaced by at least one, at least two, at least three, or at least four thymines, and / or any of the nucleotides described herein.

[0271] In some embodiments, the polynucleotide may include at least one substitution and / or insertion upstream of the start codon. For clarity, those skilled in the art will understand that the start codon is the first codon of the coding region of the protein, whereas the transcription start site is the site where transcription begins. The polynucleotide may include, but is not limited to, substitution and / or insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotide bases. The nucleotide bases may be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4, or at least 5 positions upstream of the start codon. The inserted and / or substituted nucleotides may be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T, or A, C and T), or at least four different bases.

[0272] As a non-limiting example, the guanine base upstream of the coding region of the polynucleotide can be replaced with adenine, cytosine, thymine, or any of the nucleotides described herein.In another non-limiting example, the replacement of the guanine base of the polynucleotide can be designed to leave one guanine base downstream of the transcription start site and in the region before the start codon (see Esvelt et al. Nature (2011) 472 (7344): 499-503, the contents of which are incorporated herein by reference in their entirety).As a non-limiting example, at least five nucleotides can be inserted downstream of the transcription start site but at one position upstream of the start codon, and the at least five nucleotides can be the same base type.

[0273] In some embodiments, the polynucleotide comprises 200 to 3,000 nucleotides. For example, the polynucleotide may comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0274] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of disease in humans and other mammals. The compositions may be used as therapeutic or prophylactic agents. For example, when the composition comprises a picornavirus capsid polyprotein and / or a picornavirus 3C protease, RNA encoding such picornavirus capsid polyprotein and / or picornavirus 3C protease is used to provide prophylactic or therapeutic protection against Picornaviridae infections. Prophylactic protection against Picornaviridae infections may be achieved following administration of a composition of the present disclosure (e.g., a composition comprising one or more polynucleotides encoding one or more picornavirus capsid polyproteins and / or picornavirus 3C proteases). In some embodiments, the picornavirus is a member of the Enterovirus family. In some embodiments, the Enterovirus is any virus of the Enterovirus species A-D. In some embodiments, the Enterovirus is any virus of the Rhinovirus species A-C. The composition may be administered once, twice, three times, four times or more. In some embodiments, the composition can be administered to an infected individual to achieve a therapeutic response. Dosages may need to be adjusted accordingly.

[0275] It is envisioned that there may be situations where people are at risk of infection by strains of multiple types of infectious agents. RNA (mRNA) therapy is particularly suited to a combination vaccination approach due to several factors, including but not limited to production speed, ability to rapidly tailor therapy to perceived geographic threats, etc. For protection against multiple enterovirus strains, combination therapy can be administered that includes RNA encoding at least one polypeptide (or a portion thereof) of a picornavirus capsid polyprotein and further includes RNA encoding at least one polypeptide (or a portion thereof) of a picornavirus 3C protease. The RNA (mRNA) may be, for example, co-formulated in a single lipid nanoparticle (LNP) or in separate LNPs for co-administration. In some embodiments, the enterovirus is HRV16. In some embodiments, the enterovirus is EV-D68. In some embodiments, the enterovirus is EV-A71.

[0276] A prophylactically effective dose is a therapeutically effective dose that prevents viral infection at a clinically acceptable level. In some embodiments, the therapeutically effective dose is the dose described in the package insert for the treatment. As used herein, prophylactic therapy refers to a therapy that prevents the increase of infection to some extent. Infection can be completely or partially prevented.

[0277] The method of the present invention, in some aspects, comprises passively immunizing a mammalian subject against influenza virus infection. The method comprises administering to the subject a composition comprising at least one RNA polynucleotide having an open reading frame encoding at least one picornavirus capsid polyprotein and picornavirus 3C protease. In some aspects, the method of the present disclosure provides prophylactic treatment against enterovirus infection. In some embodiments, the enterovirus is HRV16. In some embodiments, the enterovirus is EV-D68. In some embodiments, the enterovirus is EV-A71.

[0278] Therapeutic methods are also included in the present invention. A method of treating an enterovirus infection in a subject is provided in an aspect of the present disclosure. The method comprises administering a composition comprising at least one RNA polynucleotide having an open reading frame encoding at least one picornavirus capsid polyprotein and picornavirus 3C protease to a subject with an influenza virus infection. In some embodiments, the enterovirus is HRV16. In some embodiments, the enterovirus is EV-D68. In some embodiments, the enterovirus is EV-A71.

[0279] As used herein, the terms treat, treated, or treating, when used in reference to a disorder, such as a viral infection, refer to treatment that increases the subject's resistance to developing the disease, in other words, treatment that reduces the likelihood that the subject will develop the disease in response to infection with the virus, and treatment to combat or prevent the worsening of the infection after the subject has developed the disease.

[0280] An "effective amount" of an RNA therapeutic of the present disclosure is provided based, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., size and degree of modified nucleosides), and other components of the RNA therapeutic, as well as other determinants. Increased antibody production may be demonstrated by increased cellular transfection (percentage of cells transfected with the RNA therapeutic), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., as indicated by an increased duration of protein translation from a modified polynucleotide), or a change in the host cell response.

[0281] In some embodiments, RNA therapeutics (including polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used to treat such diseases.

[0282] RNA therapy may be administered prophylactically or therapeutically as part of an active immunization scheme for healthy individuals, or early in the course of infection during the latency period or during active infection after the onset of symptoms, hi some embodiments, the amount of RNA therapy of the present disclosure provided to a cell, tissue, or subject may be an amount effective for immunoprophylaxis.

[0283] RNA therapy can be administered together with other preventive or therapeutic compounds. As a non-limiting example, the preventive or therapeutic compound can be a vaccine, with or without an adjuvant or booster, including a viral therapy. As used herein, the term "booster" when referring to a preventive composition, such as a treatment or vaccine, refers to an additional administration of the preventive composition. A booster (or booster vaccine) can be administered after the previous administration of the preventive composition. The administration interval between the first administration of the prophylactic composition and the booster can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, It may be 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the interval between the first administration of the prophylactic composition and the booster administration can be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0284] In some embodiments, the RNA therapy may be administered subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection into one or more cells, tissues, or organs.

[0285] Provided herein are RNA therapeutics and pharmaceutical compositions comprising the RNA compositions and / or complexes, optionally in combination with one or more pharma- ceutically acceptable excipients.

[0286] The RNA therapy may be formulated or administered in combination with one or more pharma- ceutically acceptable excipients. In some embodiments, the composition includes at least one additional active agent, such as a therapeutic agent, a prophylactic agent, or a combination of both. The therapeutic composition may be sterile, pyrogen-free, or sterile and pyrogen-free. A general introduction to the formulation and / or manufacture of pharmaceuticals, such as therapeutic compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0287] In some embodiments, the RNA therapeutic is administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to the RNA therapeutic or a polynucleotide contained therein, such as an RNA polynucleotide (e.g., an mRNA polynucleotide) encoding a picornavirus capsid polyprotein and / or a picornavirus 3C protease.

[0288] The formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association the active ingredient (e.g., mRNA polynucleotide) with an excipient and / or one or more other accessory ingredients, and then, as necessary and / or desired, dividing, shaping and / or packaging the product into the desired single or multiple dosage units.

[0289] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100%, e.g., from 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0290] RNA therapeutics may be formulated with one or more excipients to (1) increase stability, (2) increase cellular transfection, (3) provide sustained or delayed release (e.g., from a depot formulation), (4) alter biodistribution (e.g., target to specific tissues or cell types), (5) increase translation of the encoded protein in vivo, and / or (6) alter the in vivo release profile of the encoded protein (e.g., HCAb). In addition to conventional excipients, such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, excipients may include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with RNA therapeutics (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0291] Naturally occurring eukaryotic mRNA molecules are known to contain stabilizing elements, including but not limited to untranslated regions (UTRs) at the 5' end (5'UTR) and / or 3' end (3'UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both 5'UTR and 3'UTR are usually transcribed from genomic DNA and are elements of the immature mRNA. Structural features characteristic of mature mRNAs, such as the 5'-cap and 3'-poly(A) tail, are usually added to the transcribed (immature) mRNA during mRNA processing. The 3'-poly(A) tail is usually a stretch of adenine nucleotides added to the 3' end of the transcribed mRNA. It may contain up to about 400 adenine nucleotides. In some embodiments, the length of the 3'-poly(A) tail may be an essential element for the stability of an individual mRNA.

[0292] In some embodiments, the RNA therapy may include one or more stabilizing elements. The stabilizing elements may include, for example, histone stem loops. A 32 kDa protein called stem loop binding protein (SLBP) has been identified. It associates with histone stem loops at the 3' end of histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle, peaking during S phase, when histone mRNA levels also increase. This protein has been shown to be essential for efficient 3' end processing of histone pre-mRNAs by U7 snRNP. SLBP continues to associate with stem loops after processing, and then promotes translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved throughout metazoans and protozoans, and its binding to histone stem loops depends on the structure of the loop. The minimal binding site includes at least 3 nucleotides 5' and 2 nucleotides 3' of the stem loop.

[0293] In some embodiments, the RNA therapy comprises a coding region, at least one histone stem loop, and optionally a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, β-galactosidase, EGFP), or a marker or selection protein (e.g., alpha-globin, galactokinase, and xanthine:guanine phosphoribosyltransferase (GPT)).

[0294] In some embodiments, the combination of a poly(A) sequence or a polyadenylation signal with at least one histone stem loop, both of which essentially represent alternative mechanisms, act synergistically to increase protein expression above the levels observed with either of the individual elements. The synergistic effect of the combination of poly(A) with at least one histone stem loop has been found to be independent of the order of the elements or the length of the poly(A) sequence.

[0295] In some embodiments, the polynucleotides described herein have a diameter of about 1 nm to about 100 nm, for example, but not limited to, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, Approximately 5 nm to approximately 30 nm, approximately 5 nm to approximately 40 nm, approximately 5 nm to approximately 50 nm, approximately 5 nm to approximately 60 nm, approximately 5 nm to approximately 70 nm, approximately 5 nm to approximately 80 nm, approximately 5 nm to approximately 90 nm, approximately 10 to approximately 20 nm, approximately 10 to approximately Approximately 30 nm, approximately 10 to approximately 40 nm, approximately 10 to approximately 50 nm, approximately 10 to approximately 60 nm, approximately 10 to approximately 70 nm, approximately 10 to approximately 80 nm, approximately 10 to approximately 90 nm, approximately 20 to approximately 30 nm, approximately 20 to approximately 40 nm, approximately 20 to approximately 5 0nm, about 20 to about 60nm, about 20 to about 70nm, about 20 to about 80nm, about 20 to about 90nm, about 20 to about 100nm, about 30 to about 40nm, about 30 to about 50nm, about 30 to about 60nm, about 30 to about 7 0nm, about 30 to about 80nm, about 30 to about 90nm, about 30 to about 100nm, about 40 to about 50nm, about 40 to about 60nm, about 40 to about 70nm, about 40 to about 80nm, about 40 to about 90nm, about 40 to about 1 The lipid nanoparticles may be formulated into lipid nanoparticles having a diameter of about 00 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm.

[0296] In some embodiments, the lipid nanoparticles may have a diameter of about 10-500 nm. In one embodiment, the lipid nanoparticles may have a diameter of more than 100 nm, more than 150 nm, more than 200 nm, more than 250 nm, more than 300 nm, more than 350 nm, more than 400 nm, more than 450 nm, more than 500 nm, more than 550 nm, more than 600 nm, more than 650 nm, more than 700 nm, more than 750 nm, more than 800 nm, more than 850 nm, more than 900 nm, more than 950 nm, or more than 1000 nm.

[0297] In some embodiments, the polynucleotides may be delivered using smaller LNPs. Such particles may be smaller than 0.1 μm to 100 nm in diameter, including but not limited to, smaller than 0.1 μm, smaller than 1.0 μm, smaller than 5 μm, smaller than 10 μm, smaller than 15 um, smaller than 20 um, smaller than 25 um, smaller than 30 um, smaller than 35 um, smaller than 40 um, smaller than 50 um, smaller than 55 um, smaller than 60 um, smaller than 65 um, smaller than 70 um, smaller than 75 um, smaller than 80 um, smaller than 85 um, smaller than 90 um, smaller than 95 um, smaller than 100 um, smaller than 125 um, smaller than 150 um, smaller than 175 um, smaller than 200 um, smaller than 225 um, smaller than 250 um, smaller than 27 It may consist of less than 5um, less than 300um, less than 325um, less than 350um, less than 375um, less than 400um, less than 425um, less than 450um, less than 475um, less than 500um, less than 525um, less than 550um, less than 575um, less than 600um, less than 625um, less than 650um, less than 675um, less than 700um, less than 725um, less than 750um, less than 775um, less than 800um, less than 825um, less than 850um, less than 875um, less than 900um, less than 925um, less than 950um, or less than 975um.

[0298] The nanoparticles and microparticles described herein can be geometrically engineered to modulate macrophage and / or immune response. Geometrically engineered particles can have various shapes, sizes and / or surface charges to incorporate the polynucleotides described herein into targeted delivery, for example, but not limited to, pulmonary delivery (see, for example, International Publication No. WO2013082111, which is incorporated herein by reference in its entirety). Other physical features of the geometrically engineered particles can include, but are not limited to, perforations, slanted arms, asymmetry and surface roughness, charges that can change interactions with cells and tissues.

[0299] RNA therapy may be administered by any route that results in a therapeutic outcome. These include, but are not limited to, intradermal, intramuscular, and / or subcutaneous administration. The present disclosure provides methods that include administering RNA therapy to a subject in need of RNA therapy. The exact amount required will vary from subject to subject depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its method of administration, its mode of action, and the like. RNA therapy compositions are typically formulated in unit dosage forms for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the RNA therapy composition may be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level, prophylactically effective dose level, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the particular compound used, the particular composition used, the age, weight, general health, sex, and diet of the patient, the time of administration, route of administration, and excretion rate of the particular compound used, the duration of treatment, drugs used in combination or simultaneously with the particular compound used, and similar factors well known in the medical arts.

[0300] In some embodiments, an RNA therapeutic composition is administered in an amount of from 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 0.0 The compound may be administered at a dosage level sufficient to deliver 1 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 25 mg / kg per day, multiple times per day, per week, per month, etc., to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see, e.g., unit dose ranges described in International Publication No. WO2013078199, which is incorporated herein by reference in its entirety). The desired dose may be delivered three times per day, twice per day, once per day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, etc. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are employed, split dosing regimens, such as those described herein, may be used. In some embodiments, an RNA therapeutic composition may be administered at a dosage level sufficient to deliver 0.0005 mg / kg to 0.01 mg / kg, e.g., about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg or about 0.005 mg / kg.

[0301] In some embodiments, an RNA therapeutic composition may be administered once or twice (or more) at a dosage level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0302] In some embodiments, the RNA therapeutic composition is administered in a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0. A total dose or dose level sufficient to deliver 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years). Higher and lower doses and administration frequencies are encompassed by the present disclosure. For example, the RNA therapeutic composition may be administered three or four times.

[0303] In some embodiments, an RNA therapeutic composition may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months later, 0 and 6 months later, 0 and 9 months later, 0 and 12 months later, 0 and 18 months later, 0 and 2 years later, 0 and 5 years later, or 0 and 10 years later) at a total dose or dose level sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg.

[0304] In some embodiments, the RNA for use in the method of treating a subject is administered to the subject in an amount effective to treat the subject, with a single dose of between 10 μg / kg and 400 μg / kg of nucleic acid therapy. In some embodiments, the RNA for use in the method of treating a subject is administered to the subject in an amount effective to treat the subject, with a single dose of between 10 μg and 400 μg of the nucleic acid therapy.

[0305] The RNA pharmaceutical compositions described herein can be formulated into dosage forms described herein, e.g., intranasally, intratracheally, or for injection (e.g., intravenously, intraocularly, intravitreally, intramuscularly, intradermally, intracardially, intraperitoneally, and subcutaneously).

[0306] The disclosure is not limited in its application to the details of construction or the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. Moreover, the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein means the inclusion of the items listed thereafter and equivalents thereof as well as additional items. EXAMPLES

[0307] Example 1: P1+3CD mRNA demonstrates proper P1 processing in vitro To examine whether mRNAs encoding P1 and 3CD are sufficient to produce virus-like particles (VLPs), an in vitro assay was performed as follows. Two mRNAs, one encoding human rhinovirus (HRV)-A16 P1 and the other encoding HRV-A16 3CD protease, were co-transfected into cells to determine i) whether P1 is expressed, ii) whether 3CD is expressed and can catalyze the cleavage of P1 into its components VP proteins in cells, and iii) whether an optimal P1:3CD ratio of mRNA exists that promotes more efficient cleavage of P1 while minimizing cytotoxicity by 3CD. In Figure 2A, the amount of P1 mRNA transfected was kept constant at 2 μg, and increasing amounts of 3CD mRNA were co-transfected to give the indicated molar ratios of P1:3CD. For Western blots, cell lysates were blotted with an anti-HRV-A16 VP2 antibody that also recognizes VP0 and uncleaved P1. Transfection of 3CD mRNA in any of the tested amounts resulted in cleavage of P1 in cells. Increasing the amount of 3CD resulted in a more pronounced disappearance of the high molecular weight band representing uncleaved P1 and a significant increase in the amount of low molecular weight bands representing the individual proteins (VP2) and the smaller polyprotein (VP0). These bands were consistent in size with bands seen in lysates prepared from purified wild-type HRV-A16 virions and likely represent, from largest to smallest, the VP0-VP3 precursor, VP0, and VP2 (Figure 2A).

[0308] Transfection of any amount of polyprotein encoding proteolytic 3C in the form of 3CD mRNA led to clear cleavage of P1, but increasing amounts led to a greater "conversion" of P1 to VP0, and at the highest amounts, a band corresponding in size to VP2 appeared. However, large amounts of 3CD mRNA simultaneously increased cytotoxicity in a dose-dependent manner, with a 1:1 ratio of P1:3CD showing the highest cytotoxicity. To avoid the possibility of excessive cytotoxicity of 3C, polyprotein 3CD was used instead of 3C.

[0309] Example 2: Co-expression of P1+3CD produces virus-like particles To further evaluate whether co-expression of P1 and 3CD is sufficient to produce VLPs, we performed large-scale transfection and purification experiments using two DNA plasmids encoding HRV-A16 P1 and HRV-A16 3CD, respectively. Aimed at potentially increasing VLP yields by delaying cell death due to 3CD-induced cytotoxicity, the plasmids were transfected at a mass ratio of 4 parts P1 to 1 part 3CD. VLPs were purified from the supernatant of transfected cells using PEG precipitation followed by size-exclusion chromatography. Negative staining transmission electron microscopy revealed uniform particles of the size expected for rhinovirus virions (approximately 30 nanometers in diameter) (Figure 2B). These data indicate that co-expression of P1 and 3CD results in both proper processing of P1 and organization of individual VP proteins into virus-like particles that are released into the extracellular space and morphologically resemble wild-type virions.

[0310] Example 3: Vaccines containing P1+3CD mRNA generate neutralizing antibody titers in mice To investigate whether administration of P1+3CD mRNA, an mRNA encoding two polyproteins, can result in the formation of immunogenic VLPs and in the production of HRVA-16 neutralizing antibodies, an in vivo assay was performed as follows. Mice were administered a prime-boost vaccine regimen consisting of 10 μg of P1 mRNA and, due to toxicity concerns, two different amounts of 3CD mRNA resulting in a final P1:3CD molar ratio of 10:1 and 2:1. Figure 2C shows the HRV-A16 neutralizing antibody titers 3 weeks after the first vaccination (after dose 1) and 2 weeks after the boost (after dose 2). Both the 10:1 and 2:1 ratios resulted in detectable neutralizing titers, but a single dose of the 2:1 ratio yielded higher titers than two doses at the 10:1 ratio. Serum from cotton rats injected twice with live HRV-A16 was included as a benchmark control. Surprisingly, two injections of the P1:3CD 2:1 ratio combination produced higher neutralization titers in mice than two doses of replicative HRV-A16 administered IM to cotton rats. No adverse events were observed in mice following administration of the 10:1 or 2:1 ratios, suggesting that the cytotoxicity seen following transfection of 3CD mRNA in vitro does not necessarily predict effects in vivo.

[0311] Example 4: P1+3CD mRNA is immunogenic against other species within the Enterovirus genus Following the approach used for HRV-A16 in Example 2, mRNA encoding two polyproteins, P1 and 3CD, one with enzymatic activity towards the other protein, was generated from enterovirus D68 (EV-D68) isolate US / MO / 14-18950. This isolate was collected from a human patient in Missouri during the 2014 EV-D68 outbreak in the United States and is a clade B1 isolate. In this study, mice were vaccinated with an expanded set of P1:3D ratios. The amount of P1 mRNA was kept constant at 10 μg, and increasing amounts of 3CD mRNA were added to give final P1:3CD molar ratios of 10:1, 5:1, 2:1 and 1:1. Vaccination followed the same two-dose schedule used for HRV-A16. Sera were collected 2 weeks after the boost, pooled by group, and tested for neutralization of different clade B1 (US / MO / 14-18947) and clade A2 (US / KY / 14-18953) isolates. For each isolate, neutralization titers were similar at all four ratios tested (Figure 2D). Neutralization titers were approximately one log higher for the clade B1 isolate MO / 14-18947. This was expected, as the sequence of P1 mRNA was also derived from a 2014 Missouri clade B1 isolate and shares a high degree of sequence homology. However, strong neutralization titers were also generated against the more diverse clade A2 isolates. This suggests that, in the case of EV-D68, vaccination with a single P1 mRNA generates neutralizing antibodies that are effective across clades. The results also indicate that the protease can function even when the P1 substrate is derived from different virus isolates and species.

[0312] The data from these two studies taken together suggest that administration of polyprotein P1+3CD mRNA in LNPs at a molar ratio of 2 parts P1 to 1 part 3CD is highly immunogenic and effective in eliciting neutralizing antibodies to the resulting VLPs.

[0313] To further explore the efficacy of this P1+3CD combination when delivered at lower doses (while maintaining the 2:1 molar ratio), a dose-ranging study was performed across an expanding group of enterovirus species. Table 1 shows the picornavirus species and genotypes targeted, the isolates used to design the P1 and 3CD mRNA sequences, the dose of total mRNA (P1+3CD) administered to mice, and the respective amount of P1 mRNA within the dose. In this dose-ranging study, the mRNA was diluted in a 5-fold dilution series and at least three doses were examined. [Table 1]

[0314] The vaccination schedule was the same as in previous studies. Mice were vaccinated on days 1 and 22, and sera were collected 3 weeks after dose 1 (day 21) and 2 weeks after dose 2 (day 35). Sera after dose 2 were tested for neutralizing antibody titers in the corresponding neutralization assays. For HRV-A16 and HRV-B14, the viruses used in the neutralization assays were identical to the viral sequences used for the respective P1 and 3CD mRNAs. For EV-D68 and EV-A71, sera from vaccinated mice were examined for neutralization across two or more heterologous isolates representing different clades.

[0315] Figures 3A-3D show neutralizing antibody titers 2 weeks (day 35) after the second vaccination. Figure 3A shows neutralizing antibody titers in animals vaccinated with HRV-A16 P1:3CD. Animals were administered 5 μg to 0.04 μg of HRV-A16 P1 mRNA and the corresponding amount of HRV-A16 3CD mRNA to maintain a 2:1 ratio of P1 to 3CD. Commercially available HRV-A16 antiserum from hyperimmune guinea pigs was included as a benchmark control. All animals at all doses examined produced neutralizing antibody titers above the lower limit of detection of the assay, and titers were dose-dependent. Although no doses led to titers equal to or exceeding those of guinea pig hyperimmune antiserum, the 5 μg dose produced titers higher than those produced in cotton rats administered two doses of replicative HRV-A16 (Figure 2C).

[0316] Figure 3B shows neutralizing antibody titers in animals vaccinated with HRV-B14 P1:3CD. Animals were administered doses of 5 μg to 0.2 μg of HRV-B14 P1 mRNA and corresponding amounts of HRV-B14 3CD mRNA to maintain a 2:1 ratio. Commercially available HRV-B14 antiserum from hyperimmune guinea pigs was included as a benchmark control. Surprisingly, all three doses tested here for HRV-B14 resulted in similar levels of neutralizing antibodies, and the geometric mean titers (GMTs) of all doses exceeded those of hyperimmune HRV-B14 antiserum from guinea pigs.

[0317] In Figure 3C, animals were vaccinated with EV-D68 P1+3CD mRNA using doses of P1 mRNA ranging from 5 μg to 0.2 μg, and sera from these animals were tested for neutralization of heterologous EV-D68 isolates from clades B1 and A2.

[0318] In Figure 3D, animals were vaccinated with EV-A71 P1+3CD mRNA using doses of P1 mRNA ranging from 5 µg to 0.2 µg. Sera from these animals were examined for neutralization of heterologous genotypes of EV-A71. The virus USA / 2018-23092 is a clinical isolate recovered from the stool of a patient with acute flaccid myelitis (AFM).

[0319] An effective human rhinovirus vaccine should provide protection against multiple serotypes within each of the three rhinovirus species, A, B, and C. Given the high intraspecies sequence diversity of P1, many different P1 mRNAs may be required to adequately cover even a single species. However, in contrast to P1, the amino acid sequence of the 3CD protease is well conserved among serotypes within a species and to a lesser extent across the three species. Alignments showing consensus amino acid sequences of the 3C region of the 3CD protease of 79 serotypes of rhinovirus species A and 26 serotypes of rhinovirus species B indicate that residues within 4 Å of the P1 binding site are highly conserved between HRV-A and HRV-B, and that five sites differ between HRV-A and HRV-B, with two of these sites differing within HRV-B. These putative binding residues are highly conserved within rhinovirus A and rhinovirus B, and in some cases between rhinovirus A and rhinovirus B. Such alignment further indicates that the VP2-VP3 cleavage site (QGLP) and the N-terminus of VP3 are highly conserved between both HRV-A and HRV-B. In contrast, the cleavage site and terminus of VP3 / VP1 differ between HRV-A and HRV-B but are conserved within the group, with conserved amino acid residues Q / NP-[IV]-E in HRV-A and AL-[EMPT]-E / G-[FL] in HRV-B, respectively. The 3CD protease binds to P1 and cleaves it at two locations, between VP2-VP3 and between VP3-VP1. Comparing the P1 amino acid sequences at and around these cleavage sites, there is evidence of sequence conservation. The VP3-VP1 cleavage site region shows conservation between serotypes within a species, while the VP2-VP3 cleavage site has a high degree of sequence homology between rhinovirus A and rhinovirus B species.

[0320] Example 5: [P1 B14 +3CD A16 The combination is [P1+3CD] B14demonstrated similar P1 processing in vitro and immunogenicity in vivo to a "matched" combination of The sequence conservation in both the P1 binding site of the 3CD protease and the cleavage site region of P1 itself suggests that a "matching" 3CD mRNA may not need to be provided for each P1 mRNA. It also suggests that a single 3CD protease may be capable of processing P1 proteins into VLPs across many serotypes within a species, or across serotypes in two different species. [P1 B14 +3CD A16 ] combination was previously used [P1+3CD] B14 To determine whether the "matched" combination of P1 showed similar P1 processing in vitro, an in vitro assay was performed as follows. A16 and P1 B14 Cells were transfected with mRNA alone or in combination with the indicated 3CD mRNA at a molar ratio of 2:1. Cell lysates were blotted with a pan-rhinovirus anti-VP1 antibody. Arrows indicate the low molecular weight bands corresponding to the uncleaved P1 precursor protein and VP1 (Figure 4A). P1 B14 In the case of 3CD A16 or 3CDs B14 Addition of either mRNA resulted in the disappearance of the P1 band and the appearance of VP1. The intensity of the VP1 band was compared with that of the "matched" 3CD B14 Although this indicates that P1 cleavage is likely more efficient when using mRNA, heterologous 3CD A16 Proteases are still P1 B14 can be processed into the component VP proteins.

[0321] [P1 B14 +3CD A16 ] combination was previously used [P1+3CD] B14 To determine whether the combination of [P1+3CD] has similar immunogenicity in vivo to the "matched" combination of [P1+3CD], an in vivo assay was performed as follows. A16 mRNA, or [P1 B14 +3CDA16 ] were vaccinated with a 2:1 molar ratio of P1:3CD mRNA (2.5 μg P1 + 0.94 μg 3CD). Since VLPs consist exclusively of VP proteins derived from processing and subsequent maturation of the P1 precursor protein, the serotype specificity of the neutralizing antibody response is determined exclusively by the P1 sequence and not the 3CD sequence. Thus, the matched [P1 + 3CD] A16 The combination of [P1 B14 +3CD A16 ] combinations were tested for neutralization of HRV-B14. Figure 4B shows the neutralizing antibody titers for each combination, including the respective virus-specific commercial hyperimmune antisera as benchmark controls. As expected, [P1+3CD] A16 Vaccination with 2.5 μg P1 resulted in high HRV-A16 neutralizing antibody titers. The GMT of this group (2.5 μg P1, GMT=13024) corresponds to the GMT of the 5 μg P1 and 1 μg P1 dose groups in the HRV-A16 dose-ranging study in Figure 2A (GMT=18549 and GMT=4198, respectively). B14 +3CD A16 ] vaccination also produced neutralizing antibodies to HRV-B14, but in agreement with [P1+3CD] B14 The GMT in the mixed group receiving 2.5 μg of HRV-B14 P1 mRNA was 253. In contrast, the GMTs in the groups receiving 5, 1, and 0.2 μg of HRV-B14 P1 mRNA were 1617, 1554, and 1582, respectively (Figure 2B). This is consistent with the "matched" [P1+3CD] B14 Even if the combination is less than 1 / 10 the dose, the [P1 B14 +3CD A16 This loss is due to the P1 B14This could be due to reduced processing of the rhinovirus A16 3CD or some unknown downstream effect, e.g., reduced VLP assembly. However, it is encouraging that the production of HRV-B14 neutralizing antibodies was not completely blocked by the use of HRV-A16 3CD. If the 3CD of rhinovirus A serotype can function, and to a lesser extent, by the P1 of rhinovirus B serotype, it is likely that a minimal 3CD sequence, or perhaps careful selection of the CD sequences administered, will be required to cover all serotypes within a species. Given the large amount of P1 sequences that will likely need to be included in a rhinovirus vaccine, it would be very useful to include several representative or consensus 3CD sequences in each P1 mRNA, rather than individual 3CD mRNAs that are "matched."

[0322] Example 6: HRV-A16 P1+3CD mRNA vaccination confers protection against infection in the cotton rat HRV challenge model To investigate whether the combination of P1+3CD mRNA could provide protection from infection, the effect of vaccination with HRV-A16 P1+3CD mRNA was tested in a cotton rat HRV challenge model as follows: Animals were immunized IM with decreasing amounts of HRV-A16 P1 / 3CD mRNA-LNP formulations on days 1 and 22. As a positive control, one group of animals received two IM injections of live replicating HRV-A16 virus according to the same schedule. The negative control group received PBS. On day 35, animals were inoculated intranasally with live HRV-A16 virus. Eight hours after challenge, lungs and noses were harvested and homogenized to calculate viral load. Serum was collected immediately before challenge to measure circulating neutralizing antibody levels. Vaccination with mRNA encoding P1 / 3CD LNP resulted in a dose-dependent induction of neutralizing antibodies (Figure 5A), with some animals in the 25 μg and 5 μg groups having titers approaching or exceeding those of animals administered live HRV-A16. For each of the three higher doses of P1 / 3CD, there was one animal in each group that did not have detectable titers, and none of the animals in the lowest dose group (0.2 μg) had detectable titers. Animals also showed a dose-dependent reduction in lung and nasal viral load. In the 25 μg and 5 μg groups, 4 / 5 and 3 / 5 animals, respectively, showed complete protection of lung tissue (Figure 5B). All animals in the 25 μg dose group and 2 / 5 animals in the 5 μg and 1 μg groups showed complete protection of nasal tissue (Figure 5C). Animals in the 0.2 μg dose group showed no protection, with lung and nasal viral loads comparable to the PBS negative control group. Furthermore, a significant reduction in lung viral RNA levels was detected in all animals receiving 25 μg, as well as in 4 / 5 and 3 / 5 of animals receiving 5 μg or 1 μg, respectively (Figure 5D).

[0323] Figure 6 contains the data generated per animal for each endpoint assay. These results demonstrate that vaccination with HRV-A16 P1+3CD mRNA confers dose-dependent protection from HRV-A16 infection, and that the level of protection achieved at the 25 μg dose is similar to that produced by two injections of replicative virus. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15]

[0324] Further Materials and Methods Transfection and immunoblotting 293T cells were transfected with mRNA using the Mirus Trans-It transfection kit. Cells were transfected with 2 μg of P1 mRNA and the indicated amounts of 3CD mRNA. Cells were harvested approximately 16-24 hours after transfection and lysed in RIPA buffer. Protein concentrations of lysates were quantified by BCA assay, run on 4-20% polyacrylamide gels with reducing agent (25 μg / lane), transferred to nitrocellulose membranes, and blotted overnight with primary antibodies at 1 μg / ml dilution. Primary antibodies used were mouse anti-HRV-A16 VP2 and rabbit anti-pan-HRV VP1. HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were used at 1:10,000 dilution. Signals were detected using ECL Prime detection reagent. Rhodamine-conjugated anti-GAPDH Fab was used for detection of GAPDH.

[0325] Virus-like particle production, purification and imaging Plasmids encoding HRV-A16 P1 and 3CD are co-transfected into HEK-293expi cells at a density of 3e6 / mL with a mass ratio of P1 to 3CD plasmids of 4:1 using Turbo293 transfection reagent (Speed ​​Biosystems). After 6 days, cell culture supernatants are harvested (cell pellets are discarded) and PEG-6000 and sodium chloride are added to final concentrations of 7% (w / v) and 2% (w / v), respectively. PEG (polyethylene glycol) precipitation is performed at 4°C for 18 h with stirring, followed by centrifugation at 9,500K for 30 min at 4°C. The supernatant is discarded and the precipitate is resuspended in 50 mM phosphate buffer, pH 7.0 (without sodium chloride). The resuspended solution is passed through a 0.45um filter to remove large debris. The sample is then loaded onto a HiTrap Q (Cytiva) column from Akta Pure (Cytiva) for anion exchange chromatography. The HRV VLPs bound to the HiTrap Q column are first washed with phosphate buffer pH 7.0 containing 0.1 M sodium chloride to remove impurities, and then eluted using phosphate buffer pH 7.0 containing 0.2 M sodium chloride. The eluted HRV VLP sample is used to prepare electron microscope grids with nano-W negative stain (Nanoprobes) and imaged using a Tecnai T12 electron microscope.

[0326] Vaccination and in vivo testing Approximately 16 hours prior to vaccination, formulated P1 and 3CD products were mixed and diluted in PBS to obtain the desired P1:3CD ratio and final mRNA concentration. Female 8-week-old Balb / c mice were vaccinated (50 μl injection volume, IM) on days 1 and 22. Serum was collected 3 weeks after the first injection (day 21) and 2 weeks after the second injection (day 35). Mice were sacrificed after serum collection on day 35.

[0327] Neutralization assay Viral isolates and titration: Viruses HRV-A16, HRV-B14, EV-D68 isolate US / MO / 14-18947, and EV-D68 isolate US / KY / 14-18953 were purchased from ATCC. EV-A71 isolates USA / 2018-23092 and Tainan / 4643 / 1998 were obtained from BEI Resources. Viral titers were measured by end-point dilution and calculated using the Reed-Münch statistic. Stocks of HRV-A16 and HRV-B14 were titrated on MRC-5 cells, and stocks of EV-D68 and EV-A71 isolates were titrated on RD cells. The TCID of each viral stock was calculated using the TCID of each virus stock. 50 was calculated by scoring cells for cytopathic effect (CPE) microscopically 5 days after infection.

[0328] Rhinovirus neutralization assay: 2x10 MRC-5 cells in 48-well plates 4 Cells / well were plated and incubated in MEM until the wells reached approximately 90% confluence (usually 7 days after plating). Serum from vaccinated mice was incubated at 56°C for 30 min to inactivate complement and then diluted 4-fold in a 6-fold dilution series. Diluted serum was added to 100 TCID 50 1:2 with 100 μl of virus to give a final volume of 100 μl of serum+virus. To allow neutralization, the serum+virus was incubated at 33°C for 3 hours during which the medium was aspirated from the 48-well plate and replaced with 300 μl of fresh MEM. After 3 hours of neutralization, serum+virus was added to the plate. The final volume was 400 μl / well. The plate was placed at 33°C. After 5 days, the plate was removed and the wells were scored for CPE under a microscope. Each dilution contained 8 replicate wells. The neutralizing antibody titer (IC 50 (reported as ) was calculated using the Reed-Muench method.

[0329] EV-D68 and EV-A71 neutralization assays: For EV-D68 neutralization, RD cells were plated in 96-well plates at 10 4For EV-A71 neutralization, Vero cells were plated at 1.5x10 cells / well in 100 μl of DMEM in 96-well plates and used for neutralization assays 48 hours after plating. 4 RD cells were plated at 10 cells / well in 100 μl of DMEM in a 96-well plate and used for neutralization assay the next day. 4 Cells were plated at 100 TCID 50 EV-D68 neutralization plate was placed at 33°C and EV-A71 neutralization plate at 37°C. After 5 days, plates were removed and wells were scored for CPE under a microscope. Each dilution contained 10 replicate wells. The neutralizing antibody titers (IC 50 (reported as ) was calculated using the Reed-Muench method.

[0330] Antisera: Guinea pig hyperimmune antisera against HRV-A16 and HRV-B14 were purchased from ATCC. Cotton rat HRV-A16 antisera was inoculated into 10 female cotton rats. 5 Antisera were generated by injecting PFU of replicative HRV-A16 on days 1 and 22 and collecting and pooling serum from vaccinated animals on day 35. Antisera were heat-treated at 56°C for 30 min to inactivate complement. Antisera were diluted and neutralization assays were performed as described above.

[0331] HRV-A16 inoculation study in cotton rats Animals: Forty (36) inbred, 4-6 week old female Sigmodon hispidus cotton rats (source: Sigmovir Biosystems, Inc., Rockville MD) were maintained and handled under veterinary supervision in accordance with National Institutes of Health guidelines and the Sigmovir Institutional Animal Care and Use Committee approved animal incident protocol (IACUC protocol #2). Cotton rats were housed in clear polycarbonate cages and fed standard rodent chow (Harlan #7004) and tap water ad libitum.

[0332] Inoculum virus: Human rhinovirus type 16 (HRV16) (ATCC, Manassas, VA) was propagated in HeLa Ohio cells after serial plaque purification to reduce defective interfering particles. Virus stocks were chloroform extracted for further purification. Approximately 1.0x10 8 A pool of virus containing pfu / mL, designated HRV16 p5, was used for this in vivo experiment. Stocks of this virus were stored at -80°C and were characterized in vivo in a cotton rat model, where replication in the upper and lower respiratory tract was verified.

[0333] Lung and nasal HRV16 virus titration: Lung and nasal homogenates were clarified by centrifugation and diluted in infection medium (EMEM, glutamine, pyruvate, Na-bicarbonate, Hepes) (lung, 1 / 10 to 1 / 100 dilution; nasal, neat to 1 / 10 dilution). Confluent Hela H1 monolayers are infected in 6-well plates in duplicate, with 100 μl per well of homogenate starting from the first dilution and then diluted. After 1 h incubation at 33° C. in a 5% CO2 incubator, the wells are covered with 0.75% methylcellulose medium and the plates are returned to the 33° C. incubator. After 5 days of incubation, the covers are removed and the cells are fixed with 0.1% crystal violet stain for 1 h, then rinsed and air-dried. Plaques are counted and virus titers are expressed as plaque-forming units per gram of tissue. Group virus titers are calculated as the geometric mean ± standard error of all animals in that group at a given time point.

[0334] HRV16 neutralizing antibody assay: Dilute serum samples 1:10 in infection medium and then serially dilute to 1:2. 3 TCID 50 ) for 2 hours at 33°C in a 5% CO2 incubator. The virus-antibody complexes are transferred to confluent Hela H1 cells in 96-well plates. After 5 days of incubation, the cells are fixed and stained with 0.1% crystal violet for 1 hour, then rinsed and air-dried. Neutralizing antibody titers are measured by the Reed-Münch method by identifying the cut-off dilution that completely protects the cell monolayer.

[0335] Real-time PCR viral RNA: Total RNA is extracted from homogenized lung tissue using the RNeasy purification kit (QIAGEN). 1 μg of total RNA is used to prepare cDNA using the QuantiTect reverse transcription kit (Qiagen). For real-time PCR reactions, QuantiFast SYBR Green PCR kit (Qiagen) is used in a final volume of 25 μl with a final primer concentration of 0.5 μM. Reactions are set up in 96-well trays. Amplification is performed in a Bio-Rad iCycler with one cycle of 95°C for 3 min, followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. Baseline cycles and cycle threshold (Ct) are calculated by the iQ5 software in PCR Base Line Subtracted Curve Fit mode. Relative quantification of DNA is applied to all samples. A standard curve is developed using a serially diluted cDNA sample with the highest abundance of the transcript of interest (e.g., lungs from HRV16 day 8 of infection for viral transcript qPCR). The Ct value is log 10 The Ct values ​​obtained in the various samples are plotted against the cDNA dilution factor. These curves are used to convert the Ct values ​​obtained in the various samples into relative expression units. These relative expression units are normalized to the levels of β-actin mRNA (a "housekeeping gene") expressed in the corresponding samples.

[0336] Equivalent While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision numerous other methods and / or structures for performing the functions and / or results and / or obtaining one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the present disclosure is used. Those of ordinary skill in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and equivalents thereto, the inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0337] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0338] All references, patents, and patent applications disclosed herein are each incorporated by reference with respect to the subject matter for which they are cited, and in some cases may include the entirety of that document.

[0339] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0340] The term "and / or" as used in the present specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether or not associated with the specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" can refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0341] "Or" as used herein and in the claims shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one of a plurality of elements or a list of elements, but including a plurality of them, and optionally, additional items not in the list. Only language expressly indicated to the contrary, such as "only one" or "exactly one" or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a plurality of elements or a list of elements. In general, the term "or" as used herein shall be interpreted to indicate exclusive alternatives (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either," "one," "only one," or "exactly one." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0342] As used in this specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether or not associated with the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally multiple, A and no B (and optionally including elements other than B), in another embodiment to at least one, optionally multiple, B and no A (and optionally including elements other than A), in yet another embodiment to at least one, optionally multiple, A, and at least one, optionally multiple, B (and optionally including other elements), etc.

[0343] It is also to be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0344] In the claims and above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are understood to be open-ended, i.e., meaning including but not limited to. As set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described herein using open-ended transitional phrases (e.g., "comprising") are also contemplated in alternative embodiments as "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates the alternative embodiments of "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

[Claim 1] (i) a messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding a picornavirus 3C protease; (ii) an mRNA comprising an ORF encoding a picornavirus capsid polyprotein, said capsid polyprotein comprising the viral P1 precursor polyprotein; and (iii) Lipid Nanoparticles (LNPs) An immunogenic composition comprising:

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