WO2025027492A1

WO2025027492A1 - Coronavirus antigen variants

Info

Publication number: WO2025027492A1
Application number: PCT/IB2024/057303
Authority: WO
Inventors: Ye Che; Hui Cai; Kayvon MODJARRAD; Aravinda MUNASINGHE; Kena Anne SWANSON; Ian William WINDSOR; Huixian Wu
Original assignee: Pfizer Corp Belgium; Pfizer Corp SRL
Current assignee: Pfizer Corp Belgium; Pfizer Corp SRL
Priority date: 2023-07-31
Filing date: 2024-07-29
Publication date: 2025-02-06
Anticipated expiration: 2026-01-31

Abstract

The present disclosure provides isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are variants of a native coronavirus spike protein or fragment thereof. The present disclosure also provides compositions comprising the isolated immunogenic polypeptides and methods of making and using such compositions.

Description

CORONAVIRUS ANTIGEN VARIANTS

Technical Field

This disclosure relates to the field of immunology, specifically to immunogenic compositions. More particularly, it concerns methods and compositions involving coronavirus spike protein variants, which can be used to invoke an immune response against coronaviruses.

Background

Coronaviruses are a large family of viruses that usually cause mild to moderate upper-respiratory tract illnesses, like the common cold. However, three new coronaviruses, MERS-CoV, SARS-CoV, and SARS-CoV-2, have emerged from animal reservoirs over the past two decades to cause serious and widespread illness and death. SARS coronavirus (SARS-CoV) emerged in November 2002 and caused severe acute respiratory syndrome (SARS). Middle East respiratory syndrome (MERS) is caused by the MERS coronavirus (MERS-CoV). Transmitted from an animal reservoir in camels, MERS was identified in September 2012 and continues to cause sporadic and localized outbreaks. The third novel coronavirus to emerge in this century is called SARS-CoV-2. It causes coronavirus disease 2019 (COVID-19), which emerged from China in December 2019 and was declared a global pandemic by the World Health Organization on March 11 , 2020.

Coronavirus infection is mediated by the receptor binding domain (RBD) of the coronavirus spike glycoprotein binding to the ACE2 receptor on the surface of a host cell membrane. Although the coronavirus spike sequence and structure are known and several immunogenic compositions exist to elicit an immune response to the spike, there remains a need for spike protein antigens with increased protein expression, stability, and immunogenic conformations as compared to presently-used immunogenic compositions in order to more effectively stimulate a protective immune response against coronaviruses. Summary

The present disclosure provides methods and compositions involving coronavirus spike protein variants that include subunits, domains, and/or subdomains of the spike protein. For any of the coronaviruses described herein, such variants can be in the form of protein antigens. Further, for any of the coronaviruses described herein other than SARS-CoV-2, such variants can also be in the form of RNA, such as mRNA. Specifically, the present disclosure is not directed to spike protein variants in the form of RNA, including mRNA, where the spike protein is derived from SARS- CoV-2. The variants may invoke an immune response against coronaviruses more effectively than naturally occurring coronavirus spike proteins. Increasing the immunogenicity of viral antigens by, e.g., improving expression of the viral antigens, improving the stability of the viral antigens, and/or increasing the number of neutralization-sensitive epitopes on the viral antigens, is a desirable outcome in the safety and efficacy of vaccines. The present disclosure is based, at least in part, on the discovery that one or more specific amino acid modifications can be made to native coronavirus spike protein sequences to produce variant coronavirus spike proteins having improved in vivo expression, improved stability of the prefusion conformation, and/or increased exposure of neutralization-sensitive epitopes that may result in a more immunogenic antigen.

The present disclosure is based on the principle and discovery that the multivalent presentation of the RBD and NTD of the spike protein will increase the stability, expression and immunogenicity of viral antigens that will result in the improved efficacy of vaccines against a broader set of SARS-CoV-2 variants and coronavirus species. The present disclosure is also based on the principle and discovery that an ordered and repetitive presentation of antigens — herein actualized by the trimerization of expressed coronavirus subunits, domains and subdomains — will generate greater potency and breadth of the immune response against coronavirus variants and species that are homologous and heterologous to the composition of the viral antigens. The synergy of the multivalency, repetitive array, and ordered trimerization of the RBD and NTD antigens — with or without the need for a separate self-assembling scaffold protein — will result in a directed immune response, by cross-signed B lymphocytes, against common neutralization sensitive epitopes exposed on the multiply expressed RBD and NTD antigens described herein. It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

FIG. 1 is a schematic of the various domains of a coronavirus spike protein according to some aspects disclosed herein. SS is the Signal Sequence; NTD is the N-terminal Domain; RBD is the Receptor Binding Domain; SD1 is the Spike Subdomain 1 ; SD2 is the Spike Subdomain 2; S1/S2 is the S1/S2 interface sequence; S2’ is a proteolytic cleavage site in the S2 fusion domain; FP is the Fusion Peptide sequence; HR1 is the Heptad Repeat sequence 1 ; CH is the Central Helix sequence; CD is the Cytoplasmic Domain; HR2 is the Heptad Repeat sequence 2; TM is the Transmembrane Domain; and CT is the Cytoplasmic Tail.

FIG. 2 is a schematic of the various domains (RBD) of a coronavirus spike protein expressed in sequence with a trimerization domain, according to some aspects disclosed herein.

FIG. 3 is a schematic of the various domains (RBD, NTD) of a coronavirus spike protein expressed in sequence with a trimerization domain, as well as a transmembrane domain and C-terminal domain, according to some aspects disclosed herein. FIG. 4 is a schematic of the various domains (RBD, NTD) of a coronavirus spike protein expressed in sequence with and without a trimerization domain, linked to a self-assembling protein domain, according to some aspects disclosed herein.

FIG. 5 shows the oligomeric state characterization using size-exclusion- chromatography for the secreted antigen constructs.

Detailed Description

I. Examples of Definitions

Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some aspects, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less of the measurement or quantitation.

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

The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.

The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.

The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. It is contemplated that aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.” Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of’ (and any form of consisting of, such as “consist of” and “consists of”) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

Reference throughout this specification to “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” or “a further aspect” or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.

As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and/or determined substantially simultaneously and/or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and/or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment. The term “DNA,” as used herein, means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and/or thymine (T)). For example, DNA can contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2’ position of a [3-D-ribofuranosyl group. Without any limitation, DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.

As used herein, a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues. As used herein, the term “wild-type” or “native” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wildtype activity or function in other respects, such as immunogenicity. Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state and/or that is altered or removed from the natural state through human intervention. For example, a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered.

A “nucleic acid,” as used herein, is a molecule comprising nucleic acid components and refers to DNA or RNA molecules. It may be used interchangeably with the term “polynucleotide.” A nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. Nucleic acids may also encompass modified nucleic acid molecules, such as base-modified, sugar-modified, backbone-modified, etc. DNA or RNA molecules. Nucleic acids may exist in a variety of forms such as: isolated segments and recombinant vectors of incorporated sequences and/or recombinant polynucleotides encoding polypeptides, e.g., antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide; anti-sense nucleic acids for inhibiting expression of a polynucleotide; mRNA; saRNA; and complementary sequences of the foregoing described herein. Nucleic acids may encode an epitope to which antibodies may bind.

The term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some aspects, an epitope is comprised of a plurality of chemical atoms and/or groups on an antigen. In some aspects, such chemical atoms and/or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some aspects, such chemical atoms and/or groups are physically near to each other in space when the antigen adopts such a conformation. In some aspects, at least some such chemical atoms and/or groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

Nucleic acids may be single-stranded or double-stranded and may comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids). In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow for purification of the polypeptide, transport, secretion, post-translational modification, and/or for therapeutic benefits such as targeting and/or efficacy. A tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

The term “polynucleotide” refers to a nucleic acid molecule that may be recombinant and/or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (e.g., nucleic acids 100 residues or less in length) and recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

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

The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids may be any length. They may be, for example, at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, and/or may comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some aspects, 1 , 2, 3, or more of the foregoing nucleic acid sequences can be excluded from the nucleic acid segments of the disclosure.

As used herein, the term “gene” refers to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, and/or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or a substantially similar polypeptide.

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

An immune response refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. An immune response can be measured by assays that include, but are not limited to, assays measuring the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays measuring T-cell activation or proliferation, and/or assays that measure modulation in terms of activity or expression of one or more cytokines.

II. Viruses

As contemplated herein, without any limitations, the compositions and methods herein can be used as a modality to treat and/or prevent and/or reduce the seventy of or medical/health risks of a number of diseases and/or conditions in mammals, including coronavirus infection in humans. Methods described herein comprise administration of the compositions described herein to a mammal, such as a human. For example, in one aspect, such methods of use for the compositions herein include a variant coronavirus spike protein or peptide vaccine to induce robust neutralizing antibodies and accompanying/concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses.

For example, the immunogenic polypeptide constructs, or RNA encoding such constructs, can comprise one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof that elicit an immune response. In specific aspects, the immunogenic polypeptide constructs, or RNA encoding such constructs, are variant coronavirus spike protein constructs, and the variant coronavirus spike protein constructs can comprise one or more immunogenic peptide sequences that elicit an immune response.

Conditions and/or diseases that can be treated and/or prevented with such peptide or polypeptide or RNA compositions include, but are not limited to, those caused and/or impacted by viral infection. Such viruses include, but are not limited to, coronaviruses (such as a severe acute respiratory syndrome virus (SARS) - e.g. SARS-CoV-2 in the case of polypeptide constructs (but not RNA encoding such constructs), or a Middle East Respiratory Syndrome (MERS) virus).

Variant coronavirus spike protein constructs can be based on any spike protein sequence from any Coronaviridae family virus. Coronaviridae is a family of enveloped, positive-sense, single-stranded RNA viruses. Coronavirus is the common name for Coronaviridae and Orthocoronavirinae (also referred to as Coronavirinae). The family Coronaviridae is organized in 2 sub-families, 5 genera, 23 sub-genera and approximately 40 species. They are enveloped viruses having a positive-sense single-stranded RNA genome and a nucleocapsid having helical symmetry.

Several coronaviruses utilize animals as their primary hosts and have also evolved to infect humans. There are four main sub-groupings of coronaviruses, known as alpha, beta, gamma, and delta; seven coronaviruses can infect people. The four most common coronaviruses utilize humans as their natural host and include: 229E (alpha coronavirus); NL63 (alpha coronavirus); OC43 (beta coronavirus); HKLI1 (beta coronavirus). Three other human coronaviruses are: MERS-CoV (the beta coronavirus that causes MERS); SARS-CoV (the beta coronavirus that causes SARS); and SARS-CoV-2 (the novel coronavirus that causes coronavirus disease 2019, or COVID-19).

Coronaviruses have characteristic club-shaped spikes that project from their surface, which in electron micrographs create an image reminiscent of the solar corona, from which their name derives. The average diameter of the virus particles is around 120 nm (.12 pm). The diameter of the envelope is ~80 nm (.08 pm) and the spikes are ~20 nm (.02 pm) long. Beneath the spiked exterior of the virus is a round core shrouded in a viral envelope. The core contains genetic material that the virus can inject into cells to infect them.

The viral envelope consists of a lipid bilayer where the membrane (M), envelope (E), and spike (S) structural proteins are anchored. Inside the envelope, there is the nucleocapsid of helical symmetry which is formed from multiple copies of the nucleocapsid (N) protein, which are bound to the positive-sense single-stranded RNA genome in a continuous beads-on-a-string type conformation. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases. The genome organization for a coronavirus is 5'-leader-UTR-replicase/transcriptase-spike (S)- envelope (E)-membrane (M)-nucleocapsid (N)-3'UTR-poly (A) tail. The open reading frames 1 a and 1 b, which occupy the first two-thirds of the genome, encode the replicase/transcriptase polyprotein. The replicase/transcriptase polyprotein self cleaves to form nonstructural proteins. The later reading frames encode the four major structural proteins: spike, envelope, membrane, and nucleocapsid. Interspersed between these reading frames are the reading frames for the accessory proteins. The number of accessory proteins and their function is unique depending on the specific coronavirus.

The lipid bilayer envelope, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell. The spike proteins extend from within the core to the viral surface and allow the virus to recognize and bind specific cells in the body. When the spike engages a receptor on a host cell, a cascade is triggered, resulting in the merger of the virus with the cell which allows the virus to release its genetic material and overtake the cell’s processes to produce new viruses.

Infection begins when the viral spike (S) glycoprotein attaches to its complementary host cell receptor. After attachment, a protease of the host cell (e.g., ACE2) cleaves and activates the receptor-attached spike protein. Depending on the host cell protease available, cleavage and activation allows the virus to enter the host cell by endocytosis or direct fusion of the viral envelop with the host membrane.

Binding of the S1 subunit of the S protein to the host cell receptor stabilizes the S protein in an “up” conformation, making the protein more vulnerable to cleavage by the host cell protease because the receptor binding site is exposed when the S protein is in the RBD-up conformation. Additionally, neutralization-sensitive epitopes are exposed when the RBD is in the up conformation, and thus, in some aspects of the variant coronavirus spike proteins disclosed herein, modifications are made to promote adoption of the RBD-up conformation or to inhibit adoption of the RBD-down conformation. Cleavage occurs between the S1 and S2 subunits of the S protein, and cleavage triggers conformational changes by the S2 subunit to allow insertion of the S2 subunit into the host cell membrane and mediation of fusion between the viral and host cell membranes.

On entry into the host cell, the virus particle is uncoated, and its genome enters the cell cytoplasm. The coronavirus RNA genome has a 5' methylated cap and a 3' polyadenylated tail, which allows the RNA to attach to the host cell’s ribosome for translation. The host ribosome translates the initial overlapping open reading frame of the virus genome and forms a long polyprotein. The polyprotein has its own proteases which cleave the polyprotein into multiple nonstructural proteins.

Viral entry is followed by replication of the virus. A number of the nonstructural proteins coalesce to form a multi-protein replicase-transcriptase complex (RTC). The main replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp). It is directly involved in the replication and transcription of RNA from an RNA strand. The other nonstructural proteins in the complex assist in the replication and transcription process. The exoribonuclease nonstructural protein, for instance, provides extra fidelity to replication by providing a proofreading function which the RNA-dependent RNA polymerase lacks. One of the main functions of the complex is to replicate the viral genome. RdRp directly mediates the synthesis of negative-sense genomic RNA from the positive-sense genomic RNA. This is followed by the replication of positive-sense genomic RNA from the negative-sense genomic RNA. The other important function of the complex is to transcribe the viral genome. RdRp directly mediates the synthesis of negative-sense subgenomic RNA molecules from the positive-sense genomic RNA. This is followed by the transcription of these negative-sense subgenomic RNA molecules to their corresponding positive-sense mRNAs.

The replicated positive-sense genomic RNA becomes the genome of the progeny viruses. The mRNAs are gene transcripts of the last third of the virus genome after the initial overlapping reading frame. These mRNAs are translated by the host’s ribosomes into the structural proteins and a number of accessory proteins. RNA translation occurs inside the endoplasmic reticulum. The viral structural proteins S, E, and M move along the secretory pathway into the Golgi intermediate compartment. There, the M proteins direct most protein-protein interactions required for assembly of viruses following its binding to the nucleocapsid. Progeny viruses are then released from the host cell by exocytosis through secretory vesicles.

The interaction of the coronavirus spike protein with its complement host cell receptor is central in determining the tissue tropism, infectivity, and species range of the virus. Coronaviruses mainly target epithelial cell receptors. They can be transmitted by aerosol, fomite, or fecal-oral routes, for example. Human coronaviruses infect the epithelial cells of the respiratory tract. For example, human coronaviruses can infect, via an aerosol route, human epithelial cells of the lungs by binding of the spike protein receptor binding domain (RBD) to an angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface.

The WHO has reported that the two groups most at risk of experiencing severe illness due to a coronavirus infection are adults aged 65 years or older and people who have other underlying health conditions including chronic lung disease, serious heart conditions, severe obesity, a compromised immune system, or diabetes. In humans, coronaviruses typically cause a respiratory infection with mild to severe flulike symptoms, but the exact symptoms vary depending on the type of coronavirus. The four common human coronaviruses can cause people to develop a runny nose, headache, cough, sore throat and fever. In a subset of individuals, including those with cardiopulmonary disease or a weakened immune system, the viral infection can progress to a more severe lower-respiratory infection such as pneumonia or bronchitis. In comparison, severe MERS and SARS infections often progress to pneumonia. Other symptoms of MERS include fever, coughing, and shortness of breath, while SARS can cause fever, chills and body aches.

SARS-CoV-2 causes symptoms similar to those of other coronaviruses, triggering fever, cough, and shortness of breath in most patients. Rarer symptoms include dizziness, tiredness, aches, chills, sore throat, loss of smell, loss of taste, headache, nausea, vomiting, and diarrhea. Emergency signs or symptoms can include trouble breathing, persistent chest pain or pressure, new confusion, and/or blue lips or face. Complications of SARS-CoV-2 infections can include pneumonia, organ failure, respiratory failure, blood clots, heart conditions such as cardiomyopathies, acute kidney injury, and/or further viral and bacterial infections.

The present disclosure encompasses treatment or prevention of a disease or condition caused by infection of any virus in the Coronaviridae family. In particular aspects, methods and compositions treat or prevent COVID-19 or reduce the symptoms or seventy of COVID-19, which is caused by infection from SARS-CoV-2. In certain aspects, the disclosure encompasses treatment or prevention of infection of any virus in the subfamily Coronavirinae and including the four genera, Alpha-, Beta- Gamma-, and Deltacoronavirus. In specific aspects, the disclosure encompasses treatment or prevention of infection of any virus in the genus of Betacoronavirus, including the subgenus Sarbecovirus and the species severe acute respiratory syndrome-related coronavirus; the subgenus Embecovirus and the species human coronavirus HKU1 ; and the species Betacoronavirus 1. In specific aspects, the disclosure encompasses treatment or prevention of infection of any virus in the species of severe acute respiratory syndrome-related coronavirus, including the strains severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes COVID- 19). The disclosure encompasses treatment or prevention of infection from any isolate, strain, type (including Type A, Type B and Type C; Forster et al., 2020, PNAS, available on the World Wide Web at doi.org/10.1073/pnas.2004999117), cluster, or sub-cluster of the species of severe acute respiratory syndrome-related coronavirus, including at least SARS-CoV-2. In specific aspects, the virus has a genome length between 29000 to 30000, between 29100 and 29900, between 29200 and 29900, between 29300 and 29900, between 29400 and 29900, between 29500 and 29900, between 29600 and 29900, between 29700 and 29900, between 29800 and 29900, or between 29780 and 29900 base pairs in length.

Examples of specific SARS-CoV-2 viruses include the following listed in the NCBI GenBank® Database, and these GenBank® Accession sequences are incorporated by reference herein in their entirety: (a) LC534419 and LC534418 and LC528233 and LC529905 (examples of different strains from Japan); (b) MT281577 and MT226610 and NC_045512 and MN996531 and MN908947 (examples of different strains from China); (c) MT281530 (Iran); (d) MT126808 (Brazil); (e) MT020781 (Finland); (f) MT093571 (Sweden); (g) MT263074 (Peru); (h) MT292582 and MT292581 and MT292580 and MT292579 (examples of different strains from Spain); (i) examples from the United States, such as MT276331 (TX); MT276330 (FL); MT276328 (OR) MT276327 (GA); MT276325 (WA); MT276324 (CA); MT276323 (Rl); MT188341 (MN); and (j) MT276598 (Israel). In particular aspects, the disclosure encompasses treatment or prevention of infection of any of these or similar viruses, including viruses whose genome has at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identity to any of these viruses. In particular aspects, the disclosure encompasses treatment or prevention of infection of any of these or similar viruses, including viruses whose genome has its entire sequence that is greater than 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identity to any of these viruses. As one specific example, the present disclosure includes methods of treatment or prevention of infection of a virus having a genome sequence of GenBank® Accession No. NC_045512 and any virus having a genome sequence with at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identity to said sequence.

III. Polypeptides

The isolated immunogenic polypeptide constructs of the present disclosure can comprise one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof that elicit an immune response. Thus, in certain aspects, isolated immunogenic polypeptide constructs are peptide or protein antigens derived from a pathogen associated with infectious disease, including coronaviruses. In certain aspects, the immunogenic polypeptide constructs are peptide or protein antigens derived from the spike protein of a coronavirus.

In particular aspects, the present disclosure provides immunogenic polypeptide constructs that are coronavirus spike protein variants that comprise a multivalent presentation of the RBD and NTD domains of the spike protein. Further, such variants can be: multimeric (i.e. comprise more than one of the various domains of the spike protein); heterologous (i.e. in the sense that the various domains are taken from different species of coronavirus); and are trimerized. As disclosed herein, such variants can increase the stability, expression and immunogenicity of viral antigens that can result in the improved efficacy of vaccines against a broader set of SARS- CoV-2 variants and coronavirus species. The present disclosure is based on the principle and discovery that an ordered and repetitive presentation of antigens — herein actualized by the trimerization of expressed coronavirus subunits, domains and subdomains — will generate greater potency and breadth of the immune response against coronavirus variants and species that are homologous and heterologous to the composition of the viral antigens. The synergy of the multivalency, repetitive array, and ordered trimerization of the RBD and NTD antigens - with or without the need for a separate self-assembling scaffold protein - can result in a directed immune response, by cross-signed B lymphocytes, against common neutralization sensitive epitopes exposed on the multiply expressed RBD and NTD antigens described herein. In certain aspects, peptides or proteins can exist in a variety of instances such as: isolated polypeptides or recombinant polypeptides, or a fragment, functional derivatives, muteins, or variants thereof, peptides or proteins sufficient for use as hybridization probes, peptides or proteins for inhibiting expression of a polynucleotide, and complementary amino acid sequences of the foregoing described herein. Peptides or proteins may be an epitope to which antibodies may bind. The peptides or proteins can comprise RNA and/or DNA nucleotides (e.g., peptide nucleic acids).

In certain aspects the size of a protein or peptide or derivative of a corresponding amino sequence described or referenced herein can be, for example, equal to any one of, at least any one of, at most any one of, or between any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 amino acid residues or greater. It is contemplated that proteins or peptides may be mutated by truncation, rendering them shorter than their corresponding native or wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or peptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or peptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

In some aspects, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof comprise at least a RBD. The RBD may or may not be modified with respect to the RBD of the native coronavirus spike protein. In some aspects, the RBD is modified with respect to the RBD of the native coronavirus spike protein. In some aspects, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof comprise a leader sequence. In some aspects, the leader sequence has an amino acid sequence that has equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to the leader sequence of the native coronavirus spike protein sequence. In some aspects, the leader sequence has an amino acid sequence that is at least 80% identical to the leader sequence of the native coronavirus spike protein sequence. In some aspects, inclusion of a leader sequence as part of the isolated immunogenic polypeptide sequence inhibits disulfide scrambling.

In some aspects, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof comprise a transmembrane sequence. In some aspects, the transmembrane sequence has an amino acid sequence that has equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to the transmembrane sequence of the native coronavirus spike protein sequence. In some aspects, the transmembrane sequence has an amino acid sequence that is at least 80% identical to the transmembrane sequence of the native coronavirus spike protein sequence. In some aspects, inclusion of a transmembrane sequence as part of the isolated immunogenic polypeptide sequence extends the half-life of the isolated immunogenic polypeptide.

In some aspects, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof does not comprise an endoplasmic reticulum (ER) signal sequence. In some aspects, exclusion of an ER signal sequence improves localization of the variant coronavirus spike protein to the host cell membrane.

In some aspects, the one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof comprise a trimerization domain. In some aspects, the trimerization domain is a foldon trimerization domain. In some aspects, the trimerization domain sequence has an amino acid sequence that has equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to the trimerization domain sequence of the foldon trimerization domain of T4 fibritin. In some aspects, the trimerization domain sequence has an amino acid sequence that is at least 80% identical to the trimerization domain sequence of the foldon trimerization domain of T4 fibritin.

In some aspects, one or more isolated immunogenic polypeptides comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof are encoded by a nucleic acid and elicit an immune response. The immune response may be against the immunogenic variant coronavirus spike protein constructs and/or a native coronavirus spike protein. The immunogenic variant coronavirus spike protein constructs and the native coronavirus spike protein may be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%,

65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous.

In some aspects, the variant coronavirus spike protein constructs may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more contiguous amino acids that are equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with a native coronavirus spike protein.

The immunogenic variant coronavirus spike protein constructs thereof may include equal to any one of, at least any one of, at most any one of, or between any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18 amino acid substitutions as disclosed herein compared to native, unmodified immunogenic polypeptide constructs. The immunogenic variant coronavirus spike protein constructs may equal to any one of, at least any one of, at most any one of, or between any two of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %,

32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%,

47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %,

62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%,

77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any derivable range therein) similar, identical, or homologous with native, unmodified immunogenic polypeptide constructs.

Nucleotide as well as protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

IV. Coronavirus Spike Protein Modifications

The immunogenic native coronavirus spike protein constructs of the present disclosure may be modified, such that they are substantially identical to the immunogenic variant coronavirus spike protein constructs comprised in immunogenic compositions described herein. In some aspects, the immunogenic variant coronavirus spike protein constructs continue to be bound by antibodies to elicit an immune response.

In particular aspects, it is the peptide sequences of native coronavirus spike proteins that are modified. Thus, in some aspects, variant coronavirus spike proteins comprise equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to the native coronavirus spike protein sequence. In some aspects, variant coronavirus spike proteins can comprise an amino acid sequence that is at least 70% identical to an amino acid sequence of the native coronavirus spike protein. In some aspects, variant coronavirus spike proteins can comprise an amino acid sequence that is at least 80% identical to an amino acid sequence of the native coronavirus spike protein. In some aspects, variant coronavirus spike proteins can comprise an amino acid sequence that is at least 90% identical to an amino acid sequence of the native coronavirus spike protein.

Polypeptide sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, they share at least 70% identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.

The immunogenic variant coronavirus spike protein constructs of the disclosure may include equal to any one of, at least any one of, at most any one of, or between any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18 amino acid substitutions as disclosed herein and/or be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%,

99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% similar, identical, or homologous with equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more contiguous amino acids of native, unmodified immunogenic coronavirus spike protein constructs of the present disclosure and/or of homologous peptides or proteins.

In some aspects, the immunogenic variant coronavirus spike protein constructs may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more contiguous amino acids that are equal to any one of, at least any one of, at most any one of, or between any two of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%,

27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %,

42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%,

57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %,

72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%,

87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with native, unmodified immunogenic coronavirus spike protein constructs of the present disclosure and/or of homologous peptides or proteins.

As modifications and/or changes may be made in the sequence and/or structure of proteins according to the present disclosure, while obtaining molecules having similar or improved characteristics (e.g., maintenance of antibody binding and immune response and attenuation of cross-reactivity with endogenously-expressed host proteins), such biologically functional equivalents of the immunogenic polypeptide constructs are also encompassed within the present invention.

In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins having substitutions or mutations in selected amino acids that retain the ability to be bound by an antibody and elicit an immune response and/or proteins having substitutions or mutations in selected amino acids.

In one example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, etc. A. Coronavirus Spike Proteins

The following Table 1 includes the amino acid sequences of the spike proteins of the seven coronaviruses known to infect humans. Amino acid sequences were obtained from the UniProt database, accessible via the World Wide Web at uniprot.org, or the GenBank database, accessible via the World Wide Web at ncbi.nlm.nih.gov, and the UniProt or GenBank database accession numbers of each spike protein sequence are included in the Table 1. These amino acid sequences correspond to the amino acid sequences of native coronavirus spike proteins. In some aspects, the amino acid sequences of native coronavirus spike proteins may be modified, as described herein, to produce isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof. For example, in some aspects, the amino acid sequences of native coronavirus spike proteins are substituted, as described herein, to produce isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof.

Also included in Table 1 are the amino acid sequences of the spike proteins of SARS-CoV-2 variants, including the alpha, beta, gamma, delta, and omicron variants (including omicron BA1 and BA2). Like the amino acid sequences of native coronavirus spike proteins, the amino acid sequences of spike proteins of these SARS-CoV-2 variants may be modified at the corresponding position, as described herein, to produce isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of the native variant coronavirus spike proteins or fragments thereof. For example, in some aspects, the amino acid sequences of spike proteins of these SARS-CoV-2 variants are substituted, as described herein, to produce isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of variant coronavirus spike proteins or fragments thereof. Additional variants not specifically set forth below are also contemplated. For example, any variant coronavirus spike protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the native coronavirus spike protein sequence may be modified at the corresponding position, (e.g., substituted), as described herein, to produce isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof.

Table 1

A schematic of a coronavirus spike protein having a S1 subunit and a S2 subunit is shown in FIG. 1. The S1 subunit comprises a leader, or signal, sequence (SS), a N-terminal domain (NTD) and a receptor binding domain (RBD). The S2 subunit comprises heptad repeat regions (HR1 and HR2) and a transmembrane domain (TM). Modifications to the spike protein sequence may be made anywhere within the sequence as described herein, but in some aspects, modifications are made in the NTD or RBD or a sequence linking the NTD and RBD. In some aspects, modifications are made in an amino acid sequence linking the first heptad repeat region to the second heptad repeat region. In some aspects, modifications are made in an amino acid sequence at the interface of the S1 and S2 subunits. For example, in some aspects, amino acid substitutions are made in the NTD and/or RBD and/or a sequence linking the NTD and RBD and/or amino acid substitutions are made in an amino acid sequence linking the first heptad repeat region to the second heptad repeat region and/or amino acid substitutions are made in an amino acid sequence at the interface of the S1 and S2 subunits. In further aspects, any of the regions and/or domains of the spike protein can be re-ordered from their native position within the sequence. Furthermore, any of the regions and/or domains can be duplicated so that the modified sequence contains more copies of any of the regions and/or domains than are found within the native sequence.

B. Altered Amino Acids

As used herein, an “amino molecule” refers to any amino acid, amino acid derivative, or amino acid mimic as would be known to one of ordinary skill in the art. In certain aspects, the residues of the peptide or protein are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other aspects, the sequence may comprise one or more non-amino molecule moieties. In particular aspects, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties. Peptides and proteins include the twenty “natural” amino acids, and post-translational modifications thereof. However, in vitro peptide synthesis permits the use of modified and/or unusual amino acids.

Accordingly, the term “protein,” “peptide,” or “polypeptide” encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown in the Table 2 below.

Table 2

V. Obtaining Encoded Polypeptide Aspects

In some aspects, there are nucleic acid molecules encoding peptides of interest, e.g., antigens. These nucleic acids may be generated by methods known in the art.

A. Expression The nucleic acid molecules described herein may be used to express large quantities of the polypeptide of interest, such as an antigen, such as variant coronavirus spike protein.

1. Nucleic Acid synthesis

In some aspects, contemplated are isolated nucleic acid molecules comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g., a fragment containing one or more polypeptides, or antigens) that can be used to produce the polypeptide of interest. In some aspects, nucleic acid molecules comprising nucleic acid molecules may encode antigens, fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, the nucleic acid molecules may contain nucleic acid sequences that serve other functions as well.

In some aspects, the nucleic acid molecule is an analog and may include modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and/or improve binding specificity. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Moreover, when other substitutions, such as substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the structural place of the sugar of wild type nucleic acids. Moreover, reference to inter-sugar linkages shall be taken to include moieties serving to join the sugar or sugar analog portions in the fashion of wild type nucleic acids.

Expression Systems

Numerous expression systems exist that comprise at least a part or all of the proteins, peptides, or nucleic acid molecules discussed above. Prokaryote- and/or eukaryote-based systems or cell free systems can be employed for use with an aspect to produce proteins, peptides, nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include but are not limited to bacterial, mammalian, yeast, insect cell, and cell free systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines, host systems, or expression systems can be chosen to ensure the correct modification and processing of the nucleic acid or polypeptide(s) expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.

In some aspects, immunogenic variant coronavirus spike protein constructs and/or nucleic acids encoding the immunogenic variant coronavirus spike protein constructs of the present disclosure are achieved by operably linking a nucleic acid encoding the immunogenic variant coronavirus spike protein constructs to a promoter, and incorporating the construct into an expression vector, which is taken up and expressed by cells. The vectors can be suitable for replication and, in some cases, integration in eukaryotes. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

In certain aspects the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001 ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.

Several viral based systems have been developed for gene transfer into mammalian cells. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including self-inactivating lentivirus vectors). For example, adenoviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. Thus, in some aspects, the nucleic acid encoding immunogenic polypeptide constructs of the present disclosure is introduced into cells using a recombinant vector such as a viral vector including, for example, a lentivirus, a retrovirus, gamma-retroviruses, an adeno-associated virus (AAV), a herpesvirus, or an adenovirus.

One of skill in the art would be well equipped to construct a vector comprising one or more polynucleotide sequences of interest through standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both specifically incorporated by reference herein in their entirety).

Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.

Such components also might include markers, such as detectable and/or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell’s nucleus or cytoplasm.

Eukaryotic expression cassettes included in the vectors particularly contain (in a 5'-to-3' direction) regulatory elements including a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, a transcriptional termination/polyadenylation sequence, post- transcriptional regulatory elements, and origins of replication. 2. Host Cells

In another aspect, contemplated are the use of host cells into which a nucleic acid molecule has been introduced. Nucleic acids can be transfected into cells according to a variety of methods known in the art. Nucleic acids can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some nucleic acids may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the polypeptide of interest expression construct or nucleic acid replicase can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NFAT-1 or NF-KB, both of which are transcription factors that can be activated upon T-cell activation. Control of expression allows T cells, such as tumor- targeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a nucleic acid molecule. Also understood and known are techniques and conditions that would allow large-scale production of nucleic acid molecules, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

For transfection of mammalian cells, it is known, depending upon the nucleic acid and transfection technique used, only a small fraction of cells may integrate the foreign nucleic acid into their cells. Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to identify and select these integrants, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.

In certain aspects, cells containing an exogenous nucleic acid may be identified in vitro or in vivo by including a marker in the expression vector or the exogenous nucleic acid. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker may be one that confers a property that allows for selection. A positive selection marker may be one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.

Selectable markers may include a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes; cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those cells that can grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include: the Abicr gene or Neo gene from Tn5, which confers antibiotic resistance to geneticin.

A screenable marker may comprise a reporter gene, which allows the researcher to distinguish between wanted and unwanted cells. Certain aspects of the present invention utilize reporter genes to indicate specific cell lineages. For example, the reporter gene can be located within expression elements and under the control of the ventricular- or atrial-selective regulatory elements normally associated with the coding region of a ventricular- or atrial-selective gene for simultaneous expression. A reporter allows the cells of a specific lineage to be isolated without placing them under drug or other selective pressures or otherwise risking cell viability.

Examples of such reporters include genes encoding cell surface proteins (e.g., CD4, HA epitope), fluorescent proteins, antigenic determinants and enzymes (e.g., [3- galactosidase). The vector containing cells may be isolated, e.g., by FACS using fluorescently-tagged antibodies to the cell surface protein or substrates that can be converted to fluorescent products by a vector encoded enzyme. In specific aspects, the reporter gene is a fluorescent protein. A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum (see Table 1 for non-limiting examples). Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.

In particular aspects, the cells of the disclosure may be specifically formulated and/or they may be cultured in a particular medium. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.

The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.

The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal- derived components, serum can be derived from the same animal as that of the stem cell(s). The serum-free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).

The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

In certain aspects, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HCI; Glutathione (reduced); L-Carnitine HCI; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HCI; Sodium Selenite; and/or T3 (triodo-l-thyronine). In specific aspects, one or more of these may be explicitly excluded.

In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alphatocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises proteins. In some aspects, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-l-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21 TM supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some aspects, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some aspects, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some aspects, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein and/or one or more proteins discussed herein, and/or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-l-thyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and/or phosphorus) or salts thereof, and/or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific aspects, one or more of these may be explicitly excluded.

The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and/or inorganic salts. In specific aspects, one or more of these may be explicitly excluded.

One or more of the medium components may be added at a concentration of at least, at most, or about 0.1 , 0.5, 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/L, ng/mL, pg/mL, mg/mL, or any range derivable therein.

In specific aspects, the cells of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cryopreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular aspects the cells are in a formulated cell suspension that is stable at room temperature for 1 , 2, 3, or 4 hours or more from time of thawing.

VI. RNA

With the exception of those derived from SARS-CoV-2, any of the coronavirus spike protein antigens disclosed herein can be in the form of RNA. In some aspects, the RNA molecule described herein is a coding RNA molecule. Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide. In some aspects, the coding RNA molecule includes at least one open reading frame (ORF) coding for at least one peptide or polypeptide. An open reading frame comprises a sequence of codons that is translatable into a peptide or protein. The coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) ORFs, which may be a sequence of codons that is translatable into a polypeptide or protein of interest.

The coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule, and/or self-amplifying RNA molecule (saRNA, also referred to as a replicon). In some aspects, the RNA molecule is an mRNA. Preferably, the RNA molecule of the present disclosure is an mRNA. In some aspects, the RNA molecule is a saRNA. In some aspects, the saRNA molecule may be a coding RNA molecule.

The RNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten or more polypeptides. Alternatively, or in addition, one RNA molecule may also encode more than one polypeptide of interest, such as an antigen, e.g., a bicistronic, or tricistron ic RNA molecule that encodes different or identical antigens. Bicistronic or multicistronic RNAs may include more than one polypeptide of interest with intervening sequences between the polypeptides of interest comprising an internal ribosome entry site (IRES) sequence(s) that allow for internal translation initiation between the polypeptides of interest, and/or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide. IRES sequences and 2A peptides may be used, in some aspects, to enhance expression of multiple proteins from the same vector. A variety of IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.

The sequence of the RNA molecule may be codon optimized or deoptimized for expression in a desired host, such as a human cell. In some aspects, a gene of interest (e.g., an antigen) described herein is encoded by a coding sequence which is codon-optimized and/or the guanosine/cytidine (G/C) content of which is increased compared to wild type coding sequence. In some aspects, one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In some aspects, codon-optimization and/or increasing the G/C content does not change the sequence of the encoded amino acid sequence.

As used herein, the term “codon-optimized” refers to modification of codons in the coding region of a nucleic acid molecule to accommodate the codon bias a host organism without a corresponding modification to the amino acid sequence encoded by the nucleic acid molecule. Codon optimization, in some aspects, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability and/or reduce secondary structures; minimize tandem repeat codons and/or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert and/or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove and/or shuffle protein domains; insert and/or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; and/or reduce or eliminate problem secondary structures within the polynucleotide. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing uses of codon optimization can be excluded. Within the context of the present disclosure, in some aspects, coding regions are codon-optimized for optimal expression in a subject to be treated using an RNA polynucleotide described herein.

Codon-optimization is based on the finding that the translation efficiency may be determined by a different frequency in the occurrence of transfer RNAs (tRNAs) in cells. Thus, if so-called “rare codons” are present in the coding region of the inventive artificial nucleic acid molecule as defined herein, to an increased extent, the translation of the corresponding modified nucleic acid sequence is less efficient than in the case, where codons coding for relatively “frequent” tRNAs are present. Thus, the open reading frame of the RNA molecule is modified compared to the corresponding wild type coding region such that at least one codon of the wild type sequence, which is recognized by a tRNA, and which is relatively rare in the cell, is exchanged for a codon, which is recognized by a tRNA, and which is comparably frequent in the cell and carries the same amino acid as the relatively rare tRNA. By this modification, the open reading frame of the RNA molecule is modified such that codons for which frequently occurring tRNAs are available may replace codons that correspond to rare tRNAs. Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely, is known to a person skilled in the art (see, e.g., Akashi, Curr. Opin. Genet. Dev. 2001 , 1 1 (6): 660-666), and codon optimization tools, algorithms and services are known in the art, and 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 optimization algorithms.

The sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression and/or replication of the RNA, to provide additional stability and/or resistance to degradation, and/or to reduce immunogenicity, relative to an unmodified RNA molecule. For example, the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA. In some aspects, one or more of the foregoing reasons for modification of the RNA molecule can be excluded.

In some aspects, the RNA molecule of the present disclosure comprises an open reading frame having at least one codon modified sequence. A codon modified sequence relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type coding sequence. A codon modified sequence may show improved resistance to degradation, improved stability, and/or improved translatability.

In some aspects, G/C content of a coding region (e.g., of a gene of interest sequence; open reading frame (ORF)) of an RNA is increased compared to the G/C content of the corresponding coding sequence of a wild type RNA encoding the gene of interest, wherein in some aspects, the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)ZC (cytidine) content are more stable than sequences having an increased A (adenosine)ZU (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability may be determined (so-called alternative codon usage).

Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A andZor U nucleosides may be modified by substituting these codons by other codons, which code for the same amino acids but contain no A andZor U or contain a lower content of A andZor U nucleosides. Thus, in some aspects, GZC content of a coding region of an RNA described herein is increased by at least, at most, exactly, or between (inclusive or exclusive) any two of 10%, 20%, 30%, 40%, 50%, 55%, or even more compared to the GZC content of a coding region of a wild type RNA. In some aspects, the coding region of the coronavirus RNA described herein comprises a GZC content of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some aspects, the coding region of the coronavirus RNA described herein comprises a GZC content of or of about 50% to 75%, 55% to 70%, 50% to 60%, 60% to 70%, 70% to 80%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, or 75% to 80%. In some aspects, the coding region of the coronavirus RNA described herein comprises a GZC content of or of about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, or 75%. In some aspects, the coding region of the coronavirus RNA described herein comprises a GZC content of or of about 58%, 66% or 62%.

In some aspects, the RNA molecule includes from or from about 20 to 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1 ,000, from 30 to 1 ,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1 ,000, from 100 to 1 ,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1 ,000, from 500 to 1 ,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1 ,000 to 1 ,500, from 1 ,000 to 2,000, from 1 ,000 to 3,000, from 1 ,000 to 5,000, from 1 ,000 to 7,000, from 1 ,000 to 10,000, from 1 ,000 to 25,000, from 1 ,000 to 50,000, from 1 ,000 to 70,000, from 1 ,000 to 100,000, from 1 ,500 to 3,000, from 1 ,500 to 5,000, from 1 ,500 to 7,000, from 1 ,500 to 10,000, from 1 ,500 to 25,000, from 1 ,500 to 50,000, from 1 ,500 to 70,000, from 1 ,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides).

In some aspects, the RNA molecule includes at least 100 nucleotides. For example, in some aspects, the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11 ,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; or between 7,000 and 25,000 nucleotides. In some aspects, the RNA molecule has at least, at most, exactly, between (inclusive or exclusive) any two of, or about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1000, 1050, 1100, 1150, 1200, 1250,

1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650, 12700, 12750, 12800, 12850, 12900, 12950, 13000, 13050,

13100, 13150, 13200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600,

13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150,

14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700,

14750, 14800, 14850, 14900, 14950, 15000, 16000, 18000, 20000, 22000, 24000,

26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 44000, 46000,

48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000, 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000 nucleotides.

The RNA molecules of the present disclosure may be prepared by any method know in the art, including chemical synthesis and in vitro methods, such as RNA in vitro transcription. In some of the aspects, the RNA of the present disclosure is prepared using in vitro transcription.

In some aspects, the RNA molecule of the present disclosure is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/diafiltration.

In some aspects, the RNA molecule of the present disclosure is lyophilized to be temperature stable.

In some aspects of the present disclosure, an RNA is or comprises messenger RNA (mRNA) that relates to an RNA transcript that encodes a polypeptide. In some aspects, an RNA disclosed herein comprises: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5' UTR); a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3' UTR); and/or a polyadenylate (poly-A) sequence. In some aspects, an RNA disclosed herein comprises the following components in the 5' to 3' orientation: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap proximal sequence (5' UTR), a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3' UTR); and a poly-A sequence. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing dosing regimens can be excluded.

In some aspects, an RNA disclosed herein further comprises a signal peptide. Non-limiting examples of signal peptides and amino acid and nucleic acid sequences encoding such peptides can be found in, e.g., WO 2018/170270, the disclosure of which is incorporated by reference herein in its entirety. In some aspects, an RNA disclosed herein encodes an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and/or protein fragment) joined together. Alternatively, the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to an antigen. Antigenic fusion proteins, in some aspects, retain the functional property from each original protein. In some aspects, an RNA disclosed herein encodes fusion proteins that comprise an antigen linked to a scaffold moieties. In some aspects, the RNA further encodes a linker located between at least one or each domain of the fusion protein. Non-limiting examples of such scaffold moieties and linkers can be found in, e.g., WO 2022/067010, the disclosure of which is incorporated by reference herein in its entirety.

A. Modified Nucleobases

In some aspects of the present disclosure, the RNA molecules are not chemically modified and comprise the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some aspects, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, and/or U). In some aspects, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, and/or dT).

In other aspects of the present disclosure, the RNA molecules may comprise modified nucleobases that may be incorporated into modified nucleosides and nucleotides. In some aspects, the RNA molecule may include one or more modified nucleotides. The terms "modification" and "modified", in regard to nucleic acids, refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) and/or cytidine (C) ribonucleosides and/or deoxyribonucleosides in at least one of their position, pattern, percent and/or population. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides and/or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, and/or nucleobase portion of the nucleotide and/or nucleoside as are 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. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891 ; PCT/US2014/070413;

PCT/US2015/36773; PCT/US2015/36759; and PCT/US2015/36771 ; or published international application No. PCT/IB2017/051367, all of which are incorporated by reference herein.

Hence, RNA molecules of the disclosure can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally- occurring nucleotides and nucleosides, or any combination thereof. RNA molecules, in some aspects, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some aspects, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

Modifications of RNA molecules include, without limitation, those described herein, and include, but are expressly not limited to, those modifications that comprise chemical modifications. RNA molecules may comprise modifications that are naturally- occurring or non-naturally-occurring, or the RNA molecule may comprise a combination of naturally-occurring and non-naturally-occurring modifications. RNA molecules may comprise non-natural modified nucleotides introduced during synthesis and/or post-synthesis of the RNA molecules to achieve desired functions and/or properties. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified. RNA molecules may include any useful modification, for example, of a sugar, a nucleobase, and/or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage and/or to the phosphodiester backbone).

The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a 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 “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, and/or recombinantly, to include one or more modified and/or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and/or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising nonstandard and/or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base and/or between two complementary non-standard base structures, such as, for example, in those polynucleotides having 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 base/sugar and/or linker may be incorporated into RNA molecules of the present disclosure.

In some aspects, the RNA molecule may include a modified nucleotide. Nonlimiting examples of modified nucleotides that may be included in the RNA molecule include pseudouridine, N1 -methylpseudouridine, 5-methyluridine, 3-methyl-uridine, 5- methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4- thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5- oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1 - carboxymethyl-pseudouridine, 5-carboxy hydroxymethyl-uridine, 5-carboxy hydroxy methyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5- methoxycarbonylmethyl-2 -thio-uridine, 5-aminomethyl-2 -thio-uridine, 5- methylaminomethyl-uridine, 1 -ethyl-pseudouridine, 5-methylaminomethyl-2 -thio- uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5- carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5- propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyl-uridine, 1 -taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -taurinomethyl-4-thio-pseudouridine, 5-methyl-2 -thio-uridine, 1 -methyl-4-thio-pseudouridine, 4-th io-1 -methyl- pseudouridine, 3-methyl-1 -pseudouridine, 2-thio-1 -methyl-pseudouridine, 1 -methyl-1 - deaza-pseudouridine, 2-th io-1 -methyl-1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 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, 3-(3-amino-3-carboxypropyl)uridine, 1 -methyl-3-(3-amino-3- carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5- (isopentenylaminomethyl)-2-thio-uridine, a-thio-uridine, 2'-O-methyl-uridine, 5,2'-O- dimethyl-uridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyl-uridine, 5- methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5- (isopentenylaminomethyl)-2'-O-methyl-uridine, 1 -thio-uridine, deoxythymidine, 2'-F- ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1 - E-propenylamino)uridine, any other modified uridine known in the art, or combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified nucleotides can be excluded from the RNA molecules disclosed herein.

Modifications that may be present in the RNA molecules further include, but are not limited to, e.g., the following: ms2io6A (2-methylthio-(N6-(cis- hydroxyisopentenyl)adenosine); ms2m6A (2-methylthio-N6-methyladenosine); ms2t6A 2-methylthio-N6-threonylcarbamoyladenosine; g6A (N6- glycinylcarbamoyladenosine); i6A (N6-isopentenyladenosine); m6A (N6- methyladenosine); t6A (N6-threonylcarbamoyladenosine); nTAm (1 ,2'-O- dimethyladenosine); m1A (1 -methyladenosine); 2'-O-methyladenosine; Ar(p) (2'-O- ribosyladenosine (phosphate)); 2-m ethyl adenosine; 2-methylthio-N6 isopentenyladenosine; ms2hn6A (2-methylthio-N6- hydroxynorvalylcarbamoyladenosine); 2-O-methyladenosine; Am (2-1 -0- methyladenosine); 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; io6A N6-(cis-hydroxyisopentenyl)adenosine; m6Am (N6,2'-O-dimethyladenosine); m62Am (N6,N6,2'-O-trimethyladenosine); m62A (N6,N6-dimethyladenosine); ac6A (N6- acetyladenosine); hn6A (N6-hydroxynorvalylcarbamoyladenosine); m6t6A (N6- methyl-N6-threonylcarbamoyladenosine); m2A (2-methyladenosine); ms2i6A (2- methylthio-N6-isopentenyladenosine); 7-deaza-adenosine; N1 -methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2- (alkyl)adenine; 2-(aminoalkyl)adenine; 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; 8-oxo-adenine; aza adenine; deaza adenine; 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; 2'0-methyl-N6-Bz- deoxyadenosine TP; 2'-a-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine

TP; 2-amino-ATP; 2'-a-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2'-b-

Ethynyladenosine TP; 2-bromoadenosine TP; 2'-b-trifluoromethyladenosine 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- Trifluoromethyladenosine TP; 9-deazaadenosine TP; 2-aminopurine; substituted 7- deazapurine; 7-deaza-7-substituted purine; 7-deaza-8-substituted purine; 7-deaza- 2,6-diaminopurine; 7-deaza-8-aza-2,6-diam inopurine; 7-deaza-8-aza-2-am inopurine; 2,4-diaminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine; 7-deaza-2- aminopurine; 8-azapurine; s2C (2-thiocytidine); m3C (3 -methylcytidine); f5C (5- formylcytidine); hm5C (5-hydroxymethylcytidine); m5C (5-methylcytidine); ac4C (N4- acetylcytidine); Cm (2'-O-methylcytidine); m5Cm (5,2'-O-dimethylcytidine); f5Cm (5- formyl-2'-O-methylcytidine); k2C (Lysidine); m4Cm (N4,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine); m4C (N4-methylcytidine); N4,N4-dimethyl-2'-OMe- Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; a- thio-cytidine; 2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'- deoxy-2'-a-aminocytidine TP; 2'-deoxy-2'-a-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-chlorocytosine; 5-fluorocytosine; 5- bromocytosine; 5-hydroxycytosine; 5-methylcytosine; 5-(alkyl)cytosine; 5- (alkenyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5-(propynyl)cytosine; 5- (trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6- (azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)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; Zebularine; (E)-5-(2- Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'fluor-N4-Bz- cytidine TP; 2'fluoro-N4-Acetyl-cytidine TP; 2'-O-methyl-N4-Acetyl-cytidine TP; 2'0- 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'-ethynyl cytidine TP; 5-(1 - propynyl)ara-cytidine TP; 5-(2-chloro-phenyl)-2 -thiocytidine TP; 5-(4-Amino-phenyl)- 2-thiocytidine TP; 5-Aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylara-cytidine TP; 5- Ethynylcytidine TP; 5'-Homo-cytidine TP; 5-m ethoxycytidine TP; 5-Trifluoromethyl- Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; mimG (methylguanosine); m7G (7-methylguanosine); m2Gm (N2,2'-O- dimethylguanosine); m2G (N2-methylguanosine); imG (Wyosine); m1 Gm (1 ,2'-0- dimethylguanosine); m1 G (1 -methylguanosine); 2'-O-methylguanosine; 2'-0- ribosylguanosine (phosphate); Gm (2'-O-methylguanosine); Gr(p) (2'-O-ribosyl guanosine (phosphate)); preQi (7-aminomethyl-7-deazaguanosine); preQo (7-cyano- 7 -deazaguanosine); G* (Archaeosine); methylwyosine; m2'7G (N2,7- dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); m2'2'7G (N2,N2,7- trimethylguanosine); m22G (N2,N2-dimethylguanosine); N2,7,2'-O- trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1 - methyl-guanosine; a-thio-guanosine; 2 (propyl)guanine; 2-(alkyl)guanine; 2'-amino-2'- deoxy-GTP; 2'-azido-2'-deoxy-GTP; 2'-deoxy-2'-a-aminoguanosine TP; 2'-deoxy-2'-a- azidoguanosine TP; N2-dimethylguanine; 6-(methyl)guanine; 6-(alkyl)guanine; 6- (methyl)guanine; 6-methyl-guanosine; 6-thioguanine; 7 (alkyl)guanine; 7-deaza-7- substituted guanine; 7-deaza-7-(C2-c6)alkynylguanine; 7-deaza-8-substituted guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8-azaguanine; 8-hydroxyguanine; 8-oxoguanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8- (alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8- (thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; 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; 2'0-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 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; mil (1 -methylinosine); I (Inosine); m'lm (1 ,2'-O-dimethylinosine); 2'-O- methylinosine; 7-methylinosine; Tm (2'-O-methylinosine); oQ (Epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosylqueuosine); Q (Queuosine); allyamino- thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; Um (2'-O- methyluridine); s2U (2-thiouridine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); ho5U (5-hydroxyuridine); m5U (5-methyluridine); tm5s2U (5- taurinomethyl-2-thiouridine); 5-taurinomethyluridine; D (dihydrouridine); pseudouridine; acp3U (3-(3-amino-3-carboxypropyl)uridine); 1 -methyl-3-(3-amino-5- carboxypropyl)pseudouridine; 1 -methylpseudouridine; 1 -ethyl-pseudouridine; 2'-O- methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; s2Um (2-thio-2'-O- methyluridine); 3-(3-amino-3-carboxypropyl)uridine; m3Um (3,2'-O-dimethyluridine); 3-methyl-pseudo-Uridine TP; s4U (4-thiouridine); chm5U (5- (carboxyhydroxymethyl)uridine); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); m5Um (5,2'-O-dimethyluridine); 5,6-dihydro-uridine; nm5s2U (5-aminomethyl- 2-thiouridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); ncm5U (5- carbamoylmethyluridine); 5-carboxyhydroxymethyluridine; 5- carboxyhydroxymethyluridine methyl ester; cnmm5Um (5- carboxymethylaminomethyl-2'-O-methyluridine); cmnm5s2U (5- carboxymethylaminomethyl-2 -thiouridine); 5-carboxymethylaminomethyluridine; cmnm5U (5-carboxymethylaminomethyluridine); 5-Carbamoylmethyluridine TP; mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl-2 -thiouridine); mcm5U (5-methoxycarbonylmethyluridine); mo5U (5-methoxyuridine); m5s2U (5-methyl-2-thiouridine); mnm5se2U (5- methylaminomethyl-2-selenouridine); mnm5s2U (5-methylaminomethyl-2- thiouridine); mnm5U (5-methylaminomethyluridine); m5D (5-methyldihydrouridine); 5- Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; dihydrouracil; pseudouracil; N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; cmo5U (undine 5- oxyacetic acid); mcmo5U (undine 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-propynyl uracil; a-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)- pseudouracil; 1 (aminoalkylamino-carbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminoalkylamino-carbonylethylenyl)-4 (thio)pseudouracil; 1 (aminoalkylamino- carbonylethylenyl)-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; 1-ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 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 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil; 4- (thio)uracil; 4-thiouracil; 5-aminouracil; 5 (1 ,3-diazole-1-alkyl)uracil; 5 (2- aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (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-(alkenyl)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-(methoxycarbonylmethyl)-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; allyamino- uracil; aza uracil; deaza uracil; 5-methyluracil; 5-(hydroxymethyl)uracil; 5-chlorouracil; 5-fluorouracil; 5-bromouracil; N3 (methyl)uracil; pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1 -carboxymethyl-pseudouridine; 1 -methyl-1 - deaza-pseudouridine; 1 -propynyl-uridine; 1 -taurinomethyl-1 -methyl-uridine; 1 - taurinomethyl-4-thio-uridine; 1 -taurinomethyl-pseudouridine; 2-methoxy-4-thio- pseudouridine; 2-th io-1 -methyl-1 -deaza-pseudouridine; 2-th io-1 -methylpseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio- dihydrouridine; 2-thio-pseudouridine; 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- Trimethyl-benzyl)pseudo-UTP; 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)pseudo-UTP; 1 -(3-Amino-propyl)pseudo-UTP; 1 -(3- Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1 -(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP; 1-(4-Amino-butyl)pseudo-UTP; 11 (4-Amino- phenyl)pseudo-UTP; 1-(4-azidobenzyl)pseudouridine TP; 1 -(4- Bromobenzyl)pseudouridine TP; 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)pseudo-UTP; 1 -(4-methoxy-phenyl)pseudo-UTP; 1 -(4- methylbenzyl)pseudouridine TP; 1 -(4-methyl-benzyl)pseudo-UTP; 1 -(4- nitrobenzyl)pseudouridine TP; 1 -(4-Nitro-benzyl)pseudo-UTP; 1 (4-Nitro- phenyl)pseudo-UTP; 1-(4-thiomethoxybenzyl)pseudouridine TP; 1 -(4-

Trifluoromethoxybenzyl)pseudouridine TP; 1 -(4-trifluoromethylbenzyl)pseudouridine TP; 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- allylpseudouridine TP; 1 -Aminomethyl-pseudo-UTP; 1 -benzoylpseudouridine TP; 1 - benzyloxymethylpseudouridine TP; 1-benzyl-pseudo-UTP; 1 -biotinyl-PEG2- pseudouridine TP; 1-biotinylpseudouridine TP; 1-butyl-pseudo-UTP; 1 - cyanomethylpseudouridine TP; 1 -cyclobutylmethyl-pseudo-UTP; 1 -cyclobutyl- pseudo-UTP; 1-cycloheptylmethyl-pseudo-UTP; 1-cycloheptyl-pseudo-UTP; 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 -iso-propyl-pseudo-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-pseudo-UTP; 1 -Phenyl-pseudo-UTP; 1- pivaloylpseudouridine TP; 1 -propargylpseudouridine TP; 1 -propyl-pseudo-UTP; 1 - propynyl-pseudouridine; 1 -p-tolyl-pseudo-UTP; 1-tert-butyl-pseudo-UTP; 1 - thiomethoxymethylpseudouridine TP; 1 -thiomorpholinomethylpseudouridine TP; 1 - trifluoroacetylpseudouridine 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 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-vinylarauridine 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-Ethoxy-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-lodo- pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-methoxy-pseudo-UTP; 6-methylamino- pseudo-UTP; 6-methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP;

6-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; yW (Wybutosine); OHyW (Hydroxywybutosine); imG2 (isowyosine); o2yW (Peroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG-14 (4-demethylwyosine); 2,6- (diamino)purine; 1 -(aza)-2-(thio)-3-(aza)-phenoxazin-1 -yl: 1 ,3-(diaza)-2-(oxo)- phenthiazin-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-aza- inosinyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified bases; 2'- O-methyl-ribose; 2-oxo-7-arninopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2- pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl; 3- (methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(m ethyl )indolyl; 4,6-(dimethyl)indolyl; 5 nitroindole; 5 substituted pyrimidines; 5- (methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-

7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; 7-

(aminoalkylhydroxy)-l -(aza)-2-(thio)-3-(aza)-phenthiazin-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)-phenthiazin-1 -yl; 7-

(aminoalkylhydroxy)-l ,3-(diaza)-2-(oxo)-phenoxazin-1 -yl; 7-(aza)indolyl; 7-

(guanidiniumalkylhydroxy)-1 -(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7-

(guanidiniumalkylhydroxy)-l -(aza)-2-(thio)-3-(aza)-phenthiazin-1 -yl; 7- (guanidiniumalkylhydroxy)-l -(aza)-2-(thio)-3-(aza)-phenoxazin-1 -yl; 7-

(guanidiniumalkylhydroxy)-l ,3-(diaza)-2-(oxo)-phenoxazin-1 -yl; 7-(guanidiniumalkyl- hydroxy)-1 ,3-(diaza)-2-(oxo)-phenthiazin-1 -yl; 7-(guanidiniumalkylhydroxy)-1 ,3- (diaza)-2-(oxo)-phenoxazin-1 -yl; 7-(propynyl)isocarbostyrilyl; 7-

(propynyl)isocarbostyrilyl; propynyl-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-on-3-yl; bis-ortho-substituted-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl; difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; isoguanisine; N2-substituted purines; N6-methyl-2 -am inopurine; N6-substituted purines; N-alkylated derivative; napthalenyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; 06- substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; pentacenyl; phenanthracenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2- oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; pyrrolopyrimidinyl; pyrrolopyrizinyl; stilbenzyl; substituted 1 ,2,4-triazoles; tetracenyl; tubercidine; 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 A TP; formycin B TP; pyrrolosine TP; 2'-OH-ara-adenosine TP; 2'-0H-ara-cytidine TP; 2'- OH-ara-uridine TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; N6- (19-Amino-pentaoxanonadecyl)adenosine TP; hydrogen (abasic residue); and 2'-0- methyl-U. In some aspects, RNA molecules include a combination of at least two (e.g.,

2, 3, 4, or more) of the aforementioned modified nucleobases. In some aspects, 1 , 2,

3, 4, 5, or more of the foregoing modifications can be excluded from the RNA molecules disclosed herein.

In some aspects, modified nucleobases in RNA molecules comprise pseudouridine (ip), 2-thiouridine (s2U), 4 -thiouridine, 5-methylcytosine, 2-thio-1 - methyl-1 -deaza-pseudouridine, 2-th io-1 -methyl-pseudouridine, 2-thio-5-aza-uridine,

2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy- 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 -methyl-pseudouridine, 4- thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine, 2'-O-methyl uridine, 1 -methyl-pseudouridine (m1 i ), 1 -ethyl- pseudouridine (e1 i ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), a-thio- guanosine, a-thio-adenosine, 5-cyanouridine, 4'-thio uridine 7-deaza-adenine, 1 - methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2,6- Diaminopurine, inosine (I), 1 -methyl-inosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7- deaza-guanosine (preQi), 7-methyl-guanosine (m7G), 1 -methyl-guanosine (m1 G), 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2- geranylthiouridine, 2-lysidine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6- dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-am inomethyl-2 - geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5- carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2 -thiouridine, 5-carboxymethyl- 2 -thiouridine, 5-carboxymethylaminomethyl-2 -geranylthiouridine, 5- carboxymethylam inomethyl-2 -selenouridine, 5-cyanomethyluridine, 5- hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl- demethylwyosine, 7-aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyl adenosine, N6- hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queuosine, methylated undermodified hydroxywybutosine, N4,N4,2'-O- trimethylcytidine, geranylated 5-methylam inomethyl-2 -thiouridine, geranylated 5- carboxymethylam inomethyl-2 -thiouridine, Qbase, preQObase, preQIbase, and combinations of two or more thereof. In some aspects, the RNA molecule includes a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases, including but not limited to chemical modifications. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified nucleobases can be excluded from the RNA molecules disclosed herein.

Exemplary nucleobases and nucleosides having a modified cytosine 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-hydroxymethyl-cytidine (hm5C), 1 - methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1 -methyl- pseudoisocytidine, 4-th io-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), a- thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-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. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified cytosines can be excluded from the RNA molecules disclosed herein.

In some aspects, a modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ip), 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), 5-cyanouridine, 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), undine 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 (cmnmVU), 5-propynyl-uridine, 1 - propynyl-pseudouridine, 5-taurinomethyl-uridine (xm5U), 1 -taurinomethyl- pseudouridine, 5-taurinomethyl-2 -thio-uridine (xmVu), 1 -taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine (m5U, e.g., having the nucleobase deoxythymine), 1 - methyl-pseudouridine (ml ^P), 1 -ethyl-pseudouridine (e1 i ), 5-methyl-2 -thio-uridine (m5s2U), 1 -methyl-4-thio-pseudouridine (m1s4M^J), 4-thio-1 -methyl-pseudouridine, 3- methyl-pseudouridine (mSMJ), 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, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1 -methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3 ip), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl- uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (^Pm), 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-(l-E-propenylamino)]uridine. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified uridines can be excluded from the RNA molecules disclosed herein.

In some aspects of the present disclosure, modified nucleotides include any one of N1 -methylpseudouridine and/or pseudouridine.

In some aspects, the RNA molecule comprises nucleotides that are N1 - methylpseudouridine modified. In some aspects, the RNA molecule comprises nucleotides that are pseudouridine modified.

In some aspects, an RNA comprises a modified nucleoside in place of at least one uridine. In some aspects, an RNA comprises a modified nucleoside in place of each uridine. In some aspects, the RNA molecule comprises a sequence having at least one uridine replaced by N1 -methylpseudouridine. In some aspects, the RNA molecule comprises a sequence having all uridines replaced by N1 - methylpseudouridine. N1 -methylpseudouridine is designated in sequences as “ ”. The term “uracil,” as used herein, describes one of the nucleobases that may occur in the nucleic acid of RNA. The term “uridine,” as used herein, describes one of the nucleosides that may occur in RNA. “Pseudouridine” is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

In some aspects, the RNA molecule comprises a nucleic acid sequence having at least one undine replaced by N1 -methylpseudouridine and/or pseudouridine. In some aspects, the RNA molecule comprises a nucleic acid sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or 99% of uridines replaced by N1 -methylpseudouridine and/or pseudouridine. In some aspects, the RNA molecule comprises a nucleic acid sequence having all uridines replaced by N1 -methylpseudouridine and/or pseudouridine.

In some aspects, a modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine 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 -am inopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine (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-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, a-thio-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. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified adenines can be excluded from the RNA molecules disclosed herein.

In some aspects, a modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1 -methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 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 -methylguanosine (m1 G), 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, a-thio-guanosine, 2'-O-methyl- guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O- methyl-guanosine (m22Gm), 1 -methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O- methyl-guanosine (m2'7Gm), 2'-O-methyl-inosine (Im), 1 ,2'-O-dimethyl-inosine (m1 lm), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, O6-methyl- guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing modified guanines can be excluded from the RNA molecules disclosed herein.

In some aspects, RNA molecules are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. In some aspects, the RNA molecules may be partially or fully (e.g., uniformly) modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine and/or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof. In some aspects, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, and/or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C and/or A+G+C. For example, a polynucleotide can be uniformly modified with pseudouridine, meaning that all uridine residues in the RNA sequence are replaced with pseudouridine. Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above. The modified nucleotide can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4, or more unique structures).

The RNA molecules may contain from or from about 1 % to 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, e.g., any one or more of A, G, U and/or C) (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34,

35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57,

58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80,

81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100%) or any intervening percentage e.g., from 1 % to 20%, from 1 % to 25%, from 1 % to 50%, from 1 % to 60%, from 1 % to 70%, from 1 % to 80%, from 1 % to 90%, from 1 % to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90%) to 100%), and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, and/or C.

In some aspects, the RNA molecule may include phosphoram idate, phosphorothioate, and/or methylphosphonate linkages.

In some aspects, the RNA molecules may include one or more structural and/or chemical modifications and/or alterations which impart useful properties to the polynucleotide including, in some aspects, reduced degradation in the cell or organism and/or lack of a substantial induction of the innate immune response of a cell into which the RNA molecule is introduced. As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted and/or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to affect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

In some aspects, a modified RNA molecule, introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. In some aspects, a modified RNA molecule, introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some aspects, the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some aspects, the RNA may include a 5' cap comprising a 7’-methylguanosine, and the first 1 , 2, or 3 5' ribonucleotides may be methylated at the 2’ position of the ribose.

B. 5' Cap

In some aspects, the RNA molecule described herein includes a 5' cap which generally “caps” the 5' end of the RNA and stabilizes the RNA molecule.

In some aspects, the 5' cap moiety is a natural 5' cap. A “natural 5' cap” is defined as a cap that includes 7-methylguanosine connected to the 5' end of an mRNA molecule through a 5' to 5' triphosphate linkage. In some aspects, a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose. In some aspects, a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'0MeG). In some aspects, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G). In some aspects, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3'0 methylation at a ribose (m7(3'OMeG)). The 5' cap may be incorporated during RNA synthesis (e.g., co- transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some aspects, co-transcriptional capping with a cap disclosed herein improves the capping efficiency of an RNA compared to co- transcriptional capping with an appropriate reference 5' cap. In some aspects, improving capping efficiency may increase the translation efficiency and/or translation rate of an RNA and/or increase expression of an encoded polypeptide. In some aspects, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.

In some aspects, an RNA described herein comprises a 5' cap or a 5' cap analog, e.g., a Cap 0, a Cap 1 or a Cap 2. In some aspects, a provided RNA does not have uncapped 5'-triphosphates. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide. In some aspects, the 5' cap moiety is a 5' cap analog. In some aspects, an RNA may be capped with a 5' cap analog. Cap structures include, but are not limited to, ⁷mG(5')ppp(5')NipN2p (Cap 0), ⁷mG(5')ppp(5')Ni^mpNp (Cap 1 ), and ⁷rnG(5')ppp(5')Ni^mpN2^mp (Cap 2). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cap structures can be excluded from the RNA molecules disclosed herein.

In some aspects, an RNA described herein comprises a Cap 0. In some aspects, Cap 0 is a N7-methyl guanosine, and a Cap 0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G). In some aspects, a Cap 0 structure is connected to an RNA via a 5' to 5'-triphosphate linkage and is also referred to herein as m7G, m7Gppp, and/or m7G(5')ppp(5'). A 5' cap may be methylated with the structure ⁷mG(5')ppp(5')NipN2p (Cap 0) or a derivative thereof, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5'-end of an mRNA. An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyl-transferase and guanine-7-methytransferase, which catalyzes the construction of N7-monomethylated Cap 0 structures. Cap 0 structures play an important role in maintaining the stability and translational efficacy of the RNA molecule. In the cell, the Cap 0 structure is essential for efficient translation of the mRNA that carries the cap.

In some aspects, an RNA described herein comprises a Cap 1 , e.g., as described herein. The 5' cap of the RNA molecule may be further modified on the 2'0 position by a 2'-O-methyltransferase, which results in the generation of a Cap 1 structure (m7Gppp [m2'-O] N), which may further increase translation efficacy. In some aspects, a Cap 1 structure comprises a guanosine nucleoside methylated at the 7- position of guanine (m7G) and a 2'0 methylated first nucleotide in an RNA (2'0MeNi). In some aspects, a Cap 1 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as m7GpppN^m, wherein N^m denotes any nucleotide with a 2'0 methylation, ⁷mG(5')ppp(5')Ni^mpNp, m7Gppp(2'OMeNi), and/or m7G(5')ppp(5')(2'OMeNi). In some aspects, Ni is chosen from A, C, G, or U. In some aspects, Ni is A. In some aspects, Ni is C. In some aspects, Ni is G. In some aspects, Ni is U. In some aspects, a m7G(5')ppp(5')(2'OMeNi) Cap 1 structure comprises a second nucleotide, N2, which is a cap proximal nucleotide at position 2 and is chosen from A, G, C, or U (m7G(5')ppp(5')(2'OMeNi)N2). In some aspects, N2 is A. In some aspects, N2 is C. In some aspects, N2 is G. In some aspects, N2 is U.

In some aspects, a Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and one or more additional modifications, e.g., methylation on a ribose, and a 2'0 methylated first nucleotide in an RNA. In some aspects, a Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine, a 3'0 methylation at a ribose (m7(3'OMeG)), and a 2'0 methylated first nucleotide in an RNA (2'0MeNi). In some aspects, a Cap 1 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as m7(3'OMeG)ppp(2'OMeNi) and/or m7(3'OMeG)(5')ppp(5')(2'OMeNi). In some aspects, Ni is chosen from A, C, G, or U. In some aspects, Ni is A. In some aspects, Ni is C. In some aspects, Ni is G. In some aspects, Ni is U. In some aspects, a m7(3'OMeG)(5')ppp(5')(2'OMeNi) Cap 1 structure comprises a second nucleotide, N2, which is a cap proximal nucleotide at position 2 and is chosen from A, G, C, or U (m7(3'OMeG)(5')ppp(5')(2'OmeNi)N2). In some aspects, N2 is A. In some aspects, N2 is C. In some aspects, N2 is G. In some aspects, N2 is U. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing Cap 1 structures can be excluded from the RNA molecules disclosed herein.

In some aspects, a second nucleotide in a Cap 1 structure may comprise one or more modifications, e.g., methylation. In some aspects, an RNA described herein comprises a Cap 2. In some aspects, a Cap 1 structure comprising a second nucleotide comprising a 2'0 methylation is a Cap 2 structure.

In some aspects, the RNA molecule may be enzymatically capped at the 5' end using Vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L- methionine to yield Cap 0 structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, use of a 2'0-methyltransferase with Vaccinia guanylyltransferase yields the Cap 1 structure where, in addition to the Cap 0 structure, the 2'0H group is methylated on the penultimate nucleotide. S-adenosyl- L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. Non-limiting examples of 5' cap structures are those which, among other things, have enhanced binding of cap-binding polypeptides, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping, as compared to synthetic 5' cap structures known in the art (or to a wild type, natural or physiological 5' cap structure).

For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2' O-methyltransferase enzyme may create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5'-terminal nucleotide of the mRNA includes a 2'-O-methyl. Such a structure is termed the Cap 1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5' cap analog structures known in the art.

A cap species may include one or more modified nucleosides and/or linker moieties. For example, a cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species may also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, and m27,O2'GppppG. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cap species can be excluded from the RNA molecules disclosed herein.

In some aspects, the 5' terminal cap includes a cap analog, for example, a 5' terminal cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1 -methyl-guanosine, 2'-fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing guanine analogs can be excluded from the cap structures disclosed herein.

In some aspects, the capping region may include a single cap or a series of nucleotides forming the cap. In this aspect the capping region may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1 -5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In this aspect, the capping region is at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some aspects, the cap is absent. In some aspects, the first and second operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences. In some aspects, the first and second operational regions are at least, at most, exactly, or between (inclusive or exclusive) any two of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.

Further examples of 5' cap structures include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, 1 -(beta-D- erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1 ,5- anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4- dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'- inverted abasic moiety, 1 ,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphoroth ioate, phosphorodithioate, and/or bridging or non-bridging methylphosphonate moiety. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 5' cap structures can be excluded from the RNA molecules disclosed herein.

In some aspects, the RNA molecule of the present disclosure comprises at least one 5' cap structure. In some aspects, the RNA molecule of the present disclosure does not comprise a 5' cap structure.

Numerous synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology 69 (Rabinovich, P.H. Ed), 2013). In one aspect, the 5' capping structure comprises a modified 5' Cap 1 structure (m⁷G⁺m3'-5'-ppp-5'-Am). In one aspect, the 5' capping structure comprises is (3'OMe)-m2⁷’3'-^oGppp(mi²’’°)ApG (TriLink BioTechnologies). This molecule is identical to the natural RNA cap structure in that it starts with a guanosine methylated at N7, and is linked by a 5' to 5' triphosphate linkage to the first coded nucleotide of the transcribed RNA (in this case, an adenosine). This guanosine is also methylated at the 3' hydroxyl of the ribose to mitigate possible reverse incorporation of the cap molecule. The 2’ hydroxyl of the ribose on the adenosine is methylated, conferring a Cap 1 structure.

C. Untranslated Regions (UTRs)

The 5' UTR is a regulatory region situated at the 5' end of a protein open reading frame that is transcribed into mRNA but not translated into an amino acid sequence and/or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) may be present 5' (upstream) of an open reading frame (5' UTR) and/or 3' (downstream) of an open reading frame (3' UTR).

In some aspects, the UTR is derived from an mRNA that is naturally abundant in a specific tissue (e.g., lymphoid tissue), to which the mRNA expression is targeted. In some aspects, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR may increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and/or the rate at which ribosomes initiate translation on the message (message translation efficiency). Accordingly, the UTR sequence may prolong protein synthesis in a tissue-specific manner.

In some aspects, the regulatory features of a UTR can be incorporated into the RNAs of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled downregulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5' UTR and the 3' UTR sequences are known and available in the art.

It should be understood that any UTR from any gene may be incorporated into the regions of the RNAs of the present disclosure. Furthermore, 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 which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation and/or location. Hence a 5' and/or 3' UTR may be inverted, shortened, lengthened, and/or made with one or more other 5' UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, 5' UTRs and/or 3' UTRs may be altered relative to a wild-type or native UTR by the change in orientation and/or location as taught above and/or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping, and/or transposition of nucleotides. Any of these changes produces an “altered” UTR (whether 5' and/or 3') including a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5' and/or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

RNAs may encode polypeptides of interest belonging to a family of proteins that are expressed in a particular cell, tissue and/or at some time during development. In some aspects, the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new RNA molecule. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, and/or expression pattern.

In some aspects, the 5' UTR and the 3' UTR sequences are computationally derived. In some aspects, the 5' UTR and the 3' UTRs are derived from a naturally abundant mRNA in a tissue. The tissue may be, for example, liver, a stem cell and/or lymphoid tissue. The lymphoid tissue may include, for example, any one of a lymphocyte (e.g., a B-lymphocyte, a helper T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, and/or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil and a reticulocyte. In some aspects, the 5' UTR and the 3' UTR are derived from an alphavirus. In some aspects, the 5' UTR and the 3' UTR are from a wild type alphavirus.

In some aspects, untranslated regions may also include translation enhancer elements (TEE). As a non- limiting example, the TEE may include those described in US Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.

1. 5' UTRs

In some aspects, an RNA disclosed herein comprises a 5' UTR. A 5' UTR, if present, is located at the 5' end and starts with the transcriptional start site upstream of the start codon of a protein encoding region. A 5' UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. The 5' UTR may contain various regulatory elements, e.g., 5' cap structure, stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 5' UTR may harbor signatures like Kozak sequences, which are also involved in the process by which the ribosome initiates translation of many genes.

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

In some aspects, a Cap structure comprises one or more polynucleotides of a cap proximal sequence. In some aspects, a Cap structure comprises an m7 Guanosine cap and nucleotide +1 (Ni) of an RNA polynucleotide. In some aspects, a Cap structure comprises an m7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some aspects, a Cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (N1 and N2) of an RNA polynucleotide.

Those skilled in the art, reading the present disclosure, will appreciate that, in some aspects, one or more residues of a cap proximal sequence (e.g., one or more of residues +1 , +2, +3, +4, and/or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a Cap 1 structure, etc)', alternatively, in some aspects, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified aspects where a (m2⁷’³'’^o)Gppp(m²’’°)ApG cap is utilized, +1 and +2 residues are the (m2⁷’³’’⁰) A and G residues of the cap, and +3, +4, and +5 residues are added by polymerase (e.g., T7 polymerase).

In some aspects, a cap proximal sequence comprises N1 and/or N2 of a Cap structure, wherein N1 and N2 are any nucleotide, e.g., A, C, G or U. In some aspects, N1 is A. In some aspects, N1 is C. In some aspects, N1 is G. In some aspects, N1 is U. In some aspects, N2 is A. In some aspects, N2 is C. In some aspects, N2 is G. In some aspects, N2 is U. In some aspects, a cap proximal sequence comprises N1 and N2 of a Cap structure and N3, N4 and Ns, wherein N1 to Ns correspond to positions +1 , +2, +3, +4, and/or +5 of an RNA polynucleotide. In some aspects, N1 , N2, N3, N4, or Ns are any nucleotide, e.g., A, C, G or U. In some aspects, N1 and N2 comprise any one of the following: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, or UU. In some aspects, N1N2 comprises AG and N3N4N5 comprises any one of the following:AAA, ACA, AGA, AUA, AAG, AGG, ACG, AUG, AAC, ACC, AGC, AUC, AAU, ACU, AGU, AUU, CAA, CCA, CGA, CUA, CAG, CGG, CCG, CUG, CAC, CCC, CGC, CUC, CAU, CCU, CGU, CUU, GAA, GCA, GGA, GUA, GAG, GGG, GCG, GUG, GAC, GCC, GGC, GUC, GAU, GCU, GGU, GUU, UAA, UCA, UGA, UUA, UAG, UGG, UCG, UUG, UAC, UCC, UGC, UUC, UAU, UCU, UGU, or UUU.

In some aspects, a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising: A3A4X5, wherein Xs is A, G, C, or U, and where N1 and N2 are each independently chosen from: A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, Xs is chosen from A, C, G or U. In some aspects, Xs is A. In some aspects, Xs is C. In some aspects, Xs is G. In some aspects, Xs is U.

In some aspects, a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising: C3A4X5, where Xs is A, G, C, or U, and where N1 and N2 are each independently chosen from: A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, Xs is chosen from A, C, G or U. In some aspects, Xs is A. In some aspects, Xs is C. In some aspects, Xs is G. In some aspects, Xs is U.

In some aspects, a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising X3Y4X5, where X3 or Xs are each independently chosen from A, G, C, or U, and Y4 is not C. In some aspects, N1 and N2 are each independently chosen from: A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, X3 and Xs is each independently chosen from A, C, G or U. In some aspects, X3 and/or Xs is A. In some aspects, X3 and/or Xs is C. In some aspects, X3 and/or Xs is G. In some aspects, X3 and/or Xs is U. In some aspects, Y4 is C. In other aspects, Y4 is not C. In some aspects, Y4 is A. In some aspects, Y4 is G. In other aspects, Y4 is not G. In some aspects, Y4 is U.

In some aspects, a cap proximal sequence comprises N1 and N2 of a Cap structure, and a sequence comprising A3C4A5. In some aspects, N1 and N2 are each independently chosen from: A, C, G, or U. In some aspects, N1 is A and N2 is G. In some aspects, a cap proximal sequence comprises Ni and N2 of a Cap structure, and a sequence comprising A3I G5. In some aspects, N1 and N2 are each independently chosen from: A, C, G, or U. In some aspects, N1 is A and N2 is G.

In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cap proximal sequences can be excluded from the 5' UTR of the RNA molecules disclosed herein.

In some aspects of the disclosure, a 5' UTR is a heterologous UTR, e.g., is a UTR found in nature associated with a different ORF. In another aspect, a 5' UTR is a synthetic UTR, e.g., does not occur in nature. Synthetic UTRs include UTRs that have been mutated or synthesized to improve their properties, e.g., to increase gene expression. In some aspects, the 5' UTR is functionally linked to the ORF, e.g., associated with the ORF such that it may exert a function, e.g., increasing, enhancing, stabilizing, and/or prolonging protein production from an RNA molecule and/or increasing protein expression and/or total protein production from an RNA molecule, compared to a reference RNA molecule comprising a reference 5' UTR or an RNA molecule lacking a 5' UTR. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 5' UTR functions can be excluded.

Exemplary 5' UTRs include 5' UTRs derived from Xenopus or human alpha globin or beta globin, human cytochrome b-245 a, hydroxysteroid (17b) dehydrogenase, Tobacco etch virus, the CMV immediate-early 1 (IE1 ) gene, TEV, HSP705', c-Jun, or a homolog, fragment, or variant of any of the foregoing. In some aspects, the 5' UTR is a fragment, homolog or variant of a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract), the 5' UTR derived from ribosomal protein Large 32 (L32) gene, the 5' UTR derived from the 5' UTR of an hydroxysteroid (17p) dehydrogenase 4 gene (HSD17B4), or the 5' UTR derived from the 5' UTR of ATP5A1. In some aspects, 5' UTRs are derived from SEQ ID NOs: 1 -1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application WO201 3/143700, the disclosure of which is incorporated herein by reference in its entirety, or a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing sequences.

In some aspects, the 5' UTR comprises a sequence from the 5' UTR region of a gene encoding RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS1 1 , RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21 , RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL1 1 , RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21 , RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31 , RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41 , RPLPO, RPLP1 , RPLP2, RPLP3, RPLPO, RPLP1 , RPLP2, EEF1A1 , EEF1 B2, EEF1 D, EEF1 G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1 , HNRNPA1 , TPT1 , TUBB1 , UBA52, NPM1 , ATP5G2, GNB2L1 , NME2, UQCRB, or from a homolog, fragment, or variant thereof, or a gene sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing gene sequences. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 5' UTR sequences may be excluded from the RNA molecules disclosed herein.

2. 3' UTRs

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

Natural or wild type 3' UTRs comprise stretches of adenosines and undines. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Class III ARES do not contain an AUUUA motif. Most proteins binding to AREs are known to destabilize the molecule. Accordingly, introduction, removal and/or modification of 3' UTR AREs can be used to modulate the stability of nucleic acids (e.g., RNA) of the disclosure. When engineering specific nucleic acids, in some aspects, one or more copies of an ARE can be introduced to make RNAs less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, in some aspects, AREs can be identified and removed and/or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection. In some aspects, a 3' UTR may have one or more AU-rich sequences removed. Alternatively the AU-rich sequences may remain in the 3' UTR.

A 3' UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g., a poly-A tail. A 3' UTR of the mRNA is not translated into an amino acid sequence. In some aspects, an RNA disclosed herein comprises a 3' UTR comprising an F element and/or an I element. In some aspects, a 3' UTR and/or a proximal sequence thereto comprises a restriction site. In some aspects, a restriction site is a BamHI site. In some aspects, a restriction site is a Xhol site.

In some aspects of the disclosure, a 3' UTR is a heterologous UTR, e.g., is a UTR found in nature associated with a different ORF. In another aspect, a 3' UTR is a synthetic UTR, e.g., does not occur in nature. In some aspects, the 3' UTR is functionally linked to the ORF, e.g., associated with the ORF such that it may exert a function, e.g., increasing, enhancing, stabilizing, and/or prolonging protein production from an RNA molecule and/or increasing protein expression and/or total protein production from an RNA molecule, compared to a reference RNA molecule comprising a reference 3' UTR or an RNA molecule lacking a 3' UTR. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR functions may be excluded.

Exemplary 3' UTRs include 3' UTRs derived from an albumin gene, an a-globin gene, a [3-globin gene, a ribosomal protein gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (1 ) gene, or from a homolog, fragment, or variant of a 3' UTR of a gene comprising an albumin gene, an a-globin gene, a [3- globin gene, a ribosomal protein gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and/or a collagen alpha gene, such as a collagen alpha 1 (1 ) gene according to SEQ ID NOs: 1369-1390 of the patent application WO2013/143700, the disclosure of which is incorporated herein by reference in its entirety, or a sequence having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing sequences. In some aspects, the sequence UUUGAAUU is used. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR sequences may be excluded from the RNA molecules disclosed herein.

In some aspects, the 3' UTR comprises a sequence of a transcript including NM_000661 .4, NM_001024921 .2, NM_000967.3, NM_001033853.1 , NMJD00968.3, NM_000969.3, NM_001024662.1 , NM_000970.3, NM_000971 .3, NMJD00972.2, NM_000975.3, NM_001 199802.1 , NM_000976.3, NM_000977.3, NM_033251 .2, NMJ 01243130.1 , NM_001243131 , NM_000978.3, NM_000979.3, NM_001270490.1 , NMJD00980.3, NM_000981 .3, NM_000982.3, NM_000983.3, NM_000984.5, NM_000985.4, NM_001035006.2, NM_001 199340.1 , NM_001 199341 .1 , NMJD01

199342.1 , NM_001 199343.1 , NM_001 199344.1 , NM_001 199345.1 , NM_000986.3, NM_000987.3, NM_000988.3, NM_000989.3, NM_000990.4, NM_001 136134.1 , NMJD00991 .4, NM_001 136135.1 , NM_001 136136.1 , NM_001

136137.1 , NM_000992.2, NM_000993.4, NM_001098577.2, NM_001099693.1 ,

NM_000994.3, NM_001007073.1 , NM_001007074.1 , NM_000996.2, M_000997.4, NM_000998.4, NM_000999.3, NM_001035258.1 , NM_001000.3, NM_001002.3, NM_053275.3, NM_001003.2, NM_213725.1 , NM_001004.3 , NM_001005.4, NM_00 1256802.1 , NM_001260506.1 , NM_001260507.1 , NM_001006.4, NM_00 1267699.1 , NM_001007.4, NM_001008.3, N _001009.3, NM_001010.2, NM_00101 1 .3, NM_001012.1 , NM_001013.3, NM_001203245.2, NM_001014.4, NM_00 1204091.1 , NM_001015.4, NM_001016.3, NM_001017.2, NM_001018.3, NM_00 1030009.1 , NM_001019.4, NM_001020.4, NM_001022.3, NM_001 146227.1 , NM_001023.3, NM_001024.3, NM_001025.4, NM_001028.2, NM_001029.3, NM_001030.4, NM_002954, NM_001 135592.2, NM_001 177413.1 , NM_001031 .4, NM_001032.4, NM_001030001 .2, NM_002948.3, NM_001253379.1

NM_00 1253380.1 , NM_001253382.1 , NM_001253383.1 , NM_001253384.1 , NM_002952.3, NM_001034996.2, NM_001025071 .1 , NM_001025070.1 , NM_005617.3, NM_006013.3, NM_001256577.1 , NM_001256580.1 , NM_007104.4, NM_007209.3, NM_012423.3, NM_001270491 .1 , NM_033643.2, NM_015414.3, NM_021029.5, NM_001 199972.1 , NM_021 104.1 , NM_022551 .2, NM_033022.3, NM_001 142284.1 , NM_001026.4, NM_001 142285.1 , NM_001 142283.1 , NM_001

142282.1 , NM_000973.3, NM_033301 .1 , NM_000995.3, NM_033625.2, NM_001021.3, NM_002295.4, NM_001012321 .1 , NM_001033930.1 , NM_003333.3, NM_00 1997.4, NM_001099645.1 , NM_001021 .3, NM_052969.1 , NM_080746.2, NM_001001 .4 , NM_005061.2 , NM_015920.3 , NM_016093.2 , NM_198486.2 , NG_01 1 172.1 , NG_01 1253.1 , NG_000952.4, NR_002309.1 , NG_010827.2, NG_009952.2, or NGJD09517.1 , or a sequence of a transcript having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing transcripts. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR sequences may be excluded from the RNA molecules disclosed herein.

In some aspects, the 3' UTR comprises a sequence from the 3' UTR region of a gene encoding a ribosomal protein, e.g., ribosomal protein L9 (RPL9), ribosomal protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosomal protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomal protein L7a (RPL7A), ribosomal protein L1 1 (RPL1 1 ), ribosomal protein L12 (RPL12), ribosomal protein L13 (RPL13), ribosomal protein L23 (RPL23), ribosomal protein L18 (RPL18), ribosomal protein L18a (RPL18A), ribosomal protein L19 (RPL19), ribosomal protein L21 (RPL21 ), ribosomal protein L22 (RPL22), ribosomal protein L23a (RPL23A), ribosomal protein L17 (RPL17), ribosomal protein L24 (RPL24), ribosomal protein L26 (RPL26), ribosomal protein L27 (RPL27), ribosomal protein L30 (RPL30), ribosomal protein L27a (RPL27A), ribosomal protein L28 (RPL28), ribosomal protein L29 (RPL29), ribosomal protein L31 (RPL31 ), ribosomal protein L32 (RPL32), ribosomal protein L35a (RPL35A), ribosomal protein L37 (RPL37), ribosomal protein L37a (RPL37A), ribosomal protein L38 (RPL38), ribosomal protein L39 (RPL39), ribosomal protein, large, P0 (RPLPO), ribosomal protein, large, P1 (RPLP1 ), ribosomal protein, large, P2 (RPLP2), ribosomal protein S3 (RPS3), ribosomal protein S3A (RPS3A), ribosomal protein S4, X-linked (RPS4X), ribosomal protein S4, Y-linked 1 (RPS4Y1 ), ribosomal protein S5 (RPS5), ribosomal protein S6 (RPS6), ribosomal protein S7 (RPS7), ribosomal protein S8 (RPS8), ribosomal protein S9 (RPS9), ribosomal protein S10 (RPS10), ribosomal protein S1 1 (RPS1 1 ), ribosomal protein S12 (RPS12), ribosomal protein S13 (RPS13), ribosomal protein S15 (RPS15), ribosomal protein S15a (RPS15A), ribosomal protein S16 (RPS16), ribosomal protein S19 (RPS19), ribosomal protein S20 (RPS20), ribosomal protein S21 (RPS21 ), ribosomal protein S23 (RPS23), ribosomal protein S25 (RPS25), ribosomal protein S26 (RPS26), ribosomal protein S27 (RPS27), ribosomal protein S27a (RPS27a), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), ribosomal protein L15 (RPL15), ribosomal protein S2 (RPS2), ribosomal protein L14 (RPL14), ribosomal protein S14 (RPS14), ribosomal protein L10 (RPL10), ribosomal protein L10a (RPL10A), ribosomal protein L35 (RPL35), ribosomal protein L13a (RPL13A), ribosomal protein L36 (RPL36), ribosomal protein L36a (RPL36A), ribosomal protein L41 (RPL41 ), ribosomal protein S18 (RPS18), ribosomal protein S24 (RPS24), ribosomal protein L8 (RPL8), ribosomal protein L34 (RPL34), ribosomal protein S17 (RPS17), ribosomal protein SA (RPSA) or ribosomal protein S17 (RPS17), or a sequence of a gene encoding a ribosomal protein having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing ribosomal gene protein sequences. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR sequences may be excluded from the RNA molecules disclosed herein.

In some aspects, the 3' UTR comprises a sequence from the 3' UTR region of a gene encoding a ribosomal protein or from a gene comprising ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52), Finkel-B iskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU), ribosomal protein L22-like 1 (RPL22L1 ), ribosomal protein L39-like (RPL39L), ribosomal protein L10-like (RPL10L), ribosomal protein L36a-like (RPL36AL), ribosomal protein L3-like (RPL3L), ribosomal protein S27-like (RPS27L), ribosomal protein L26-like 1 (RPL26L1 ), ribosomal protein L7-like 1 (RPL7L1 ), ribosomal protein L13a pseudogene (RPL13AP), ribosomal protein L37a pseudogene 8 (RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomal protein S26 pseudogene 1 1 (RPS26P1 1 ), ribosomal protein L39 pseudogene 5 (RPL39P5), ribosomal protein, large, PO pseudogene 6 (RPLP0P6) and ribosomal protein L36 pseudogene 14 (RPL36P14), and/or a sequence of a gene encoding a protein having at least, at most, exactly, or between (inclusive or exclusive) any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with any of the foregoing gene protein sequences. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing 3' UTR sequences may be excluded from the RNA molecules disclosed herein.

Those of ordinary skill in the art will understand that 5' UTRs that are heterologous and/or synthetic may be used with any desired 3' UTR sequence, and vice versa. For example, a heterologous 5' UTR may be used with a synthetic and/or heterologous 3' UTR. D. Open Reading Frame (ORF)

The 5' and 3' UTRs may be operably linked to an open reading frame (ORF), which may be a sequence of codons that is capable of being translated into a polypeptide of interest. An open reading frame may be a sequence of several DNA or RNA nucleotide triplets, which may be translated into a peptide or protein. An ORF may begin with a start codon, e.g., a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG or AUG), at its 5' end and a subsequent region, which usually exhibits a length that is a multiple of 3 nucleotides. An open reading frame may terminate with at least one stop codon, including but not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof. In some aspects, an open reading frame may terminate with one, two, three, four or more stop codons, including but not limited to TAATAA, TAATAG, TAATGA, TAGTGA, TAGTAA, TAGTAG, TGATGA), TGATAG, TGATAA or UAAUAA, UAAUAG, UAAUGA, UAGUGA, UAGUAA, UAGUAG, UGAUGA, UGAUAG, UGAUAA, or any combination thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing stop codons may be excluded. An open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, e.g., in a vector or an mRNA. An open reading frame may also be termed “(protein) coding region” or “coding sequence”.

As stated herein, the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames.

In some aspects, the ORF encodes a non-structural viral gene. In some aspects, the ORF further includes one or more subgenomic promoters. In some aspects, the RNA molecule includes a subgenomic promoter operably linked to the ORF. In some aspects, a first RNA molecule does not include an ORF encoding any polypeptide of interest, whereas a second RNA molecule includes an ORF encoding a polypeptide of interest. In some aspects, the first RNA molecule does not include a subgenomic promoter.

The present disclosure provides for an RNA molecule comprising at least one open reading frame encoding a coronavirus polypeptide (other than polypeptides from SARS-CoV-2).

E. Genes of Interest The RNA molecules described herein may include a gene of interest. The gene of interest encodes a polypeptide of interest. Non-limiting examples of polypeptides of interest include, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties, those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery, or combinations thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing polypeptides of interest may be excluded. The sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GENBANK®.

In some aspects, the RNA molecules include a coding region for a gene of interest. In some aspects, a gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some aspects, an antigenic polypeptide comprises one epitope from an antigen. In some aspects, an antigenic polypeptide comprises a plurality of distinct epitopes from an antigen. In some aspects, an antigenic polypeptide comprising a plurality of distinct epitopes from an antigen is polyepitopic. In some aspects, an antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing antigenic polypeptides may be excluded.

The term “antigen” may refer to a substance, which is capable of being recognized by the immune system, e.g., by the adaptive immune system, and which is capable of eliciting an antigen-specific immune response, e.g., by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response. An antigen may be or may comprise a peptide or protein, which may be presented by the MHC to T cells. An antigen may be the product of translation of a provided nucleic acid molecule, e.g., an RNA molecule comprising at least one coding sequence as described herein. In addition, fragments, variants and derivatives of an antigen, such as a peptide or a protein, comprising at least one epitope are understood as antigens. In some aspects, an RNA encoding a gene of interest, e.g., an antigen, is expressed in cells of a subject treated to provide a gene of interest, e.g., an antigen. In some aspects, the RNA is transiently expressed in cells of the subject. In some aspects, expression of a gene of interest, e.g., an antigen, is at the cell surface. In some aspects, a gene of interest, e.g., an antigen, is expressed and presented in the context of MHC. In some aspects, expression of a gene of interest, e.g., an antigen, is into the extracellular space, e.g., the antigen is secreted.

In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen. In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen, that is derived from a pathogen associated with an infectious disease. In some aspects, the RNA molecules include a coding region for a gene of interest, e.g., an antigen, that is derived from a coronavirus (other than SARS-CoV-2).

In some aspects, the RNA molecule encodes a coronavirus (other than SARS- CoV-2) protein or a fragment or a variant thereof.

In some aspects, an RNA polynucleotide described herein or a composition or medical preparation comprising the same comprises a nucleotide sequence disclosed herein. In some aspects, an RNA polynucleotide comprises a sequence having at least 80% identity to a nucleotide sequence disclosed herein. In some aspects, an RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some aspects, an RNA polynucleotide described herein or a composition or medical preparation comprising the same is transcribed by a DNA template. In some aspects, a DNA template used to transcribe an RNA polynucleotide described herein comprises a sequence complementary to an RNA polynucleotide. In some aspects, a gene of interest described herein is encoded by an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein. In some aspects, an RNA polynucleotide encodes a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some aspects, a polypeptide described herein is encoded by an RNA polynucleotide transcribed by a DNA template comprising a sequence complementary to an RNA polynucleotide.

F. Poly-A Tail In some aspects, RNA molecules disclosed herein comprise a poly-adenylate (poly-A) sequence, e.g., as described herein. In some aspects, a poly-A sequence is situated downstream of a 3' UTR, e.g., adjacent to a 3' UTR. A “poly-A tail” or “poly-A sequence” refers to a stretch of consecutive adenine residues, e.g., of up to or up to about 400 adenosine nucleotides, e.g., from or from about 20 to about 400, preferably from or from about 50 to about 400, more preferably from or from about 50 to about 300, even more preferably from or from about 50 to about 250, most preferably from or from about 60 to about 250 adenosine nucleotides, which may be attached to the 3' end of the RNA molecule. Poly-A sequences are known to those of skill in the art and may follow the 3' UTR in the RNA molecules described herein. The poly-A tail may increase the stability, half-life, and/or translational efficiency of the RNA molecule.

After cleavage, most pre-m RNAs, with exceptions that include replicationdependent histone transcripts that terminate with a histone stem -loop instead of a poly-A sequence, acquire a polyadenylated tail. In this context, 3'-end processing is a nuclear co-transcriptional process that promotes transport of m RNAs from the nucleus to the cytoplasm and affects the stability and the translation of mRNAs. Formation of this 3' end occurs in a two-step reaction directed by the cleavage/polyadenylation machinery and depends on the presence of two sequence elements in mRNA precursors (pre-m RNAs); a hexanucleotide polyadenylation signal and a downstream G/U-rich sequence. In a first step, pre-mRNAs are cleaved between these two elements to a free 3' hydroxyl. In a second step, the newly formed 3' end is extended by polyadenylation or addition of a poly-A sequence.

Polyadenylation refers to the addition of a poly-A sequence to an RNA molecule, e.g., to a premature mRNA. Polyadenylation may be induced by a so-called polyadenylation signal. This signal may be located within a stretch of nucleotides close to or at the 3'-end of an RNA molecule to be polyadenylated. A polyadenylation signal may also be comprised by the 3' UTR of the artificial nucleic acid molecule. A polyadenylation signal typically comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA, though other sequences, preferably hexamer sequences, are also conceivable. Polyadenylation typically occurs during processing of a pre-m RNA (also called premature-mRNA). Typically, RNA maturation (from pre-m RNA to mature mRNA) comprises the step of polyadenylation. Poly-A tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly-T tract into the DNA template or by post-transcriptional addition using poly-A polymerase. The term may relate to polyadenylation of RNA as a cellular process or to polyadenylation carried out by enzymatic reaction in vitro with a suitable enzyme, such as E. coli poly-A polymerase, or by chemical synthesis.

RNA molecules disclosed herein may have a poly-A sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. In some aspects, a poly-A sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.

The DNA sequence encoding a poly-A sequence (coding strand) is referred to as poly-A cassette. In some aspects, the poly-A cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such a random sequence may be at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. Such a cassette is disclosed in, e.g., WO 2016/005324 A1 , hereby incorporated by reference. Any poly-A cassette disclosed in WO 2016/005324 A1 may be used in the present disclosure. A poly-A cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides, shows, on a DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on an RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency. In some aspects, the poly-A sequence contained in an RNA polynucleotide described herein consists essentially of adenosine nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such a random sequence may be at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

The poly-A sequence may be located at any position within the 3' UTR. In some aspects, no nucleotides other than adenosine nucleotides flank a poly-A sequence at its 3'-end, e.g., the poly-A sequence is not masked or followed at its 3'-end by a nucleotide other than adenosine. In some aspects, the poly-A sequence may be located at the 3' terminus of the 3' UTR, e.g., the 3' UTR does not contain more than 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides located 3' of the poly-A sequence; more preferably the 3' UTR does not contain further elements located 3' to the poly-A sequence. In some aspects, poly-A sequence is located at the 3' terminus of the RNA molecule, e.g., the artificial nucleic acid molecule does not contain more than 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides located 3' of the poly-A sequence. Alternatively, the poly-A sequence may be located at the 5' terminus of the 3' UTR, e.g., immediately 3' of the ORF of the artificial nucleic acid molecule, or located within the 3' UTR, e.g., flanked on the 5' and on the 3' side by other 3' UTR elements. In some aspects, the poly-A sequence is flanked on the 3' side by a poly-C sequence and/or a histone stem-loop sequence. In addition or alternatively, the poly-A sequence can be flanked on the 5' side by a 3' UTR element derived from, e.g., a human albumin or globin gene.

In some aspects, the RNA molecule may further include an endonuclease recognition site sequence immediately downstream of the poly-A tail sequence. The RNA molecule may further include a poly-A polymerase recognition sequence (e.g., a polyadenylation signal) (e.g., AAUAAA) near its 3' end. In some aspects, the polyadenylation signal is located 3' of the poly-A sequence comprised in the 3' UTR. In some aspects, the poly-A sequence is separated from the polyadenylation signal by a nucleotide sequence comprising or consisting of at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides, wherein the nucleotide sequence preferably does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides. In some aspects, the nucleotide sequence that separates the poly-A sequence and the polyadenylation signal comprises from or from about 1 to about 200 nucleotides, e.g., from 10 to 90, from 20 to 85, from 30 to 80, from 40 to 80, from 50 to 75 or from 55 to 85 nucleotides, more preferably from 55 to 80 nucleotides, and the nucleotide sequence does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides.

In some aspects, the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N = A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA. Such a consensus sequence may be recognized by most animal and bacterial cellsystems, for example, by the polyadenylation-factors, such as cleavage/polyadenylation specificity factor (CPS F) cooperating with CstF, PAP, PAB2, CFI and/or CFII. In some aspects, the polyadenylation signal (e.g., the consensus sequence NNUANA) is located less than or less than about 50 nucleotides, e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, downstream of the 3'-end of the 3' UTR element as defined herein such that transcription of an RNA molecule will result in a premature- RNA containing the polyadenylation signal downstream of its 3' UTR and subsequent attachment of a poly-A sequence to the premature-RNA. Accordingly, a resulting RNA may comprise a 3' UTR, which comprises at least one poly-A sequence, and wherein the 3' UTR is followed by an additional poly-A sequence.

The poly-A sequence may be of any length. In some aspects, the poly-A tail may be 5 to 300 nucleotides in length. In some aspects, the RNA molecule includes a poly-A tail that comprises, consists essentially of, or consists of a sequence of or of about 25 to about 400 adenosine nucleotides, a sequence of or of about 50 to about 400 adenosine nucleotides, a sequence of or of about 50 to about 300 adenosine nucleotides, a sequence of or of about 50 to about 250 adenosine nucleotides, a sequence of or of about 60 to about 250 adenosine nucleotides, or a sequence of or of about 40 to about 100 adenosine nucleotides. In some aspects, the poly-A tail comprises, consists essentially of, or consists of at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,

220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,

305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,

390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470,

475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555,

560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640,

645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725,

730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810,

815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895,

900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980,

985, 990, 995, or 1000 adenosine nucleotides. In this context, “consists essentially of” means that most nucleotides in the poly-A sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A sequence are adenosine nucleotides, but permits remaining nucleotides to be nucleotides other than adenosine nucleotides, such as uridine, guanosine, and/or cytosine. In this context, “consists of” means that all nucleotides in the poly-A sequence, i.e., 100% by number of nucleotides in the poly-A sequence, are adenosine nucleotides.

In some aspects, the RNA molecule includes a poly-A tail that includes a sequence of greater than 30 adenosine nucleotides. In some aspects, the RNA molecule includes a poly-A tail that includes or includes about 40 adenosine nucleotides. In some aspects, the RNA molecule includes a poly-A tail that includes or includes about 80 adenosine nucleotides. In some aspects, the 3' poly-A tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some specific aspects, the RNA molecule includes or includes about 40 consecutive adenosine residues. In some aspects, the RNA molecule includes or includes about 80 consecutive adenosine residues. Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation.

In some aspects, a poly-A tail may be located within an RNA molecule or other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, e.g., an mRNA, e.g., by transcription of the vector. In some aspects, the RNA molecule may not include a poly-A tail.

G. Other Elements

In some aspects of the present disclosure, the RNA molecules additionally include a chain terminating nucleoside. For example, a chain terminating nucleoside may include those nucleosides deoxygenated at the 2’ and/or 3' positions of their sugar group. Such species may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxycytosine, 3' deoxyguanosine, 3' deoxythymine, and 2', 3' dideoxynucleosides, such as 2', 3' dideoxyadenosine, 2', 3' dideoxyuridine, 2', 3' dideoxycytosine, 2', 3' dideoxyguanosine, and 2', 3' dideoxythymine. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing chain terminating nucleosides may be excluded from the RNA molecules disclosed herein. In some aspects, incorporation of a chain terminating nucleotide into an mRNA, for example at the 3'-terminus, may result in stabilization of the mRNA, as described, for example, in International Patent Publication No. WO 2013/103659.

In some aspects of the present disclosure, the RNA molecules additionally include a stem loop, such as a histone stem loop. A stem loop may include 2, 3, 4, 5,

6, 7, 8, or more nucleotide base pairs. For example, a stem loop may include 4, 5, 6,

7, or 8 nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in, before, or after an untranslated region (a 5' UTR or a 3' UTR), a coding region, or a poly-A sequence or tail. In some aspects, a stem loop may affect one or more function(s) of an mRNA, such as initiation of translation, translation efficiency, and/or transcriptional termination. Such histone stem-loop sequences may be histone stem-loop sequences disclosed in WO 2012/019780, the disclosure of which is incorporated herein by reference in its entirety. Other non-limiting examples of histone stem loop structures and nucleic acid sequences encoding such structures can be found in, e.g., WO 2016/091391 , the disclosure of which is incorporated by reference herein in its entirety.

In some aspects, the combination of a poly-A sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. In some aspects, the synergistic effect of the combination of poly-A and at least one histone stem-loop does not depend on the order of the elements and/or the length of the poly-A sequence.

In some aspects, the RNA does not comprise a histone downstream element (HDE). An HDE includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.

In some aspects, the histone stem-loop is generally derived from histone genes, and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. Stability of the stem -loop structure generally depends on the length, number of mismatches or bulges, and/or base composition of the paired region. In some aspects, wobble base pairing (non- Watson-Crick base pairing) may result. In some aspects, the at least one histone stem- loop sequence comprises a length of 15 to 45 nucleotides.

In some aspects, the RNA molecules include (e.g., within the 3' UTR) a poly(C) sequence. In some aspects, the poly-C sequences has at least, at most, exactly, or between (inclusive or exclusive) any two of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 cytidines. In some aspects, the poly-C sequences has or has about 30 cytidines.

In some aspects, the RNA molecules include an internal ribosome entry site (IRES) sequence or IRES-motif. In some aspects, an IRES sequence separates ORFs, e.g., if the RNA encodes two or more peptides or proteins. An IRES-sequence may therefore be useful if the RNA molecule is a bi- or multicistronic nucleic acid molecule.

In some aspects, the RNA does not comprise an intron. In some aspects, the RNA may instead or additionally include a microRNA binding site.

Representative RNA molecules including a combination of the elements disclosed herein can include, without limitation, in 5'-to-3'-direction, the following:

ORF - poly-A sequence;

ORF - IRES - ORF - poly-A sequence;

ORF - 3' UTR - poly-A sequence;

ORF - poly-A sequence - 3' UTR;

ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stem-loop;

ORF - 3' UTR - poly-A sequence - poly(C) sequence - poly-A sequence;

ORF - 3' UTR - poly-A sequence - histone stem-loop - poly-A sequence;

5' UTR - ORF - 3' UTR;

5' UTR - ORF - poly-A sequence;

5' UTR - ORF - poly-A sequence - poly(C) sequence - histone stem-loop;

5' UTR - ORF - poly-A sequence - poly(C) sequence - poly-A sequence;

5' UTR - ORF - poly-A sequence - histone stem-loop - poly-A sequence;

5' UTR - ORF - 3' UTR - poly-A sequence;

5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence

5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stemloop;

5'-cap - 5' UTR - ORF - 3' UTR;

5'-cap - 5' UTR - ORF - poly-A sequence; 5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence;

5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence; or

5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stem- loop.

In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing elements may be excluded from the RNA molecules disclosed herein.

H. Self-Amplifying RNA (saRNA)

In some aspects, the RNA molecule may be an saRNA. “Self-amplifying RNA,” “saRNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, and/or may be transcribed to provide further transcripts with the same sense as the delivered RNA that are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNA molecules, and consequently, the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.

In some aspects, the self-amplifying RNA includes at least one or more genes including any one of viral replicases, viral proteases, viral helicases and other nonstructural viral proteins, or combination thereof. In some aspects, 1 , 2, 3, or more of the foregoing genes may be excluded from the self-amplifying RNA molecules disclosed herein. In some aspects, the self-amplifying RNA may also include 5'- and 3'-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and/or may be under the control of an internal ribosome entry site (IRES).

In some aspects, a self-amplifying RNA molecule described herein encodes (i) an RNA-dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen. In some aspects, the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus proteins nsP1 , nsP2, nsP3, nsP4, or any combination thereof. In some aspects, 1 , 2, 3, or more of the foregoing alphavirus proteins may be excluded from the RNA molecules disclosed herein.

In some aspects, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame may encode a replicase; the second (3') open reading frame may encode a polypeptide comprising an antigen of interest. In some aspects the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides.

In some aspects, the saRNA molecule further includes (1 ) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some aspects, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

In some aspects, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g., linked in series) when expressed as an amino acid sequence. The polypeptides generated from the selfamplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.

In some aspects, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. In some aspects, the selfamplifying RNA described herein may encode epitopes capable of eliciting either a helper T cell response or a cytotoxic T cell response or both.

VII. RNA Encapsulation

The RNA in an RNA product solution may be encapsulated, and the RNA solution may further comprise at least one encapsulating agent. In one aspect, the encapsulating agent comprises a lipid, a lipid nanoparticle (LNP), lipoplexes, polymeric particles, polyplexes, monolithic delivery systems, or a combination thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing elements may be excluded as an encapsulating agent.

In one aspect, the encapsulating agent is a lipid, and produced is lipid nanoparticle (LNP)-encapsulated RNA. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and/or the cationic polymer combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester- linked fatty acids and polymerizable lipids, and combinations thereof. A lipid is a substance that is insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids and are encompassed by the compositions and methods of the present disclosure. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently.

In some aspects, LNPs may be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular Rnases and/or may be engineered for systemic delivery of the RNA to target cells. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intravenously administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intramuscularly administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intradermally administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules (e.g., saRNA, mRNA) when RNA molecules are intranasally administered to a subject in need thereof.

In one aspect, the RNA in the RNA product solution is at a concentration of < 1 mg/mL. In another aspect, the RNA is at a concentration of at least or at least about 0.05 mg/mL. In another aspect, the RNA is at a concentration of at least or at least about 0.5 mg/mL. In another aspect, the RNA is at a concentration of at least or at least about 1 mg/mL. In another aspect, the RNA concentration is from or from about 0.05 mg/mL to about 0.5 mg/mL. In another aspect, the RNA is at a concentration of at least 10 mg/mL. In another aspect, the RNA is at a concentration of at least 50 mg/mL. In some aspects, the RNA is or is not at a concentration of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.05 mg/mL, 0.5 mg/mL, 1 mg/mL, 10 mg/mL, 50 mg/mL, 75 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 400 mg/mL, or more.

The present disclosure provides for an RNA product solution and a lipid preparation mixture or compositions thereof comprising at least one RNA encoding, e.g., an antigen (e.g., a coronavirus (other than SARS-CoV-2) polypeptide) complexed with, encapsulated in, and/or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes and/or nanoliposomes. In some aspects, the composition comprises a lipid nanoparticle.

A lipid nanoparticle or LNP refers to particles of any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and/or in the presence of RNA. In some aspects, lipid nanoparticles are included in a formulation that may be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some aspects, the lipid nanoparticles of the present disclosure comprise a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids, or combinations thereof. In some aspects, the LNPs comprise at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and/or at least one polymer conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, 1 , 2, 3, or more of the foregoing excipients may be excluded from the LNPs.

In some aspects, the LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipid(s). For example, the LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise or do not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 45 to 55 mole percent (mol%) cationic (e.g., ionizable) lipid(s). For example, LNPs may comprise or not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipid(s).

In some aspects, the LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipid(s). For example, the LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, I Q- 25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 5 to 15 mol% neutral (e.g., non-cationic) lipid(s). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 mol% neutral (e.g., non-cationic) lipid(s).

In some aspects, the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid). For example, the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 35 to 40 mol% structural lipid(s) (e.g., a steroid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, or 40 mol% structural lipid(s) (e.g., a steroid).

In some aspects, the LNPs comprise 0.5-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1 -15 mol%, 1-10 mol%, 1 -5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, the lipid LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, the LNPs comprise 1 to 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1 , 1.5, or 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid).

In some aspects, the LNPs comprise 20-75 mol% cationic (e.g., ionizable) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) any two of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%), 0.5-25 mol% neutral (e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11 %, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25%, and 25%), 5-55 mol% structural lipid(s) (e.g., a sterol) e.g., non-cationic) lipid(s) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5-20 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-modified lipid) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11 %, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some aspects, 1 , 2, 3, or more of the lipids may be excluded from the LNPs.

In some non-limiting aspects, the molar lipid ratio is 50/10/38.5/1.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 60/7.5/31/1.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 57.5/7.5/31.5/3.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 57.2/7.1/34.3/1.4 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 40/15/40/5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 50/10/35/4.5/0.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 50/10/35/5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 40/10/40/10 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), 35/15/40/10 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid), or 52/13/30/5 (mol% cationic lipid/neutral lipid/structural lipid/polymer conjugated lipid).

In some aspects, the active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle and/or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. The nucleic acid (e.g., mRNA) or a portion thereof may also be associated and complexed with the lipid nanoparticle. A lipid nanoparticle may comprise any lipid capable of forming a particle to which the nucleic acids are attached, and/or in which the one or more nucleic acids are encapsulated.

In some aspects, provided RNA molecules (e.g., saRNA, mRNA) may be formulated with LNPs. In some aspects, the lipid nanoparticles may or may not have a mean diameter of or of about 1 to 500 nm (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1 , 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, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some aspects, the lipid nanoparticles have a mean diameter of or of from about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most, exactly, or between (inclusive or exclusive) of 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. The term “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321 ). Here, “mean diameter,” “diameter,” or “size” for particles is used synonymously with the value of the Z-average.

LNPs described herein may exhibit a polydispersity index less than or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, exactly, or between (inclusive or exclusive) of 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. The polydispersity index is, in some aspects, calculated based on dynamic light scattering measurements by the so-called cumulant analysis referred to in the definition of “average diameter.” Under certain prerequisites, it may be taken as a measure of the size distribution of an ensemble of nanoparticles. In some aspects, an LNP of the disclosure comprises or does not comprise an N:P ratio of or of from about 2:1 to about 30:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 2:1, 3:1, 4:1 , 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1 , 25:1 , 26:1 , 27:1, 28:1, 29:1, or 30:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 6:1. In some aspects, an LNP of the disclosure comprises an N:P ratio of or of about 3:1.

In some aspects, an LNP of the disclosure comprises or does not comprise a wt/wt ratio of the cationic lipid component to the RNA of or of from about 5:1 to about 100:1, e.g., at least, at most, exactly, or between (inclusive or exclusive) of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1,

38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,

53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1 , 65:1 , 66:1 , 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1,

83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1,

98:1, 99:1, or 100:1. In some aspects, an LNP of the disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of or of about 20:1. In some aspects, an LNP of the disclosure comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of or of about 10:1.

In certain aspects, nucleic acids (e.g., RNA molecules), when present in provided LNPs, are resistant in aqueous solution to degradation with a nuclease. In some aspects, LNPs are liver-targeting lipid nanoparticles. In some aspects, LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some aspects, cationic LNPs may comprise at least one cationic lipid, at least one polymer conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

In certain aspects, the RNA solution and lipid preparation mixture or compositions thereof may have at least, at most, exactly, between (inclusive or exclusive) of, or about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,

29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,

44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a particular lipid, lipid type, or non-lipid component such as lipid-like materials and/or cationic polymers and/or an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or as would be known to one of skill in the art.

LNPs described herein can be generated using components, compositions, and methods as are generally known in the art, see, , e.g., 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; PCT/US2014/027077:

PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610:

PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety. Other non-limiting examples of methods for preparing LNPs can be found in, e.g., WO 2022/032154, the disclosure of which is incorporated by reference herein in its entirety.

For example, methods of preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term “colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” refers only to the particles in the mixture and not the entire suspension.

For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer, methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).

In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included. Reverse phase evaporation is an alternative method to film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that subsequently turns into a liposomal suspension.

The term “ethanol injection technique” refers to a process in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example, lipid vesicle formation such as liposome formation. Generally, the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some aspects, formed as follows: an ethanol solution comprising lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In some aspects, the RNA lipoplex particles described herein are obtainable without a step of extrusion. The term “extruding” or “extrusion” refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.

Other methods for preparing a colloid having organic solvent free characteristics may also be used according to the present disclosure.

In some aspects, LNP-encapsulated RNA may be produced by rapid mixing of an RNA solution described herein (e.g., the RNA product solution) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some aspects, suitable buffering agents comprise tris, histidine, citrate, acetate, phosphate, and/or succinate. In some aspects, 1 , 2, 3, or more of the foregoing buffering agents are excluded. The pH of a liquid formulation relates to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which, for example, in some aspects, may result in much higher encapsulation efficiency than is achieved in the absence of interactions between nucleic acids and at least one of the lipid components. In certain aspects, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub. Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species, such as RNA species. In an LNP formulation, it is possible that each nucleic acid species is separately formulated as an individual LNP formulation. In that case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

In some aspects, a composition such as a pharmaceutical composition comprises more than one individual LNP formulation. Respective pharmaceutical compositions are referred to as mixed LNP formulations. Mixed LNP formulations according to the invention are obtainable by forming, separately, individual LNP formulations, as described above, followed by a step of mixing of the individual LNP formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing LNPs is obtainable. Individual LNP populations may be together in one container, comprising a mixed population of individual LNP formulations.

Alternatively, it is possible that different nucleic acid species are formulated together as a combined LNP formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. As opposed to a mixed LNP formulation, a combined LNP formulation will typically comprise LNPs that comprise more than one RNA species. In a combined LNP composition, different RNA species are typically present together in a single particle.

A. Cationic Polymeric Materials

Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some aspects herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. In some aspects, such synthetic materials may be suitable for use as cationic materials herein.

A “polymeric material,” as used herein, is given its ordinary meaning, e.g., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some aspects, such repeat units may all be identical; alternatively, in some cases, there may be more than one type of repeat unit present within the polymeric material. In some cases, a polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties may also be present in the polymeric material, for example targeting moieties such as those described herein.

Those skilled in the art are aware that, when more than one type of repeat unit is present within a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is said to be a “copolymer.” In some aspects, a polymer (or polymeric moiety) utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer may be arranged in any fashion. For example, in some aspects, repeat units may be arranged in a random order; alternatively or additionally, in some aspects, repeat units may be arranged in an alternating order, or as a “block” copolymer, e.g., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

In certain aspects, a polymeric material for use in accordance with the present disclosure is biocompatible. Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations. In certain aspects, a biocompatible material is biodegradable, e.g., is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. In certain aspects, a polymeric material may be or comprise protamine or polyalkylene imine, in particular protamine.

As those skilled in the art are aware, the term “protamine” is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (e.g., fish). In particular, the term “protamine” is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

In some aspects, the term “protamine” as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and/or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides that are artificial and designed for specific purposes and cannot be isolated from native or biological sources.

In some aspects, a polyalkylene imine comprises polyethylenimine and/or polypropylenimine. In some aspects, the polyalkylene imine is polyethyleneimine (PEI). In some aspects, the polyalkylene imine is a linear polyalkylene imine, e.g., linear polyethyleneimine (PEI).

Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some aspects, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid may be associated, e.g., by forming complexes with the nucleic acid and/or forming vesicles in which the nucleic acid is enclosed or encapsulated.

In some aspects, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and/or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.

B. Lipids and Lipid-Like Materials

The terms “lipid” and “lipid-like material” are used herein to refer to molecules that comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

The term “lipid” refers to a group of organic compounds that are characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterol lipids as well as sterol-containing metabolites such as cholesterol, and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramides phosphosphingolipids (e.g., sphingomyelins, phosphocholine), and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives. In some aspects, 1 , 2, 3, 4, 5, or more of the lipids may be excluded from the LNPs of the present disclosure.

The term “lipid-like material,” “lipid-like compound,” or “lipid-like molecule” relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment, and includes surfactants or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules that comprise hydrophilic and hydrophobic moieties with different structural organization that may or may not be similar to that of lipids.

In some aspects, the RNA product solution and lipid preparation mixture or compositions thereof may comprise cationic lipids, neutral lipids, cholesterol, and/or polymer (e.g., polyethylene glycol)-conjugated lipids which form lipid nanoparticles that encompass the RNA molecules. Therefore, in some aspects, the LNP may comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer conjugated lipids (e.g., PEG-lipid), or combinations thereof. In some aspects, 1 , 2, 3, or more of the foregoing excipients may be excluded from the LNPs of the present disclosure. In some aspects, the lipids are present in a composition in an amount that is effective to form a lipid nanoparticle and deliver a therapeutic agent, e.g., an RNA molecule, for treating a particular disease or condition of interest, e.g., those related to a coronavirus other than SARS-CoV-2. In some aspects, the LNPs encompass, or encapsulate, the nucleic acid molecules.

C. Cationic Lipids

Cationic or cationically ionizable lipids or lipid-like materials refer to a lipid or lipid-like material capable of being positively charged and able to electrostatically bind nucleic acid. As used herein, a “cationic lipid” or “cationic lipid-like material” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl, or more acyl chains, and the head group of the lipid typically carries the positive charge. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Cationic lipids may encapsulate negatively charged RNA.

In some aspects, cationic lipids are ionizable such that they may exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. Without wishing to be bound by theory, this ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH. For purposes of the present disclosure, such “cationically ionizable” lipids or lipid-like materials are comprised by the term “cationic lipid” or “cationic lipid-like material” unless contradicted by the circumstances.

In some aspects, a cationic lipid may comprise from or from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle. In some aspects, a cationic lipid may or may not be at least, at most, exactly, or between (inclusive or exclusive) of 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, 90 mol %, or 100 mol %, or any range or value derivable therein, of the total lipid present in the particle.

Examples of cationic lipids include, but are not limited to: ((4- hydroxybutyl)azanediyl)bis(hexane-6, 1 -diyl)bis(2-hexyldecanoate), 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-( N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, 1 ,2-dialkyloxy-3- dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1 ,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl- (2-hydroxyethyl)-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC), 1 ,2-dimyristoyl-3-trimethylammonium propane

(DMTAP), 1 ,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide

(DORIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1 - propanamium trifluoroacetate (DOSPA), 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta- oxybutan-4-oxy)-1 -(cis, cis-9, 12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'- (cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3- dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1 ,2-N,N'-dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP), 1 ,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1 ,3]- dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-K- XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2- DMA), heptatriaconta-6,9,28,31 -tetraen-19-yl-4-(dimethylamino)butanoate (DLin- MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1 - propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9- tetradecenyloxy)-1 -propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)- N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3- aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1 -propanaminium bromide (GAP- DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (bAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan- 1 -aminium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z, 12Z)-octadeca-9, 12-dien-1 -yloxy]propan-1 -amine (Octyl-CLinDMA), 1 ,2- dimyristoyl-3-dimethylammonium-propane (DMDAP), 1 ,2-dipalm itoy I-3- dimethylammonium-propane (DPDAP), N1 -[2-((1 S)-1 -[(3-aminopropyl)amino]-4-[di(3- amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1 ,2- dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2- hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1 -aminium bromide (DMORIE), di((Z)- non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1 -amine (DLDMA), N,N-dimethyl- 2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2- dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl- ethyl)-[2-(2-dodecylcarbamoyl-ethylam ino)-ethy l]-am ino}-ethylam ino)propionam ide (I ipidoid 98N 12-5), 1 -[2-[bis(2-hydroxydodecyl)am ino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid 02- 200); C 12-200; or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate (SM-102). In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cationic lipids may be excluded from the LNPs of the present disclosure.

In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cationic lipids may be excluded from the LNPs of the present disclosure.

In some aspects, the RNA-LNPs comprise a cationic lipid, an RNA molecule as described herein, and one or more of neutral lipids, steroids, pegylated lipids, or combinations thereof. In one aspect, the cationic lipid is or is not present in the LNP in an amount such as at least, at most, exactly, between (inclusive or exclusive) of, or about 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent (mol %). In some aspects, two or more cationic lipids are incorporated within the LNP. If more than one cationic lipid is incorporated within the LNP, the foregoing percentages apply to the combined cationic lipids.

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

D. Polymer Conjugated Lipids

In some aspects, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid (e.g., polyethylene glycol-lipid, PEG-lipid). In certain aspects, the LNP comprises an additional, stabilizing lipid that is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.

Pegylated lipids are known in the art and include, but are not limited to, PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide, and mixtures thereof. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, PEG-DSG, PEG-DPG, and PEG-s- DMG (1 -(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol). In one aspect, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2000)carbamoyl]-1 ,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one aspect, the polyethylene glycol-lipid is PEG-2000-DMG. In one aspect, the polyethylene glycol-lipid is PEG-c-DOMG. In other aspects, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1 - (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1 -O-((O- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG- cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N- (2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co- methoxy(polyethoxy)ethyl)carbamate. PEG-lipids are disclosed in, e.g., U.S. 9,737,619, the full disclosures of which is herein incorporated by reference in its entirety for all purposes. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing pegylated lipids may be excluded from the LNPs of the present disclosure.

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

In certain aspects, the PEG-lipid is or is not present in the LNP in an amount from or from about 1 to about 10 mole percent (mol %) (e.g., at least, at most, exactly, or between (inclusive or exclusive) of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %), relative to the total lipid content of the nanoparticle.

In some aspects, the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulations.

E. Additional Lipids

In certain aspects, the LNP comprises one or more additional lipids or lipid-like materials that stabilize particles during their formation. Suitable stabilizing or structural lipids include non-cationic lipids, e.g., neutral lipids and anionic lipids. Without being bound by any theory, optimizing the formulation of LNPs by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery.

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

Representative neutral lipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, ceramides, sphingomyelins, dihydro-sphingomyelins, cephalins, and cerebrosides. Exemplary phospholipids include, for example, phosphatidylcholines, e.g., diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC); and phosphatidylethanolamines, e.g., diacylphosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), 1 ,2-diundecanoyl-sn-glycero- phosphocholine (DLIPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), distearoyl-phosphatidylethanolamine (DSPE), 1 -phytanoyl- phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoylphosphatidyethanolamine (SOPE), 1 ,2-dielaidoyl-sn- glycero-3-phosphoethanolamine (transDOPE), 1 ,2- dilinolenoyl-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 (ME 16.0 PE), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2- dilinoleoyl-sn-glycero-3-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. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure.

In one aspect, the neutral lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), having the formula:

In some aspects, the LNPs comprise a neutral lipid, and the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and/or SM. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing neutral lipids may be excluded from the LNPs of the present disclosure.

In various aspects, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

In certain aspects, the steroid or steroid analogue is cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing steroid or steroid analogues may be excluded from the LNPs of the present disclosure. In certain aspects, the steroid or steroid analogue is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2 -hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In some aspects, 1 , 2, 3, 4, 5, or more of the foregoing cholesterol derivatives may be excluded from the LNPs of the present disclosure. In one aspect, the cholesterol has the formula:

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

In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol), may comprise from or from about 0 mol % to about 90 mol %, from or from about 0 mol % to about 80 mol %, from or from about 0 mol % to about 70 mol %, from or from about 0 mol % to about 60 mol %, or from or from about 0 mol % to about 50 mol %, of the total lipid present in the particle. In some aspects, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol), may or may not be at least, at most, exactly, or between (inclusive or exclusive) of 0 mol %, 10 mol %, 20 mol %, 30 mol %, 40 mol %, 50 mol %, 60 mol %, 70 mol %, 80 mol %, or 90 mol % of the total lipid present in the particle.

VIII. Immune Response and Assays

As discussed herein, the disclosure concerns evoking or inducing an immune response in a subject against a coronavirus protein, e.g., a native or variant coronavirus spike protein. In one aspect, the immune response can protect against or treat a subject having, suspected of having, or at risk of developing an infection or related disease, particularly those related to coronaviruses. One use of the immunogenic compositions of the disclosure is to prevent coronavirus infections by inoculating a subject.

A. Immunoassays

The present disclosure includes the implementation of serological assays to evaluate whether and to what extent an immune response is induced or evoked by compositions of the disclosure. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the present disclosure include, but are not limited to, those described in U.S. Patent 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.

Immunoassays generally are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one example, antibodies or antigens are immobilized on a selected surface, such as a well in a polystyrene microtiter plate, dipstick, or column support. Then, a test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen or antibody, that is linked to a detectable label. This type of ELISA is known as a “sandwich ELISA.” Detection also may be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

Competition ELISAs are also possible implementations in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal. Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes.

Antigen or antibodies may also be linked to a solid support, such as in the form of plate, beads, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely-adsorbed material. Any remaining available surfaces of the wells are then “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

B. Diagnosis of Coronavirus Infection In addition to the use of proteins, polypeptides, and/or peptides to treat, prevent, or reduce the seventy of illness from infection as described above, the present disclosure contemplates the use of these polypeptides, proteins, and/or peptides in a variety of ways, including the detection of the presence of coronavirus to diagnose an infection. In accordance with the disclosure, a preferred method of detecting the presence of infections involves the steps of obtaining a sample suspected of being infected by one or more coronavirus strains, such as a sample taken from an individual, for example, from one’s blood, saliva, tissues, bone, muscle, cartilage, or skin. Following isolation of the sample, diagnostic assays utilizing the polypeptides, proteins, and/or peptides of the present disclosure may be carried out to detect the presence of coronavirus, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassay, western blot analysis and ELISA assays.

In general, in accordance with the disclosure, a method of diagnosing an infection is contemplated wherein a sample suspected of being infected with coronavirus has added to it the polypeptide, protein, or peptide, in accordance with the present disclosure, and coronaviruses are indicated by antibody binding to the polypeptides, proteins, and/or peptides, or polypeptides, proteins, and/or peptides binding to the antibodies in the sample.

Accordingly, polypeptides, proteins, and/or peptides in accordance with the disclosure may be used for to treat, prevent, or reduce the severity of illness from infection due to coronavirus infection (7.e. , active or passive immunization) or for use as research tools.

Any of the above described polypeptides, proteins, and/or peptides may be labeled directly with a detectable label for identification and quantification of coronavirus. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

C. Protective Immunity

In some aspects of the disclosure, proteinaceous compositions confer protective immunity to a subject. Protective immunity refers to a body’s ability to mount a specific immune response that protects the subject from developing a particular disease or condition that involves the agent against which there is an immune response. An immunogenically effective amount is capable of conferring protective immunity to the subject.

As used herein, the term polypeptide or peptide refers to a stretch of amino acids covalently linked by peptide bonds. Different polypeptides have different functionalities according to the present invention. While according to one aspect, a polypeptide is derived from an immunogen designed to induce an active immune response in a recipient, according to another aspect of the invention, a polypeptide is derived from an antibody which results following the elicitation of an active immune response in, for example, an animal, and which can serve to induce a passive immune response in the recipient. In both cases, however, the polypeptide is encoded by a polynucleotide according to any possible codon usage.

As used herein the phrase “immune response” or its equivalent “immunological response” refers to the development of a humoral (antibody mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against a protein, peptide, carbohydrate, or polypeptide of the invention in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody, antibody containing material, or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules, to activate antigen-specific CD4 (+) T helper cells and/or CD8 (+) cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity. As used herein “active immunity” refers to any immunity conferred upon a subject by administration of an antigen.

As used herein “passive immunity” refers to any immunity conferred upon a subject without administration of an antigen to the subject. “Passive immunity” therefore includes, but is not limited to, administration of activated immune effectors including cellular mediators or protein mediators (e.g., monoclonal and/or polyclonal antibodies) of an immune response. A monoclonal or polyclonal antibody composition may be used in passive immunization to treat, prevent, or reduce the severity of illness caused by infection by organisms that carry the antigen recognized by the antibody. An antibody composition may include antibodies that bind to a variety of antigens that may in turn be associated with various organisms. The antibody component can be a polyclonal antiserum. In certain aspects the antibody or antibodies are affinity purified from an animal or second subject that has been challenged with an antigen(s). Alternatively, an antibody mixture may be used, which is a mixture of monoclonal and/or polyclonal antibodies to antigens present in the same, related, or different microbes or organisms, such as viruses, including but not limited to coronaviruses.

Passive immunity may be imparted to a patient or subject by administering to the patient immunoglobulins (Ig) and/or other immune factors obtained from a donor or other non-patient source having a known immunoreactivity. In other aspects, an immunogenic composition of the present disclosure can be administered to a subject who then acts as a source or donor for globulin, produced in response to challenge with the immunogenic composition (“hyperimmune globulin”), that contains antibodies directed against a coronavirus or other organism. A subject thus treated would donate plasma from which hyperimmune globulin would then be obtained, via conventional plasma-fractionation methodology, and administered to another subject in order to impart resistance against or to treat coronavirus infection.

For purposes of this specification and the accompanying claims the terms “epitope” and “antigenic determinant” are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by ³H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion.

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

As used herein, the terms “antibody” or “immunoglobulin” are used interchangeably and refer to any of several classes of structurally related proteins that function as part of the immune response of an animal or recipient, which proteins include IgG, IgD, IgE, IgA, IgM and related proteins. Under normal physiological conditions antibodies are found in plasma and other body fluids and in the membrane of certain cells and are produced by lymphocytes of the type denoted B cells or their functional equivalent.

As used herein the terms “immunogenic agent” or “immunogen” or “antigen” are used interchangeably to describe a molecule capable of inducing an immunological response against itself on administration to a recipient, either alone, in conjunction with an adjuvant, or presented on a display vehicle.

IX. Compositions

In one aspect, the disclosure relates to an immunogenic composition for administration to a host. In some aspects, the host is a human. In other aspects, the host is a non-human. In some aspects, RNA molecules and/or RNA-LNPs disclosed herein may be administered in a pharmaceutical composition or a medicament and may be administered in the form of any suitable pharmaceutical composition. In some aspects, a pharmaceutical composition is for therapeutic and/or prophylactic treatment. In one aspect, the disclosure relates to a composition for administration to a host. In some aspects, the host is a human. In other aspects, the host is a non- human.

In some aspects, the composition comprises an immunogenic polypeptide construct. In some aspects, the immunogenic polypeptide construct of the composition is an isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof. In some aspects, the composition comprises a variant coronavirus spike protein construct. The variant coronavirus spike protein construct can differ from a native, unmodified coronavirus spike protein construct at one or more amino acids. In some aspects, the variant coronavirus spike protein construct has at least about 50% amino acid sequence identity with the native, unmodified coronavirus spike protein construct.

A. Vaccines

In some instances, the compositions described herein are immunogenic compositions. In some instances, the compositions described herein include at least one isolated polypeptide molecule as described herein and/or at least one RNA molecule (and/or RNA-LNP) as described herein. In some instances, the compositions described herein are vaccines. In yet other aspects, the immunogenic compositions comprise a polypeptide, and vaccines are polypeptide vaccines. Conditions and/or diseases that can be treated with the peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of protein or nucleic acids.

In some aspects, the composition is substantially free of one or more impurities or contaminants and, for instance, includes polypeptide molecules that are equal to any one of, at least any one of, at most any one of, or between any two of 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure, or at least 99% pure.

The present disclosure includes methods for preventing or ameliorating coronavirus infections. As such, the invention contemplates vaccines for use in both active and passive immunization aspects. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared from native or variant coronavirus polypeptide(s), such as a native or variant coronavirus spike proteins. In other aspects coronavirus spike proteins can be used in combination with other secreted virulence proteins, surface proteins, or immunogenic fragments thereof. In certain aspects, antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.

The preparation of vaccines that contain peptide or polypeptide as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines. In specific aspects, vaccines are formulated with a combination of substances, as described in U.S. Patents 6,793,923 and 6,733,754, which are incorporated herein by reference.

Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 % to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.

The polypeptides may be formulated into a vaccine as neutral or salt forms. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.

Typically, vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including the capacity of the individual’s immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application within a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.

In certain instances, it will be desirable to have multiple administrations of the vaccine, e.g., 2, 3, 4, 5, 6 or more administrations. The vaccinations can be at 1 , 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 ,10, 11 , 12 twelve week intervals, including all ranges there between. Periodic boosters at intervals of 1 -5 years will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies against the antigens, as described in U.S. Patents 3,791 ,932; 4,174,384 and 3,949,064.

B. Carriers

A given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide to a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde, and bis-biazotized benzidine.

C. Adjuvants

The immunogenicity of polypeptide or peptide compositions and/or RNA compositions as described herein can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. A number of adjuvants can be used to enhance an antibody response against a variant SpA polypeptide or coagulase, or any other bacterial protein or combination contemplated herein. Adjuvants can (1 ) trap the antigen in the body to cause a slow release; (2) attract cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of the antigen throughout the subject’s body.

Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include IL-1 , IL-2, IL-4, IL-7, IL-12, □-interferon, GMCSP, BCG, aluminum salts, such as aluminum hydroxide or other aluminum compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). R IB I , which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used. Others adjuvants or methods are exemplified in U.S. Patents 6,814,971 , 5,084,269, 6,656,462, each of which is incorporated herein by reference).

Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1 % solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101 °C for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of Gramnegative bacteria; emulsion in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed to produce an adjuvant effect.

Examples of and often preferred adjuvants include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund’s adjuvants, and aluminum hydroxide.

In some aspects, it is preferred that the adjuvant be selected to be a preferential inducer of either a Th1 or a Th2 type of response. High levels of Th1 -type cytokines tend to favor the induction of cell mediated immune responses to a given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses to the antigen. The distinction of Th1 and Th2-type immune response is not absolute. In reality an individual will support an immune response which is described as being predominantly Th1 or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4+ T cell clones by Mosmann and Coffman (Mosmann, and Coffman, 1989). Traditionally, Th1 -type responses are associated with the production of the INF-y and IL-2 cytokines by T- lymphocytes. Other cytokines often directly associated with the induction of Th1 -type immune responses are not produced by T-cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL- 4, IL-5, IL-6, IL-10.

In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) to enhance immune responses. BRMs have been shown to upregulate T cell immunity or downregulate suppresser cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low- dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/ Mead, NJ) and cytokines such as y-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

D. General Pharmaceutical Compositions

In some aspects, the compositions further comprise one or more stabilizing agents and one or more buffers. A nucleic acid molecule, e.g., a naked or encapsulated nucleic acid, or a polypeptide as disclosed herein may be comprised in a solution comprising the one or more stabilizing agents and one or more buffers. In some aspects, the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinylpyrolidone, glycine, or a combination thereof. In some aspects, the stabilizing agent is a disaccharide, or sugar. In one aspect, the stabilizing agent is sucrose. In another aspect, the stabilizing agent is trehalose. In a further aspect, the stabilizing agent is a combination of sucrose and trehalose. In some aspects, the total concentration of the stabilizing agent(s) in the composition is about 5% to about 10% w/v. For example, the total concentration of the stabilizing agent can be equal to any one of, at least any one of, at most any one of, or between any two of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v or any range or value derivable therein. In specific aspects, the total concentration of the stabilizing agent(s) in the composition is 10% w/v. In specific aspects, the amino acid concentration is 5% w/v. Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, and/or combinations thereof. In some aspects, the buffer is a HEPES buffer, a Tris buffer, or a PBS buffer. In one aspect, the buffer is Tris buffer. In another aspect, the buffer is a HEPES buffer. In a further aspect, the buffer is a PBS buffer. In some aspects, the concentration of the buffer in the composition is about 10 mM. For example, the buffer concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein. In specific aspects, the buffer concentration is 10 mM. The buffer can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the buffer can be at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In specific aspects, the buffer is at pH 7.4.

The compositions may further include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents, and/or excipients, in some cases. In some aspects, the composition further includes a pharmaceutically acceptable vehicle. In some aspects, each of a buffer, stabilizing agent, salt, surfactant, preservative, and excipient are included in the compositions. In other aspects, any one or more of a buffer, stabilizing agent, salt, surfactant, preservative, excipient, carrier, diluent, or vehicle may be excluded from compositions.

Examples of salts include but not limited to sodium salts and/or potassium salts. In some aspects, the sodium salt comprises sodium chloride. In some aspects, the potassium salt comprises potassium chloride. The concentration of the salts in the composition can be about 70 mM to about 140 mM. For example, the salt concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or any range or value derivable therein. In specific aspects, the salt concentration is 70 mM. In specific aspects, the salt concentration is 140 mM. The salt can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the salt can be at a pH equal to any one of, at least any one of, at most any one of, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.

Examples of excipients, which refer to ingredients in the compositions that are not active ingredients, include but are not limited to carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants. Preservatives for use in the compositions disclosed herein include but are not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal. As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), nonaqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Diluents, or diluting or thinning agents, include but are not limited to ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, corn rice, and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10% to about 90% by weight of the total composition, about 25% to about 75%, about 30% to about 60% by weight, or about 12% to about 60%. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic, prophylactic and/or therapeutic compositions is contemplated.

E. Combination Therapy

The compositions and related methods of the present invention, particularly administration of a coronavirus protein, including an isolated polypeptide comprising a native or variant coronavirus spike protein or fragment thereof, may also be used in combination with the administration of traditional therapies. These include, but are not limited to, the administration of antiviral therapies such as nirmatrelvir/ritonavir, remdesivir, or various combinations of antivirals. Also included are the administration of steroids including corticosteroids, e.g., dexamethasone, anti-inflammatories including acetaminophen or ibuprofen, or various combinations thereof.

In one aspect, it is contemplated that a vaccine and/or therapy is used in conjunction with antiviral treatment. Alternatively, the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In aspects where the other agents and/or vaccines are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and immunogenic composition would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer both modalities within about 12 to 24 hours of each other or within about 6 to 12 hours of each other. In some situations, it may be desirable to extend the time period for administration significantly, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, for example antiviral therapy is “A” and the immunogenic polypeptide given as part of an immune therapy regime is “B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the immunogenic compositions of the present disclosure to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the coronavirus spike protein composition, or other compositions described herein. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.

F. Administration

Administration of the compositions described herein can be carried out via any of the accepted modes of administration of agents for serving similar utilities. Pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. Pharmaceutical compositions described herein are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound in aerosol form may hold a plurality of dosage units. The composition to be administered will, in any event, contain a therapeutically and/or prophylactical ly effective amount of a compound within the scope of this disclosure, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings described herein.

A pharmaceutical composition within the scope of this disclosure may be in the form of a solid or liquid. In one aspect, the camer(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid, or an aerosol, which is useful in, for example, inhalator administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present or exclude: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch, lactose, or dextrins; disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate, or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil. The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant, and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included or exclude.

A liquid pharmaceutical composition, whether they be solutions, suspensions, or other like form, may include or exclude one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a compound such that a suitable dosage will be obtained. The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the nucleic acid or polypeptide with sterile, distilled water or other carrier so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a compound consistent with the teachings herein so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

The compositions within the scope of the disclosure are administered in a therapeutically and/or prophylactically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic and/or prophylactic agent employed; the metabolic stability and length of action of the therapeutic and/or prophylactic agent; the age, body weight, general health, gender, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.

Examples

The following examples are included to demonstrate aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1 : Coronavirus spike protein constructs

Several engineered coronavirus spike protein constructs are shown below in Table 3.

Table 3

Example 2: Additional Coronavirus spike protein constructs

Several engineered transmembrance (TM) coronavirus spike protein constructs are shown below in Table 4. Table 4

Example 3: Additional Coronavirus spike protein constructs Several engineered secreted coronavirus spike protein constructs are shown below in Table 5.

Table 5

Example 4: DNA-Transfected HeLa Cell in vitro Expression (IVE) Imaging Assay for Covid Truncated Spike Antigen Screening

To characterize and evaluate COVID antigens, an in vitro expression (IVE) imaging assay was developed using DNA-transfected HeLa cells. Cells were plated in 384 well PDL coated imaging plates and transfected with DNA encoding antigen constructs in pcDNA3.1 (+) vector by Lipofectamine-LTX+Plus. Plasmid DNAs were diluted in Opti-MEM media to create an 11 point 2-fold dilution series for each construct. Antigen construct expression was examined at 24 hours post transfection by immunofluorescence imaging using mAb0403 (Pfizer) which binds to class 2 epitope of RBD. To image the plate, cells were fixed with 4% paraformaldehyde, washed, and blocked with PBS buffer containing 3% BSA. Subsequently, plates were incubated with mAb0403 at 0.2 pg/mL overnight at 4 °C, followed by PBS wash and anti-human AlexaFluor-488-labeled-secondary antibody (0.2 pg/mL) incubation for 2 hours at room temperature. Hoechst nuclear stain is included at 0.2 pg/mL to allow cell count. The plates were subjected to final washes by PBS to remove excess amount of secondary antibody before imaging on the Opera Phenix High Content Imager. The images were analyzed with Signals Image Artist software and multiple endpoints were calculated including MFI (mean fluorescence intensity), cell count (as a measure of toxicity/cell death) and %Antigen-positive cells. For %Antigen-positive cells readout, the WT full-length S(P2) at 20 ng/well was used as the 100% control and Lipofectamine-LTX+Plus alone without DNA was used as the negative control. ECso curves were generated using Signals GeneData Screener software. ECso of MFI readout was used for antigen selection, where mean values for constructs are show below in Table 6. Table 6: IVE EC50 (g/well) of Transmembrane Constructs

Example 5: Secreted Antigen Oligomeric State Characterization

The secreted antigens were expressed by DNA-transfected Expi293 cells. Upon expression, the antigen proteins were secreted into cell media and were purified using affinity chromatography in PBS buffer for further characterization. Purified antigen proteins were profiled by size-exclusion-chromatography (SEC) and the protein retention time off SEC were used to evaluate antigen oligomeric state. These results are shown in Figure ## and below in Table 7.

Table 7

EMBODIMENTS

The foregoing describes embodiments of the present disclsoure along with possible alternatives. These embodiments, however, are merely for example and the disclosure is not restricted thereto.

1. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, wherein said variant comprises an amino acid sequence that is at least 70% identical to any of SEQ ID NOs: 15 to 96.

2. The isolated immunogenic polypeptide of Embodiment 1 , wherein said amino acid sequence is at least 80% identical to any of SEQ ID NOs: 15 to 96. 3. The isolated immunogenic polypeptide of Embodiment 1 , wherein said amino acid sequence is at least 90% identical to any of SEQ ID NOs: 15 to 96.

4. The isolated immunogenic polypeptide of Embodiment 1 , wherein said amino acid sequence is at least 95% identical to any of SEQ ID NOs: 15 to 96.

5. The isolated immunogenic polypeptide of Embodiment 1 , wherein said amino acid sequence is at least 98% identical to any of SEQ ID NOs: 15 to 96.

6. The isolated immunogenic polypeptide of Embodiment 1 , wherein said amino acid sequence is at least 99% identical to any of SEQ ID NOs: 15 to 96.

7. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, wherein said variant comprises an amino acid sequence selected from any of SEQ ID NOs: 15 to 96.

8. The isolated immunogenic polypeptide of any one of Embodiments 1 to 7, further comprising a leader sequence having an amino acid sequence that is at least 80% identical to a leader sequence of the native coronavirus spike protein.

9. The isolated immunogenic polypeptide of Embodiment 8, wherein the leader sequence inhibits disulfide scrambling.

10. A plurality of isolated immunogenic polypeptides comprising variant coronavirus spike proteins that are variants of a native coronavirus spike protein or fragments thereof, wherein the isolated immunogenic polypeptide comprising the plurality of isolated immunogenic polypeptides comprises the isolated immunogenic polypeptide of any one of Embodiments 1 to 9.

11. A pharmaceutically acceptable composition comprising an isolated immunogenic polypeptide comprising a variant of a native coronavirus spike protein according to any one of Embodiments 1 to 10.

12. A pharmaceutically acceptable composition comprising an isolated immunogenic polypeptide comprising a variant of a native coronavirus spike protein according to any one of Embodiments 1 to 10.

13. A vaccine comprising the isolated immunogenic polypeptide of any one of Embodiments 1 to 10. 14. A method of preventing or treating coronavirus infection comprising the step of administering the vaccine or composition of any of Embodiments 11 to 13 to a subject in need thereof.

15. A use of the isolated immunogenic polypeptide of any one of Embodiments 1 to 10 or the composition of any one of Embodiments 11 to 12 in the manufacture of a vaccine for treatment or prevention of coronavirus infection.

16. A use of the isolated immunogenic polypeptide of any one of Embodiments 1 to 10, or the composition of any one of Embodiments 11 to 12 for bioinformatics analyses.

17. A method of synthesizing the isolated immunogenic polypeptide of any one of Embodiments 1 -10, or the composition of any one of Embodiments 11 to 12.

18. Formulations comprising the isolated immunogenic polypeptide of any one of Embodiments 1 to 10, or the composition of any one of Embodiment 11 to 12.

19. A device for delivery of the isolated immunogenic polypeptide of any one of Embodiments 1 to 10, or the composition of any one of Embodiment 11 to 12.

20. The device of Embodiment 19, wherein the device comprises a syringe.

21 . An RNA molecule comprising at least one open reading frame encoding at least one variant coronavirus spike protein, wherein said variant is not derived from SARS- CoV-2.

22. The RNA molecule of of Embodiment 21 , further comprising a 5’ untranslated region (5’ UTR).

23. The RNA molecule of Embodiment 21 , further comprising a 3' untranslated region (3' UTR).

24. The RNA molecule of Embodiment 21 , wherein the RNA molecule further comprises a 5' cap moiety and/or a 3' poly-A tail.

25. The RNA molecule of Embodiment 21 , wherein the open reading frame comprises a G/C content of at least 55%, 60%, 65%, 70%, or 75%, or of or of about 50% to 75% or 55% to 70%.

26. 26. The RNA molecule of Embodiment 21 , wherein the encoded variant coronavirus spike protein localizes in the cellular membrane, localizes in the Golgi and/or is secreted.

27. The RNA molecule of Embodiment 21 , wherein the RNA comprises at least one modified nucleotide.

28. The RNA molecule of Embodiment 21 , wherein each uridine is replaced by N1 -methylpseudouridine ( ).

29. The RNA molecule of Embodiment 21 , wherein the RNA is a mRNA.

30. A composition comprising the RNA molecule of Embodiment 21 , wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

31. The composition of Embodiment 30, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, and a steroid or steroid analog.

32. A method of inducing an immune response against a coronavirus (other than SARS-CoV-2) in a subject, comprising administering to the subject an effective amount of the RNA molecule of Embodiment 21 .

33. A method of preventing, treating, and/or ameliorating an infection, disease, or condition associated with a coronavirus (other than SARS-CoV-2) in a subject, comprising administering to a subject an effective amount of the RNA molecule of Embodiment 21 .

34. The method of Embodiment 32, wherein the RNA molecule is administered as a vaccine.

35. The method of Embodiment 32, wherein the subject is administered a single dose, two doses, three doses, or more, and optionally, a booster dose of the RNA molecule.

36. A method of inducing an immune response against a coronavirus (other than SARS-CoV-2) in a subject, comprising administering to the subject an effective amount of the composition of Embodiment 30.

37. A method of preventing, treating, and/or ameliorating an infection, disease, or condition associated with a coronavirus (other than SARS-CoV-2) in a subject, comprising administering to a subject an effective amount of the composition of Embodiment 30. 38. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, wherein said variant comprises an amino acid sequence of any of SEQ ID NOs: 28 to 40, but where the signal peptide has been removed.

39. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, wherein said variant comprises an amino acid sequence of any of SEQ ID

NOs: 28 to 40, but where the GS-linked Strep purification tags have been removed.

40. An isolated immunogenic polypetide comprising a variant coronavirus spike protein, wherein said variant comprises an amino acid sequence that is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to any of SEQ ID NOs: 15 to 96, wherein the signal peptide and/or the GS-linked Strep purification tags (if present) have been removed from said SEQ ID Nos: 15 to 96.

Claims

1. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, wherein said variant comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 15.

The isolated immunogenic polypeptide of claim 1 , wherein said amino acid sequence is at least 80% identical to SEQ ID NO: 15.

3. The isolated immunogenic polypeptide of claim 1 , wherein said amino acid sequence is at least 90% identical to SEQ ID NO: 15.

4. The isolated immunogenic polypeptide of claim 1 , wherein said amino acid sequence is at least 95% identical to SEQ ID NO: 15.

5. The isolated immunogenic polypeptide of claim 1 , wherein said amino acid sequence is at least 98% identical to SEQ ID NO: 15.

6. The isolated immunogenic polypeptide of claim 1 , wherein said amino acid sequence is at least 99% identical to SEQ ID NO: 15.

7. An isolated immunogenic polypeptide comprising a variant coronavirus spike protein, wherein said variant comprises the amino acid sequence of SEQ ID NO: 15.

8. The isolated immunogenic polypeptide of any one of claims 1 to 7, further comprising a leader sequence having an amino acid sequence that is at least 80% identical to a leader sequence of the native coronavirus spike protein.

9. The isolated immunogenic polypeptide of claim 8, wherein the leader sequence inhibits disulfide scrambling.

10. A pharmaceutically acceptable composition comprising an isolated immunogenic polypeptide comprising a variant of a native coronavirus spike protein according to any one of claims 1 to 9.

11. A vaccine comprising the isolated immunogenic polypeptide of any one of claims 1 to 9.

12. A method of preventing or treating coronavirus infection comprising the step of administering the vaccine or composition of any one of claims 10 or 11 to a subject in need thereof.

13. A use of the isolated immunogenic polypeptide of any one of claims 1 to 9 or the composition of claim 10 in the manufacture of a vaccine for treatment or prevention of coronavirus infection.

14. A use of the isolated immunogenic polypeptide of any one of claims 1 to 9 for bioinformatics analyses.