Immunogenic compositions and uses thereof

Abstract

The present disclosure provides compositions, pharmaceutical preparations, and methods relating to circular polyribonucleotides encoding immunogens and multimerization domains useful in vaccine development and production.

Description

[Background technology]

[0001] Vaccination has made a great contribution to both human and animal health. Since the invention of the first vaccine in 1796, vaccines have become regarded as the most successful method of preventing many infectious diseases by inducing an immune response in subjects. According to the World Health Organization, immunization currently prevents 2 to 3 million deaths annually across all age groups. Today, vaccines have been developed to prevent and control the spread of more than 20 infectious diseases, including diphtheria, tetanus, whooping cough, influenza, and measles, and have led to the complete eradication of smallpox. However, there remains a need to develop new and improved immunogenic compositions and their uses. Summary of the Invention [Means for solving the problem]

[0002] The present disclosure provides compositions, pharmaceutical preparations, and methods relating to cyclic polyribonucleotides encoding one or more immunogens comprising a multimerization domain. The present disclosure also provides methods of using cyclic polyribonucleotides encoding one or more immunogens comprising a multimerization domain. The present disclosure also provides a cyclic polyribonucleotide comprising a first expressed sequence encoding an immunogen comprising a multimerization domain and a second expressed sequence encoding an adjuvant. The present disclosure also provides a cyclic polyribonucleotide comprising an expressed sequence encoding an immunogen comprising a multimerization domain and a non-coding sequence that stimulates the innate immune system. The cyclic polyribonucleotide compositions and pharmaceutical preparations described herein can induce an immune response in a subject upon administration. The cyclic polyribonucleotide compositions and pharmaceutical preparations described herein can be used to treat or prevent a disease, disorder, or condition in a subject.

[0003] In a first aspect, the disclosure provides a circular polyribonucleotide comprising an open reading frame comprising a sequence encoding an immunogen and a sequence encoding a multimerization domain.

[0004] In some embodiments, the open reading frame comprises, in the following 5' to 3' order: a first sequence encoding an immunogen and a second sequence encoding a multimerization domain.

[0005] In some embodiments, the open reading frame includes, in the following 5' to 3' order: a first sequence encoding an immunogen, a second sequence encoding a multimerization domain, and a third sequence encoding an immunogen.

[0006] In some embodiments, the open reading frame includes, in the following 5' to 3' order, a first sequence encoding an immunogen, a second sequence encoding a multimerization domain, a third sequence encoding an immunogen, and a fourth sequence encoding a multimerization domain.

[0007] In some embodiments, the open reading frame includes, in the following 5' to 3' order: a first sequence encoding an immunogen, a second sequence encoding a multimerization domain, and a third sequence encoding a multimerization domain.

[0008] In some embodiments, the open reading frame comprises, in the following 5' to 3' order, a first sequence encoding a multimerization domain and a second sequence encoding an immunogen.

[0009] In some embodiments, the open reading frame comprises, in the following 5' to 3' order: a first sequence encoding a multimerization domain, a second sequence encoding an immunogen, and a third sequence encoding a multimerization domain.

[0010] In some embodiments, the open reading frame includes, in the following 5' to 3' order: a first sequence encoding a multimerization domain, a second sequence encoding an immunogen, a third sequence encoding a multimerization domain, and a fourth sequence encoding an immunogen.

[0011] In some embodiments, the open reading frame includes, in the following 5' to 3' order: a first sequence encoding a multimerization domain, a second sequence encoding a multimerization domain, and a third sequence encoding an immunogen.

[0012] In some embodiments, the or each multimerization domain comprises a T4 Foldon domain. In some embodiments, the or each multimerization domain comprises a ferritin domain. In some embodiments, the or each multimerization domain comprises a β-annulus peptide. In some embodiments, the or each multimerization domain comprises an AaLS peptide. In some embodiments, the or each multimerization domain comprises a lumazine synthase domain.

[0013] In some embodiments, the open reading frame comprises, in the following 5' to 3' order, a first sequence encoding an immunogen and a second sequence encoding a T4 Foldon domain:

[0014] In some embodiments, the open reading frame comprises, in the following 5' to 3' order, a first sequence encoding an immunogen and a second sequence encoding a ferritin domain.

[0015] In some embodiments, the open reading frame comprises, in the following 5' to 3' order, a first sequence encoding an immunogen and a second sequence encoding a β-annulus peptide.

[0016] In some embodiments, the open reading frame comprises, in the following 5' to 3' order, a first sequence that encodes an immunogen and a second sequence that encodes an AaLS peptide:

[0017] In some embodiments, the open reading frame comprises, in the following 5' to 3' order: a first sequence encoding an immunogen, a second sequence encoding a T4 Foldon domain, and a third sequence encoding an immunogen.

[0018] In some embodiments, the open reading frame comprises, in the following 5' to 3' order: a first sequence encoding an immunogen, a second sequence encoding a T4 Foldon domain, and a third sequence encoding a ferritin domain.

[0019] In some embodiments, the open reading frame comprises, in the following 5' to 3' order: a first sequence encoding an immunogen, a second sequence encoding a ferritin domain, and a third sequence encoding a T4 Foldon domain.

[0020] In some embodiments, each immunogen is independently operably linked to a secretory signal sequence.

[0021] In some embodiments, the open reading frame is operably linked to an IRES.

[0022] In some embodiments, the circular polyribonucleotide further comprises a second open reading frame encoding a second polypeptide operably linked to a second IRES.

[0023] In some embodiments, the second polypeptide is a polypeptide immunogen. In some embodiments, the second polypeptide is a polypeptide adjuvant. In some embodiments, the polypeptide adjuvant is a cytokine, a chemokine, a costimulatory molecule, an innate immune stimulator, a signal transduction molecule, a transcription activator, a cytokine receptor, a bacterial component, a viral component, or a component of the innate immune system.

[0024] In some embodiments, the circular polyribonucleotide further comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator. In some embodiments, the innate immune system stimulator is selected from a GU-rich motif, an AU-rich motif, a structured region comprising dsRNA, or an aptamer.

[0025] In another aspect, the present disclosure provides an immunogenic composition comprising the cyclic polyribonucleotide described herein and a pharma- ceutically acceptable excipient.In some embodiments, the composition further comprises a second cyclic polyribonucleotide.In some embodiments, the second cyclic polyribonucleotide comprises an open reading frame that encodes an immunogen.In some embodiments, the second cyclic polyribonucleotide comprises an open reading frame that encodes a polypeptide adjuvant.In some embodiments, the second cyclic polyribonucleotide comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator.

[0026] In another aspect, the disclosure provides a method of inducing an immune response to an immunogen in a subject, the method comprising administering to the subject a cyclic polyribonucleotide or an immunogenic composition described herein.

[0027] In another aspect, the disclosure provides a method of treating or preventing a disease, condition, or disorder in a subject, comprising administering to the subject a cyclic polyribonucleotide or immunogenic composition described herein.

[0028] In some embodiments, the subject is a human subject.

[0029] In some embodiments, the methods further comprise administering to the subject an adjuvant.

[0030] In some embodiments, the methods further comprise administering to the subject a polypeptide immunogen.

[0031] definition The present disclosure will be described with respect to particular embodiments and with reference to certain figures but the disclosure is not limited thereto but only by the claims. Terms used hereinafter generally should be understood in their ordinary sense unless otherwise indicated.

[0032] As used herein, the term "adaptive immune response" refers to either a humoral or a cellular immune response. For the purposes of this disclosure, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T lymphocytes and / or other white blood cells.

[0033] As used herein, the term "adjuvant" refers to a composition (e.g., a compound, a polypeptide, a nucleic acid, or a lipid) that increases an immune response, e.g., increases a specific immune response to an immunogen. Increasing an immune response includes enhancing or broadening the specificity of either or both the antibody response and the cellular immune response.

[0034] As used herein, the term "carrier" refers to a compound, composition, reagent, or molecule that facilitates the transport or delivery of a composition (e.g., polyribonucleotide) to a subject, tissue, or cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles encapsulated or covalently attached to cyclic polyribonucleotides), liposomes, fusosomes, exosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently attached to polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0035] As used herein, the terms "circRNA", "circular polyribonucleotide", "circular RNA", and "circular polyribonucleotide molecule" are used interchangeably and refer to polyribonucleotide molecules having a structure with no free ends (i.e., free 3' and / or 5' ends), e.g., polyribonucleotide molecules that form a circular or endless structure through covalent (e.g., covalently closed) or non-covalent bonds. A circular polyribonucleotide can be a covalently closed circular polyribonucleotide.

[0036] As used herein, the term "circularization efficiency" is a measure of the resulting circular polyribonucleotide relative to its non-circular starting material.

[0037] The term "diluent" refers to a vehicle that includes an inert solvent capable of diluting or dissolving a composition described herein (e.g., a composition including a cyclic polyribonucleotide). The diluent may be an RNA solubilizing agent, a buffer, an isotonic agent, or a mixture thereof. The diluent may be a liquid diluent or a solid diluent. Non-limiting examples of liquid diluents include water or other solvents, solubilizing agents, and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and 1,3-butanediol. Non-limiting examples of solid diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, or powdered sugar.

[0038] As used herein, the terms "disease," "disorder," and "condition" each refer to a state of less than optimal health, e.g., a condition that is or would be normally diagnosed or treated by a medical professional.

[0039] As used herein, the term "epitope" refers to a portion or entirety of an immunogen that is recognized, targeted, or bound by an antibody or T-cell receptor. An epitope can be a linear epitope, e.g., a continuous sequence of nucleic acids or amino acids. An epitope can be a conformational epitope, e.g., an epitope that includes amino acids that form an epitope in a folded conformation of a protein. A conformational epitope can include non-contiguous amino acids from a primary amino acid sequence. As another example, a conformational epitope includes a nucleic acid that forms an epitope in a folded conformation of an immunogenic sequence based on its secondary or tertiary structure.

[0040] As used herein, the term "expressed sequence" refers to a nucleic acid sequence that encodes a product, such as a peptide or polypeptide (e.g., an immunogen), or a regulatory nucleic acid. An exemplary expressed sequence that encodes a peptide or polypeptide can include multiple nucleotide triplets, each of which can encode an amino acid, and are referred to as a "codon."

[0041] As used herein, the term "fragment" in reference to a polypeptide or nucleic acid sequence (e.g., a polypeptide immunogen or a nucleic acid sequence encoding a polypeptide immunogen) refers to a contiguous portion of less than the entire polypeptide or nucleic acid sequence. A fragment of a polypeptide immunogen or a nucleic acid sequence encoding a polypeptide immunogen refers to a contiguous percentage (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the full length) of a sequence, such as, for example, a sequence disclosed herein. It is understood that the present disclosure contemplates all fragments (e.g., immunogenic fragments) of all immunogens disclosed herein.

[0042] As used herein, the term "GC content" refers to the percentage of guanine (G) and cytosine (C) in a nucleic acid sequence. The formula for calculating GC content is (G+C) / (A+G+C+U)×100% (for RNA) or (G+C) / (A+G+C+T)×100% (for DNA). Similarly, the term "uridine content" refers to the percentage of uridine (U) in a nucleic acid sequence. The formula for calculating uridine content is U / (A+G+C+U)×100%. Similarly, the term "thymidine content" refers to the percentage of thymidine (T) in a nucleic acid sequence. The formula for calculating thymidine content is T / (A+G+C+T)×100%.

[0043] As used herein, the term "innate immune system stimulator" refers to a substance that induces an innate immune response, in part, by inducing expression of one or more genes involved in innate immunity, including, but not limited to, type I interferons (e.g., IFNα, INFβ, and / or IFNγ), proinflammatory cytokines (e.g., IL-1, IL-12, IL-18, TNF-α, and / or GM-CSF), retinoic acid-inducible gene-I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, also known as IFIH1), 2'-5' oligoadenylate synthase 1 (OAS1), OAS-like protein (OASL), and / or protein kinase R (PKR). Innate immune system stimulators may act as adjuvants (e.g., when administered in combination with or formulated with a ribonucleotide encoding an immunogen). The innate immune system stimulator may be a separate (e.g., not encoded by or incorporated as a sequence in a polyribonucleotide) molecular entity, such as STING (e.g., caSTING), TLR3, TLR4, TLR9, TLR7, TLR8, TLR7, RIG-I / DDX58, and MDA-5 / IFIH1 or a constitutively active mutant thereof. The innate immune system stimulator may be encoded by (e.g., expressed from) a polyribonucleotide. The polyribonucleotide may alternatively or additionally include a ribonucleotide sequence that acts as an innate immune system stimulator (e.g., a structural region that includes a GU-rich motif, an AU-rich motif, dsRNA, or an aptamer).

[0044] As used herein, the term "impurity" refers to an undesired substance present in a composition (e.g., a pharmaceutical composition described herein). In some embodiments, the impurity is a process-related impurity. In some embodiments, the impurity is a product-related substance other than the desired product in the final composition (e.g., other than an active drug component, e.g., a cyclic polyribonucleotide described herein). As used herein, the term "process-related impurity" refers to a substance used, present, or produced in the manufacture of a composition, preparation, or product that is undesirable in the final composition, preparation, or product other than the linear polyribonucleotide described herein. In some embodiments, the process-related impurity is an enzyme used in the synthesis or cyclization of polyribonucleotides. As used herein, the term "product-related substance" refers to a substance or by-product produced during the synthesis of a composition, preparation, or product, or any intermediate thereof. In some embodiments, the product-related substance is a deoxyribonucleotide fragment. In some embodiments, the product-related substance is a deoxyribonucleotide monomer. In some embodiments, product-related materials are derivatives or fragments of one or more of the polyribonucleotides described herein (e.g., fragments of 10, 9, 8, 7, 6, 5, or 4 ribonucleic acids, monoribonucleic acids, diribonucleic acids, or triribonucleic acids).

[0045] As used herein, the term "immunogen" refers to any molecule or molecular structure that contains one or more epitopes that are recognized, targeted, or bound by an antibody or T-cell receptor. In particular, an immunogen induces an immune response in a subject (e.g., is immunogenic as defined herein). An immunogen can induce an immune response in a subject, where an immune response refers to a series of molecular, cellular, and biological events that are elicited when the immune system encounters an immunogen. An immune response can be a humoral and / or cellular immune response. These can include the production of antibodies and the proliferation of B cells and T cells. To determine whether an immune response has occurred and to monitor its progress, an immunized subject can be monitored for the appearance of immune reactants against a particular immunogen. An immune response against most immunogens induces the production of both specific antibodies and specific effector T cells. In some embodiments, the immunogen is foreign to the host. In some embodiments, the immunogen is not foreign to the host. An immunogen can include all or part of a polypeptide, polysaccharide, polynucleotide, or lipid. The immunogen may also be a mixture of polypeptides, polysaccharides, polynucleotides, and / or lipids. For example, the immunogen may be a translationally modified polypeptide. A "polypeptide immunogen" refers to an immunogen that comprises a polypeptide. The polypeptide immunogen may also include one or more post-translational modifications, and / or may be complexed with one or more additional molecules, and / or may have a selected tertiary or quaternary structure, each of which may determine or affect the immunogenicity of the polypeptide.

[0046] As used herein, the term "immunogenicity" refers to the potential to elicit a response above a given threshold to a substance in a particular immune response assay. The assay can be, for example, the expression of a particular inflammatory marker, the production of antibodies, or an assay of immunogenicity as described herein. In some embodiments, an immune response can be elicited when an organism's immune system or a particular type of immune cell is exposed to the immunogen.

[0047] Immunogenic responses can be evaluated by assessing antibodies in the plasma or serum of a subject using total antibody assays, confirmatory tests, antibody titration and isotyping, and neutralizing antibody assessment. Total antibody assays measure all antibodies generated as part of an immune response in the serum or plasma of a subject to which an immunogen has been administered. The most commonly used test to detect antibodies is the ELISA (enzyme-linked immunosorbent assay), which detects antibodies in the serum being tested that bind to the antibody of interest, including IgM, IgD, IgG, IgA, and IgE. Immunogenic responses can be further evaluated by confirmatory assays. Following total antibody assessment, confirmatory assays can be used to confirm the results of the total antibody assay. Competitive assays can be used to confirm that the antibodies are specifically binding to the target and that a positive finding in a screening assay is not the result of a non-specific interaction of the test serum or detection reagent with other substances in the assay.

[0048] Immunogenic response can be evaluated by isotyping and titration. Isotyping assay can be used to evaluate only relevant antibody isotypes. For example, expected isotypes can be IgM and IgG, which can be specifically detected and quantified by isotyping and titration, and compared to total antibodies present.

[0049] Immunogenic responses can be assessed by neutralizing antibody assays (nAbs). Neutralizing antibody assays (nAbs) can be used to determine whether antibodies produced in response to an immunogen neutralize the immunogen, thereby inhibiting the immunogen from affecting the target and causing aberrant pharmacokinetic behavior. Often, nAb assays are cell-based assays in which target cells are incubated with the antibody. A variety of cell-based nAb assays can be used, including but not limited to cell proliferation, viability, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), cytopathic effect inhibition (CPE), apoptosis, ligand-stimulated cell signaling, enzyme activity, reporter gene assays, protein secretion, metabolic activity, stress, and mitochondrial function. Detection readouts include absorbance, fluorescence, luminescence, chemiluminescence, or flow cytometry. Ligand-binding assays can also be used to measure the binding affinity of an immunogen to an antibody in vitro to assess neutralizing efficacy.

[0050] Furthermore, induction of a cellular immune response can be assessed by measuring T cell activation in a subject using cell markers on T cells obtained from the subject. Blood samples, lymph node biopsies, or tissue samples can be collected from the subject, and T cells from the samples can be assessed for one or more (e.g., two, three, four, or more) activation markers: CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, CD134, CD69, CD62L, or CD44. T cell activation can also be assessed using the same method in an in vivo animal model. This assay can also be performed by adding an immunogen to T cells in vitro (e.g., T cells obtained from a subject, an animal model, a repository, or a commercial source) and measuring the aforementioned markers to assess T cell activation. Similar approaches can be used to assess the effect on activation of other immune cells, such as eosinophils (markers: CD35, CD11b, CD66, CD69, and CD81), dendritic cells (markers: IL-8, MHC class II, CD40, CD80, CD83, and CD86), basophils (CD63, CD13, CD4, and CD203c), and neutrophils (CD11b, CD35, CD66b, and CD63). These markers can be assessed using flow cytometry, immunohistochemistry, in situ hybridization, and other assays that allow for the measurement of cell markers. Comparison of results before and after administration of the immunogen can be used to determine the effect.

[0051] As used herein, the term "eliciting an immune response" refers to initiating, amplifying, or maintaining an immune response by a subject. Eliciting an immune response may refer to an adaptive immune response or an innate immune response. Eliciting an immune response can be measured as described above.

[0052] As used herein, the term "linear counterpart" refers to a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) and has two free ends (i.e., the non-cyclic form of a circular polyribonucleotide (and fragments thereof)). In some embodiments, a linear counterpart (e.g., a form prior to circularization) is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) and the same or similar nucleic acid modifications as a circular polyribonucleotide and has two free ends (i.e., the non-cyclic form of a circular polyribonucleotide (and fragments thereof)). In some embodiments, a linear counterpart is a polyribonucleotide molecule (and fragments thereof) that has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) and a different nucleic acid modification, or has the same or similar nucleotide sequence as a circular polyribonucleotide (e.g., 100%, 95%, 90%, 85%, 80%, 75%, or any percentage sequence identity therebetween) but no nucleic acid modification and has two free ends (i.e., a non-cyclic form of a circular polyribonucleotide (and fragments thereof)). In some embodiments, a fragment of a polyribonucleotide molecule that is a linear counterpart is any portion of the linear counterpart polyribonucleotide molecule that is shorter than the linear counterpart polyribonucleotide molecule. In some embodiments, the linear counterpart further comprises a 5' cap. In some embodiments, the linear counterpart further comprises a polyadenosine tail. In some embodiments, the linear counterpart further comprises a 3' UTR. In some embodiments, the linear counterpart further comprises a 5' UTR.

[0053] As used herein, the terms "linear RNA", "linear polyribonucleotide", and "linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having a 5' and a 3' end. One or both of the 5' and 3' ends may be free ends or may be attached to another moiety. Linear RNA includes RNA that has not been circularized (e.g., not previously circularized) and can be used as starting material for circularization, for example, by splint ligation or chemical, enzymatic, ribozyme or splicing catalyst circularization methods.

[0054] As used herein, the term "modified ribonucleotide" means a nucleotide having at least one modification to the sugar, nucleobase, or internucleoside linkage.

[0055] As used herein, the term "multimerization domain" refers to a polypeptide domain that self-assembles to form a multimer (e.g., a dimer, trimer, tetramer, or oligomer). In particular embodiments, the multimerization domain can be fused to a polypeptide (e.g., a polypeptide immunogen). In such cases, fusion to the multimerization domain results in the formation of a multimeric immunogen complex having two or more immunogens upon expression of a polypeptide that includes an immunogen covalently linked to the multimerization domain.

[0056] As used herein, the term "naked delivery" refers to a formulation for delivery to cells without the aid of a carrier or covalent modification of moieties that aid in delivery to cells. Naked delivery formulations do not include any transfection reagent, cationic carrier, carbohydrate carrier, nanoparticle carrier, or protein carrier. For example, a naked delivery formulation of a cyclic polyribonucleotide is a formulation that includes cyclic polyribonucleotide without covalent modification and does not include a carrier.

[0057] As used herein, the terms "nicked RNA," "nicked linear polyribonucleotide," and "nicked linear polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having 5' and 3' ends that result from cleavage or degradation of a circular RNA.

[0058] As used herein, the term "non-circular RNA" refers to all nicked RNA and linear RNA.

[0059] The term "pharmaceutical composition" is also intended to disclose that the cyclic polyribonucleotide contained within the pharmaceutical composition can be used for the treatment of the human or animal body by therapy. Thus, a pharmaceutical composition is meant to be equivalent to "cyclic polyribonucleotide for use in therapy."

[0060] As used herein, the term "polynucleotide" refers to a molecule that includes one or more nucleic acid subunits or nucleotides, and can be used interchangeably with "nucleic acid" or "oligonucleotide." A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, ten, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. Polyribonucleotide or ribonucleic acid, or RNA, can refer to a polymer that includes multiple ribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0061] "Polydeoxyribonucleotide", "deoxyribonucleic acid", and "DNA" refer to a polymer comprising a plurality of deoxyribonucleotides polymerized through phosphodiester bonds. Nucleotides can be nucleoside monophosphates or nucleoside polyphosphates. Nucleotides refer to deoxyribonucleoside polyphosphates, such as deoxyribonucleoside triphosphates (dNTPs), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, including detectable tags (e.g., fluorophores), such as luminescent tags and markers. Nucleotides can include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or any other subunits that are specific to one or more complementary A, C, G, T, or U, or that are complementary to purines (i.e., A or G, or variants thereof) or pyrimidines (i.e., C, T, or U, or variants thereof). In some instances, the polynucleotide is a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a derivative or variant thereof. In some instances, the polynucleotide is a small interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a small hairpin RNA (shRNA), a small nuclear RNA (snRNA), a messenger RNA (mRNA), a pre-mRNA (pre-mRNA), an antisense RNA (asRNA), to name a few, and includes both nucleotide sequences and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In some instances, the polynucleotide molecule is circular. The polynucleotide may have a variety of lengths. The nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. Polynucleotides can be isolated from cells or tissues.As embodied herein, polynucleotide sequences can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0062] A polynucleotide (e.g., a polyribonucleotide or a polydeoxyribonucleotide) may include one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylketone, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguan ... -methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. In some cases, nucleotides can include modifications of their phosphate moieties, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains having 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications of the thiol moiety (e.g., α-thiotriphosphate and β-thiotriphosphate).Nucleic acid molecules can also be modified at the base moiety, sugar moiety, or phosphate backbone (e.g., at one or more atoms typically available to form hydrogen bonds with complementary nucleotides, and / or at one or more atoms typically not available to form hydrogen bonds with complementary nucleotides). Nucleic acid molecules can also include amine-modifying groups such as aminoallyl-dUTP (aa-dUTP) and aminohexylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS). Alternatives to standard DNA or RNA base pairs in the oligonucleotides of the present disclosure can provide high bit density per cubic mm, higher safety (resistance to accidental or deliberate synthesis of natural toxins), easier identification in photoprogrammed polymerases, or subsecondary structures. Such alternative base pairs compatible with native and mutant polymerases for de novo and / or amplicon synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, incorporated herein by reference for all purposes.

[0063] As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) linked together, most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs thereof. Polypeptides can be single molecules or multi-molecular complexes such as dimers, trimers, or tetramers. Polypeptides can also include single chain or multi-chain polypeptides such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0064] As used herein, the term "prevent" means reducing the likelihood of a disease, disorder, or condition occurring, or reducing the severity or frequency of symptoms of a disease or disorder that subsequently occurs. Therapeutic agents can be administered to subjects who are at increased risk of developing a disease or disorder compared to members of the general population to prevent the onset of the disease or condition or to reduce its severity. Therapeutic agents can be administered prophylactically (e.g., prior to the onset of symptoms or manifestations of the disease or disorder).

[0065] As used interchangeably herein, the terms "polyA" and "polyA sequence" refer to a contiguous untranslated region of a nucleic acid molecule consisting of adenosine residues, at least 5 nucleotides in length. In some embodiments, the polyA sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some embodiments, the polyA sequence is located 3' to (e.g., downstream of) an open reading frame (e.g., an open reading frame encoding a polypeptide) and the polyA sequence is 3' to a termination element (e.g., a stop codon), such that the polyA is not translated. In some embodiments, the polyA sequence is located 3' to the termination element and the 3' untranslated region.

[0066] As used herein, the term "regulatory element" refers to a moiety, such as a nucleic acid sequence, that modulates the expression of an expression sequence within a circular polyribonucleotide.

[0067] As used herein, the term "replication elements" are sequences and / or motifs useful for replication or that initiate transcription of a circular polyribonucleotide.

[0068] As used herein, the term "systemic delivery" or "systemic administration" refers to a route of administration of a pharmaceutical composition or other substance into the circulatory system (e.g., the blood system or lymphatic system). Systemic administration can include oral administration, parenteral administration, intranasal administration, sublingual administration, rectal administration, transdermal administration, or any combination thereof. As used herein, the term "non-systemic delivery" or "non-systemic administration" can refer to any other route of administration of a pharmaceutical composition or other substance other than systemic delivery (e.g., the substance being delivered does not enter the circulatory system (e.g., the blood system and lymphatic system) of the subject's body).

[0069] As used herein, the term "sequence identity" is determined by alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm. Thus, sequences can be called "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (when optimally aligned, for example, by the programs GAP or BESTFIT, using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the default parameters of GAP are used: gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is the nwsgapdna.cmp scoring matrix, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and sequence identity percentage scores can be determined using computer programs such as GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program "Needle"). Alternatively or additionally, percent identity can be determined by searching in a database using algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

[0070] "Signal sequence" refers to a polypeptide sequence (eg, 10-45 amino acids in length) present at the N-terminus of a polypeptide sequence of a nascent protein that targets the polypeptide sequence to the secretory pathway.

[0071] As used herein, the term "treat" or "treating" refers to the therapeutic treatment of a disease or disorder (e.g., an infection, cancer, toxicity, or allergic response) of a subject. The effect of treatment can include reversing, alleviating, reducing the severity of, curing, inhibiting progression of, reducing the likelihood of recurrence of, the condition of the disease or disorder (i.e., not worsening), and / or preventing the spread of the disease or disorder compared to the condition and / or symptoms of the disease or disorder in the absence of therapeutic treatment.

[0072] As used herein, the term "termination element" is a portion, such as a nucleic acid sequence, that terminates translation of an expressed sequence in a circular polyribonucleotide.

[0073] As used herein, the term "total ribonucleotide molecules" refers to the total amount of any ribonucleotide molecules, including linear polyribonucleotide molecules, cyclic polyribonucleotide molecules, monomeric ribonucleotides, other polyribonucleotide molecules, fragments thereof, and modified forms thereof, as measured by the total amount of ribonucleotide molecules.

[0074] As used herein, the term "translation efficiency" refers to the rate or amount of protein or peptide production from a ribonucleotide transcript. In one embodiment, translation efficiency can be expressed as the amount of protein or peptide produced per given amount of transcript encoding the protein or peptide (e.g., in a given translation system, e.g., in an in vitro translation system such as a rabbit reticulocyte lysate, or in an in vivo translation system such as a eukaryotic or prokaryotic cell, for a given period of time).

[0075] As used herein, the term "translation initiation sequence" is a nucleic acid sequence that initiates translation of an expression sequence in a circular polyribonucleotide. [Brief description of the drawings]

[0076] [Figure 1]1 is a schematic diagram of an exemplary polyribonucleotide construct encoding an immunogen and one or more multimerization domains, and an exemplary corresponding immunogen complex. [Diagram 2] FIG. 1 is a schematic diagram of an exemplary circular RNA comprising two expression sequences, each expression sequence operably linked to an IRES, and at least one expression sequence being an immunogen that includes a multimerization domain. [Diagram 3] FIG. 1 is a schematic diagram of an exemplary circular RNA comprising two expressed sequences separated by a cleavage domain (e.g., 2A, furin site, or furin-2A), at least one of the expressed sequences being an immunogen comprising a multimerization domain, and all operably linked to an IRES. [Figure 4] 1 shows a schematic diagram of a circular RNA comprising an ORF encoding an immunogen containing a multimerization domain and a polynucleotide adjuvant sequence (eg, a non-coding nucleotide sequence that stimulates the innate immune system). [Diagram 5] FIG. 1 shows a schematic diagram of multiple circular RNAs, where a first circular RNA contains an ORF encoding an immunogen that includes a multimerization domain, and a second circular RNA contains an ORF encoding either a second immunogen or a polypeptide adjuvant. [Figure 6] Western blot showing RBD and RBD-Foldon expression by circular RNA encoding SARS-CoV-2 RBD immunogen (circRNA-RBD) or circular RNA encoding SARS-CoV-2 RBD immunogen fused to the Foldon multimerization domain (circRNA-RBD-Foldon) in HEK293T cells 24 hours after transfection. Asterisks indicate that the samples were run under denaturing conditions. Figure 6 shows that circRNA-RBD expresses RBD monomers and circRNA encoding SARS-CoV-2 RBD immunogen fused to the T4 Foldon multimerization domain expresses and forms trimeric structures in vitro (trimers). [Figure 7]Figure 1 shows expression of SARS-CoV-2 RBD immunogen in serum of mice following administration of either circular RNA encoding the SARS-CoV-2 RBD immunogen or circular RNA encoding the SARS-CoV-2 RBD immunogen fused to the Foldon multimerization domain. [Figure 8] Figure 2 shows that binding antibodies were produced in the serum of mice 14, 27, 35, and 42 days after administration of the first dose (post-priming dose) of either circular RNA encoding a SARS-CoV-2 RBD immunogen or circular RNA encoding a SARS-CoV-2 RBD immunogen fused to a Foldon multimerization domain. [Figure 9] Figure 2 shows that T cell responses were primed in mice 42 days after administration of an initial dose (post-priming dose) of either a circular RNA encoding a SARS-CoV-2 RBD immunogen or a circular RNA encoding a SARS-CoV-2 RBD immunogen fused to a Foldon multimerization domain. [Figure 10] We show that neutralizing antibodies against SARS-CoV-2 were produced in the serum of mice 42 days after administration of an initial dose (post-priming dose) of either a circular RNA encoding a SARS-CoV-2 RBD immunogen or a circular RNA encoding a SARS-CoV-2 RBD immunogen fused to a Foldon multimerization domain. [Figure 11] Figure 1 shows expression of SARS-CoV-2 RBD immunogen fused to the T4 Foldon multimerization domain in serum of cynomolgus monkeys following intramuscular injection of a 100μg dose of LNP-formulated circular RNA or a 1000μg dose of LNP-formulated circular RNA. [Figure 12] Figure 1 shows that RBD-specific antibodies were primed in cynomolgus monkeys 42 days after administration of a first dose (post-priming dose) of either LNP-formulated cyclic polyribonucleotide encoding a SARS-CoV-2 RBD immunogen fused to a T4 Foldon multimerization domain or adjuvanted cyclic polyribonucleotide encoding a SARS-CoV-2 RBD immunogen fused to a T4 Foldon multimerization domain. [Figure 13] 1 shows that neutralizing antibodies were primed in cynomolgus monkeys 42 days after administration of a first dose (post-priming dose) of either LNP-formulated cyclic polyribonucleotide encoding a SARS-CoV-2 RBD immunogen fused to a T4 Foldon multimerization domain or adjuvanted cyclic polyribonucleotide encoding a SARS-CoV-2 RBD immunogen fused to a T4 Foldon multimerization domain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The present disclosure provides compositions, pharmaceutical preparations, and methods relating to cyclic polyribonucleotides encoding one or more immunogens comprising a multimerization domain. The present disclosure also provides methods of using cyclic polyribonucleotides encoding one or more immunogens comprising a multimerization domain. The cyclic polyribonucleotide compositions and pharmaceutical preparations described herein can induce an immune response in a subject upon administration. The cyclic polyribonucleotide compositions and pharmaceutical preparations described herein can be used to treat or prevent a disease, disorder, or condition in a subject.

[0078] Circular Polyribonucleotides The circular polyribonucleotides described herein may comprise any one or more of the elements described herein and an expression sequence encoding an immunogen comprising a multimerization domain. In some embodiments, the circular polyribonucleotide comprises any feature or any combination of features as disclosed in WO 2019 / 118919 (hereby incorporated by reference in its entirety).

[0079] In some embodiments, a cyclic polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

[0080] In some embodiments, the cyclic polyribonucleotide is 500 to 20,000 nucleotides, 1,000 to 20,000 nucleotides, 2,000 to 20,000 nucleotides, or 5,000 to 20,000 nucleotides. In some embodiments, the cyclic polyribonucleotide is 500 to 10,000 nucleotides, 1,000 to 10,000 nucleotides, 2,000 to 10,000 nucleotides, or 5,000 to 10,000 nucleotides.

[0081] immunogen The circular polyribonucleotides described herein include at least one expression sequence encoding an immunogen comprising a multimerization domain. The circular polyribonucleotides described herein may include a plurality of expression sequences, where at least one expression sequence encodes an immunogen comprising a multimerization domain. The circular polyribonucleotides described herein may include two or more (2, 3, 4, 5, 6 or more) expression sequences, where each expression sequence encodes an immunogen comprising a multimerization domain. The circular polyribonucleotides described herein may include a first expression sequence encoding an immunogen comprising a multimerization domain and a second expression sequence encoding an adjuvant. The circular polyribonucleotides described herein may include an expression sequence encoding an immunogen comprising a multimerization domain and a non-coding sequence that stimulates the innate immune system.

[0082] An immunogen includes one or more epitopes that are recognized, targeted, or bound by a given antibody or T cell receptor. An epitope can be a linear epitope, such as a nucleic acid or a continuous sequence of amino acids. An epitope can be a conformational epitope, such as an epitope that includes amino acids that form an epitope in a folded conformation of a protein. A conformational epitope can include non-contiguous amino acids from a primary amino acid sequence. As another example, a conformational epitope includes a nucleic acid that forms an epitope in a folded conformation of an immunogenic sequence based on its secondary or tertiary structure.

[0083] In some embodiments, the immunogen comprises all or a portion of a protein, peptide, glycoprotein, lipoprotein, phosphoprotein, ribonucleoprotein, carbohydrate (e.g., polysaccharide), lipid (e.g., phospholipid or triglyceride), or nucleic acid (e.g., DNA, RNA).

[0084] In other embodiments, the immunogen includes a protein immunogen or epitope (e.g., a peptide immunogen or peptide epitope derived from a protein, glycoprotein, lipoprotein, phosphoprotein, or ribonucleoprotein). The immunogen may include an amino acid, sugar, lipid, phosphoryl, or sulfonyl group, or a combination thereof.

[0085] In certain embodiments, the immunogen is a polypeptide immunogen.

[0086] Polypeptide immunogens can include post-translational modifications, such as glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation.

[0087] In certain embodiments, the immunogen comprises an epitope that comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids, or more. In certain embodiments, the epitope comprises 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, or 30 or less amino acids, or fewer. In certain embodiments, the epitope comprises or contains 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, or 30 amino acids. In certain embodiments, the epitope contains 5 amino acids. In certain embodiments, the epitope contains 6 amino acids. In certain embodiments, the epitope contains 7 amino acids. In certain embodiments, the epitope contains 8 amino acids. In certain embodiments, the epitope can be about 8 to about 11 amino acids. In certain embodiments, the epitope can be about 9 to about 22 amino acids.

[0088] The immunogen may comprise an immunogen recognized by B cells, an immunogen recognized by T cells, or a combination thereof. In some embodiments, the immunogen comprises an immunogen recognized by B cells. In some embodiments, the immunogen is an immunogen recognized by B cells. In some embodiments, the immunogen comprises an immunogen recognized by T cells. In some embodiments, the immunogen is an immunogen recognized by T cells.

[0089] The epitope may include those recognized by B cells, immunogens recognized by T cells, or a combination thereof. In some embodiments, the epitope includes an epitope recognized by B cells. In some embodiments, the epitope is an epitope recognized by B cells. In some embodiments, the epitope includes an epitope recognized by T cells. In some embodiments, the epitope is an epitope recognized by T cells.

[0090] Techniques for identifying immunogens and epitopes in silico are described, for example, in Sanchez-Trincado JL, et al., Fundamentals and methods for T- and B-cell epitope prediction, J. Immunol. Res., 2017:2680160. doi:10.1155 / 2017 / 2680160(2017)); Grifoni, A, et al., A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2, Cell Host Microbe, 27(4):671-80(2020)); Russi RC et al., In silico prediction of epitopes recognized by T cells and B cells in PmpD: First step towards the design of a Chlamydia trachomatis vaccine, Biomedical J.,41(2):109-17(2018));Baruah V,et al.,Immunoinformatics-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV,J.Med.Virol.,92(5),doi:10.1002 / jmv.25698(2020)).

[0091] In some embodiments, the immunogen comprises a polynucleotide. In some embodiments, the immunogen is a polynucleotide. In some embodiments, the immunogen comprises RNA. In some embodiments, the immunogen is RNA. In some embodiments, the immunogen comprises DNA. In some embodiments, the immunogen is DNA. In some embodiments, the polynucleotide is encoded by a circular or linear polyribonucleotide.

[0092] The circular or linear polyribonucleotides of the present disclosure may comprise or encode a variety of immunogens. The circular or linear polyribonucleotides may comprise or encode at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, or more immunogens.

[0093] In certain embodiments, a circular or linear polyribonucleotide comprises or encodes, for example, 1 or less, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 100 or less, 120 or less, 140 or less, 160 or less, 180 or less, 200 or less, 250 or less, 300 or less, 350 or less, 400 or less, 450 or less, 500 or less, or less than this number of immunogens.

[0094] In one embodiment, a circular or linear polyribonucleotide comprises or encodes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 immunogens.

[0095] In some embodiments, the circular or linear polyribonucleotide encodes multiple immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 1-100 immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 1-50 immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 1-10 immunogens; for example, the circular or linear polyribonucleotide encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 2 immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 3 immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 4 immunogens. In some embodiments, the circular or linear polyribonucleotide comprises or encodes 5 immunogens.

[0096] In some embodiments, each of the multiple immunogens identifies the same target. In other words, a single target may include each of the multiple immunogens, each of the multiple immunogens may be derived from the same target, and / or each of the multiple immunogens may share a high degree of similarity with part or all of the target. For example, the target may be a cell, and each of the immunogens may match a protein of that cell. For example, the target may be a specific cancer cell, and each of the immunogens may match a tumor antigen associated with that cancer. Thus, in some embodiments, each of the multiple immunogens is derived from a different protein from the same target.

[0097] In some embodiments, the immunogens are derived from different targets. In some embodiments, the multiple immunogens may be derived from various capsid proteins of a given virus. For example, one immunogen may be derived from an Orthopoxvirus, another from a Hepadnavirus, and a third from a Flavivirus. For example, a polyribonucleotide may encode multiple immunogens, each from Yellow Fever, Chikungunya, Zika, Hepatitis A, and Hepatitis B. A polyribonucleotide may encode an immunogen from each of Yellow Fever, Chikungunya, Zika, Hepatitis A, and Hepatitis B. The polyribonucleotide may encode an immunogen derived from each of Japanese encephalitis, Chikungunya virus, Zika, Hepatitis A, and Hepatitis B. The polyribonucleotide may encode a plurality of immunogens, each of which is derived from SARS-CoV-2, a poxvirus, a respiratory syncytial virus, or a human papillomavirus. The polyribonucleotide may encode an immunogen derived from each of SARS-CoV-2, a poxvirus, a respiratory syncytial virus, and a human papillomavirus. The polyribonucleotide may encode a plurality of immunogens, each of which is derived from a herpesvirus: CMV, EBV, or VZV. The polyribonucleotide may encode an immunogen derived from each of the herpesviruses: CMV, EBV, or VZV. The polyribonucleotide may encode a plurality of immunogens, each of which is derived from a Singles or West Nile virus. The polyribonucleotides may encode immunogens derived from each of the Shingles and West Nile viruses.

[0098] In some embodiments, the immunogens have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the immunogens also have less than 100% sequence identity. This may indicate that the immunogens are closely related to each other due to genetic drift, so that a single circular or linear polyribonucleotide composition or immunogenic composition may be capable of inducing an immune response against targets that exist in a population in various mutational states, or that the immunogens may induce an immune response against multiple targets with the same immunogens that are closely related by genetic drift. For example, the immunogens may be closely related to each other due to genetic drift of the target virus. In some embodiments, the multiple immunogens may be derived from receptor binding domains (RBDs) from unique, but closely related, viruses.

[0099] In some embodiments, the circular or linear polyribonucleotide encodes a variant of the immunogen. The variant may be a naturally occurring variant (e.g., a variant identified in sequence data from different viral genera, species, isolates, or quasi-species) or may be a derivative sequence as disclosed herein that has been created in silico (e.g., an immunogen or epitope having one or more amino acid insertions, deletions, substitutions, or combinations thereof compared to the wild-type immunogen or epitope).

[0100] The immunogen is derived from a virus, such as, for example, a viral surface protein, a viral membrane protein, a viral envelope protein, a viral capsid protein, a viral nucleocapsid protein, a viral spike protein, a viral entry protein, a viral membrane fusion protein, a viral structural protein, a viral nonstructural protein, a viral regulatory protein, a viral accessory protein, a secreted viral protein, a viral polymerase protein, a viral DNA polymerase, a viral RNA polymerase, a viral protease, a viral glycoprotein, a viral fusogen, a viral helical capsid protein, a viral icosahedral capsid protein, a viral matrix protein, a viral replicase, a viral transcription factor, or a viral enzyme.

[0101] In some embodiments, the immunogen is derived from one of these viruses: Orthomyxovirus: Useful immunogens can be derived from influenza A, B, or C viruses, such as the hemagglutinin, neuraminidase, or matrix M2 proteins. If the immunogen is an influenza A virus hemagglutinin, it can be from any subtype (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16).

[0102] Paramyxoviridae Family Viruses: Viral immunogens include, but are not limited to, those derived from pneumoviruses (e.g., respiratory syncytial virus (RSV)), rubulaviruses (e.g., mumps virus), paramyxoviruses (e.g., parainfluenza virus), metapneumoviruses, and morbilliviruses (e.g., measles virus), henipaviruses (e.g., Nipah virus).

[0103] Poxviridae family: Viral immunogens include, but are not limited to, those derived from orthopoxviruses such as smallpox viruses, including but not limited to variola major and variola minor.

[0104] Picornavirus: Viral immunogens include, but are not limited to, those derived from picornaviruses, such as enterovirus, rhinovirus, heparnavirus, cardiovirus, and aphthovirus. In one embodiment, the enterovirus is a poliovirus (e.g., poliovirus type 1, 2, and / or 3). In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is a Coxsackie A or B virus.

[0105] Bunyaviruses: Viral immunogens include, but are not limited to, those derived from an Orthobunyavirus, such as California encephalitis virus, a Phlebovirus, such as Rift Valley fever virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever virus.

[0106] Heparnavirus: Viral immunogens include, but are not limited to, those derived from a heparnavirus, such as Hepatitis A virus (HAV).

[0107] Filoviruses: Viral immunogens include, but are not limited to, those derived from a filovirus, such as Ebola virus (including Zaire, Côte d'Ivoire, Reston, or Sudan Ebola virus) or Marburg virus.

[0108] Togavirus: Viral immunogens include, but are not limited to, those derived from a togavirus, such as a rubivirus, an alphavirus, or an arterivirus, including rubella virus.

[0109] Flaviviruses: Viral immunogens include, but are not limited to, tick-borne encephalitis (TBE) virus, Dengue (type 1, 2, 3, or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus, and Zika virus.

[0110] Pestivirus: Viral immunogens include, but are not limited to, those derived from a pestivirus, such as bovine viral diarrhea (BVDV), classical swine fever (CSFV), or border disease (BDV).

[0111] Hepadnavirus: Viral immunogens include, but are not limited to, those derived from a hepadnavirus, such as Hepatitis B. The Hepatitis B virus immunogen can be a Hepatitis B virus surface immunogen (HBsAg).

[0112] Other Hepatitis Viruses: Viral immunogens include, but are not limited to, those derived from Hepatitis C virus, Hepatitis Delta virus, Hepatitis E virus, or Hepatitis G virus.

[0113] Rhabdoviruses: Viral immunogens include, but are not limited to, those derived from rhabdoviruses, such as lyssaviruses (e.g., rabies virus and vesiculovirus (VSV)).

[0114] Caliciviridae: Viral immunogens include, but are not limited to, those derived from the Caliciviridae family, such as Norwalk virus (Norovirus), and Norwalk-like viruses, such as Hawaii virus and Snow Mountain virus.

[0115] Retroviruses: Viral immunogens include, but are not limited to, those derived from an oncovirus, a lentivirus (eg, HIV-1 or HIV-2), or a spumavirus.

[0116] Reovirus: Viral immunogens include, but are not limited to, those derived from an orthoreovirus, a rotavirus, an orbivirus, or a coltivirus.

[0117] Parvovirus: Viral immunogens include, but are not limited to, those derived from parvovirus B19.

[0118] Bocavirus: Viral immunogens include, but are not limited to, those derived from bocavirus.

[0119] Herpesviruses: Viral immunogens include, but are not limited to, those derived from human herpes viruses, such as herpes simplex virus (HSV) (e.g., HSV types 1 and 2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV6), human herpes virus 7 (HHV7), and human herpes virus 8 (HHV8).

[0120] Papovavirus: Viral immunogens include, but are not limited to, those derived from papillomavirus and polyomavirus. The (human) papillomavirus can be serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63, or 65 (e.g., one or more of serotypes 6, 11, 16, and / or 18).

[0121] Orthohantavirus: Viral immunogens include, but are not limited to, those derived from Hantaviruses.

[0122] Arenaviruses: Viral immunogens include, but are not limited to, those derived from Guanarito virus, Junin virus, Lassa virus, Lujo virus, Machupo virus, Sabia virus, or Whitewater Arroyo virus.

[0123] Adenovirus: Viral immunogens include those derived from adenovirus serotype 36 (Ad-36).

[0124] Community acquired respiratory viruses: Viral immunogens include those derived from community acquired respiratory viruses.

[0125] Coronavirus: Viral immunogens include, but are not limited to, those derived from SARS coronaviruses (e.g., SARS-CoV-1 and SARS-CoV-2), MERS coronaviruses, infectious bronchitis avian (IBV), mouse hepatitis virus (MHV), and transmissible gastroenteritis virus of porcine (TGEV). Coronavirus immunogens can be spike polypeptides or receptor binding domains (RBDs) of spike proteins. Coronavirus immunogens can also be envelope polypeptides, membrane polypeptides, or nucleocapsid polypeptides.

[0126] In some embodiments, the immunogen is derived from a virus that infects fish. In some embodiments, the immunogen induces an immune response against a virus that infects fish. For example, the virus that infects fish is selected from infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), piscine lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), carp herpesvirus, salmon picorna-like virus (also known as Atlantic salmon picorna-like virus), landlocked salmon virus (LSV), Atlantic salmon rotavirus (ASR), trout strawberry disease virus (TSD), coho salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

[0127] In some embodiments, the immunogen is derived from a host cell of the subject. For example, antibodies that block viral entry can be generated by using immunogens or epitopes from components of the host cell that the virus uses as an entry factor.

[0128] The immunogen is derived from bacteria, such as, for example, a bacterial surface protein, a bacterial membrane protein, a bacterial envelope protein, a bacterial inner membrane protein, a bacterial outer membrane protein, a bacterial periplasmic protein, a bacterial entry protein, a bacterial membrane fusion protein, a bacterial structural protein, a bacterial nonstructural protein, a secreted bacterial protein, a bacterial polymerase protein, a bacterial DNA polymerase, a bacterial RNA polymerase, a bacterial protease, a bacterial glycoprotein, a bacterial transcription factor, a bacterial enzyme, or a bacterial toxin.

[0129] In some embodiments, the immunogen elicits an immune response from one of these bacteria: Streptococcus agalactiae (also known as group B streptococcus or GBS), Streptococcus pyogenes (also known as group A Streptococcus (GAS)); Staphylococcus aureus; Methicillin-resistant Staphylococcus aureus (MRSA); Staphylococcus epidermis; Treponema pallidum; Francisella tularensis; Rickettsia spp.; Yersinia pestis; Neisseria meningitidis. meningitidis: immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron acquisition proteins, and factor H binding proteins; Streptococcus pneumoniae; Moraxella catarrhalis; Bordetella pertussis: immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous hemagglutinin (FHA), pertactin, agglutinogens 2 and 3; Clostridium tetani: a typical immunogen is tetanus toxoid; Cornynebacterium diphtheriae: a typical immunogen is diphtheria toxoid; Haemophilus influenzae; Pseudomonas aeruginosa; Chlamydia trachomatis trachomatis); Chlamydia pneumoniae; Helicobacter pylori;Escherichia coli (immunogens include, but are not limited to, immunogens derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), pathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC), and / or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and meningitis / hemorrhage associated E. coli (MNEC). Also, Bacillus anthracis; Clostridium perfringens or Clostridium botulinums; Legionella pneumophila; Coxiella burnetii burnetiid; Brucella spp., e.g., B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, and B. pinnipediae; Francisella spp., e.g., F. novicida, F. philomiragia, and F. tularensis; Neisseria gonorrhoeae; Haemophilus ducreyi; Enterococcus faecalis or Enterococcus faecium. faecium; Staphylococcus saprophyticus; Yersinia enterocolitica; Mycobacterium tuberculosis; Listeria monocytogenes; Vibrio cholerae;These include Salmonella typhi, Borrelia burgdorferi, Porphyromonas gingivalis, and Klebsiella species.

[0130] The immunogen is derived from a fungus, such as, for example, a fungal surface protein, a fungal membrane protein, a fungal envelope protein, a fungal inner membrane protein, a fungal outer membrane protein, a fungal periplasmic protein, a fungal invasion protein, a fungal membrane fusion protein, a fungal structural protein, a fungal nonstructural protein, a secreted fungal protein, a fungal polymerase protein, a fungal DNA polymerase, a fungal RNA polymerase, a fungal protease, a fungal glycoprotein, a fungal transcription factor, a fungal enzyme, or a fungal toxin.

[0131] In some embodiments, the fungal immunogen is derived from a dermatophyte, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton nigricans ... megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton fabiforme. faviforme; or Aspergillus fumigatus, Aspergillus flavus, Aspergillus nigerniger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoide stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalisintestinalis, and Enterocytozoon bieneusi; less commonly, Brachiola spp., Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp., Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, Saccharomyces cerevisiae, cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp. spp.), Conidiobolus spp., Rhizopus spp., Mucor spp., Absidia spp., Mortierella spp., Cunninghamella spp., Saksenaea spp., Alternaria spp.spp.), Curvularia spp., Helminthosporium spp., Fusarium spp., Aspergillus spp., Penicillium spp., Monolinia spp., Rhizoctonia spp., Paecilomyces spp., Pithomyces spp., and Cladosporium spp.

[0132] The immunogen is derived, for example, from a eukaryotic parasite surface protein, a eukaryotic parasite membrane protein, a eukaryotic parasite envelope protein, a eukaryotic parasite invasion protein, a eukaryotic parasite membrane fusion protein, a eukaryotic parasite structural protein, a eukaryotic parasite nonstructural protein, a secreted eukaryotic parasite protein, a eukaryotic parasite polymerase protein, a eukaryotic parasite DNA polymerase, a eukaryotic parasite RNA polymerase, a eukaryotic parasite protease, a eukaryotic parasite glycoprotein, a eukaryotic parasite transcription factor, a eukaryotic parasite enzyme, or a eukaryotic parasite toxin.

[0133] In some embodiments, the immunogen induces an immune response against a parasite from the genus Plasmodium, such as P. falciparum, P. vivax, P. malariae, or P. ovale. In some embodiments, the immunogen induces an immune response against a parasite from the family Caligidae, particularly the genera Lepeophtheirus and Caligus (e.g., parasites such as fish lice, such as Lepeophtheirus salmonis or Caligus rogercresseyi). In some embodiments, the immunogen induces an immune response against the parasite Toxoplasma gondii.

[0134] In some embodiments, the immunogen is a cancer immunogen (such as a neoepitope). For example, the immunogen may be an immunogen selected from the group consisting of acute leukemia, astrocytoma, biliary cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchioloalveolar cell lung cancer, adrenal gland cancer, anal region cancer, bladder cancer, endocrine system cancer, esophageal cancer, head and neck cancer, kidney cancer, parathyroid cancer, penile cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureter cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, cutaneous melanoma, ependymoma, epidermoid tumor, Ewing's sarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, glioma, hematologic malignancies, hepatocellular (liver) carcinoma, hepatoma, Hodgkin's and / or neoepitopes associated with tumors and their metastases, including ovarian cancer, ovarian cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, schwannoma, skin cancer, spinal axis tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, uterine cancer, or refractory forms of any of the above cancers, or any combination thereof.

[0135] In some embodiments, the immunogen is a tumor antigen selected from: (a) cancer testis antigens such as NY-ESO-1, SSX2, SCP1, and RAGE, BAGE, GAGE, and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used to address, e.g., melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors); (b) mutant Antigens such as p53 (e.g., associated with various solid tumors such as colorectal cancer, lung cancer, head and neck cancer), p21 / Ras (e.g., associated with melanoma, pancreatic cancer, and colorectal cancer), CDK4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (e.g., associated with head and neck cancer), CIA0205 (e.g., associated with bladder cancer), HLA-A2-R1701, beta-catenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin's lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triosephosphate isomerase, KIA0205, CDC-27, and LDLR-FUT; (c) overexpressed antigens, such as galectin 4 (e.g., associated with colon cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT1 (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., associated with breast, colon, lung, and ovarian cancer), mammaglobin, alpha-fetoprotein (e.g., associated with liver cancer), KSA (e.g., associated with colon cancer), gastrin (e.g., associated with pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (e.g., associated with breast and ovarian cancer), G-250 (e.g., associated with renal cell carcinoma), p53 (e.g., associated with breast, colon cancer), and carcinoembryonic antigen (e.g., associated with cancers of the gastrointestinal tract such as breast, lung, and colon cancer);(d) shared antigens, e.g., melanoma-melanocyte differentiation antigens such as MART-l / Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1, and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma); (e) prostate-associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2 (e.g., associated with prostate cancer); (f) immunoglobulin idiotypes (e.g., associated with myeloma and B-cell lymphoma); (g) neoantigens. In certain embodiments, tumor immunogens include, but are not limited to, pi5, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, human papillomavirus (HPV) antigens including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23HI, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, pl6, TAGE, P These include SCA, CT7, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29YBCAA), CA195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein cyclophilin C-related protein), TAAL6, TAG72, TLP, and TPS;

[0136] In some embodiments, the immunogen elicits an immune response against: pollen allergens (tree, herb, weed, and grass pollen allergens); insect or arachnid allergens (inhalant, saliva, and venom allergens, e.g., mite allergens, cockroach, and midge allergens, hymenopthera venom allergens); animal hair and burrowing allergens (e.g., from dogs, cats, horses, rats, mice, etc.); and food allergens (e.g., gliadin). Important pollen allergens from trees, grasses, and herbs include, but are not limited to, the taxonomic orders Fagales, Oleales, Pinales, and Platanaceae, including birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus), and olive (Olea), cedars (Cryptomeria and Juniperus), Platanus, Lolium, It is from the order Poales, which includes the genera Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, grasses, and the orders Asterales and Urticales, which include the genera Ambrosia, Artemisia, and Parietaria, herbs.Other important inhalant allergens are those from house mites of the genera Dermatophagoides and Euroglyphus, from storage mites (e.g. Lepidoglyphys, Glycyphagus, and Tyrophagus), cockroaches, small insects, and fleas (e.g. Blatella germanica, Blattella americana, Blattella spp., and Blattella spp.), and from insects such as cockroaches, small insects, and fleas (e.g. Blattella germanica, Blattella spp., and Blattella spp.). venom allergens including those from stinging and biting insects such as those from the genera Periplaneta, Chironomus, and Ctenocepphalides, from mammals such as cats, dogs, and horses, and from the taxonomic order Hymenoptera, which includes honeybees (Apidae), wasps (Vespidea), and ants (Formicoidae).

[0137] In some embodiments, the immunogen may be an immunogen that is specific to, for example, a snake (e.g., most species of rattlesnake (e.g., eastern diamondback rattlesnake), brown snake species (e.g., king brown snake and eastern brown snake), Russell's viper, cobra (e.g., Indian cobra, king cobra), certain species of krait (e.g., common krait), mamba (e.g., black mamba), carpet viper, boomslang, dubois sea snake, taipan species (e.g., coastal taipan and inland taipan snakes), lancehead species (e.g., fer de lance and terciopelo), bushmaster, copperhead, cottonmouth, coral snake, death adder, Belcher's sea snake, tiger snake, Australian black snake, spiders (e.g., brown recluse spider, black widow spider, Brazilian wandering spider, funnel-web spider, button spider, Australian redback spider, katipo, false black widow, Chilean recluse spider, mouse spider, Macrothele species, Sicarius species, Exophthalmic species, certain species of tarantula), scorpions and other arachnids (e.g., fat-tailed scorpion, deathstalker scorpion, Indian red scorpion, scorpion, Centruroides species, Tityus species such as the Brazilian yellow scorpion), insects (e.g. bee species, wasp species, certain ants such as the fire ant, some species of Lepidoptera caterpillars, certain species of centipedes, Remipede xibarbanus turmensis, etc.),Xibalbanus tulumensis, fish (e.g., certain species of catfish (e.g., striped eel catfish and other eeltail catfish), certain species of stingrays (e.g., blue-spotted stingray), lionfish, stonefish, scorpionfish, pufferfish, rabbitfish, goblinfish, stonefish, striped blenny, stargazer, chimaera, weever, dogfish shark), cnidaria (e.g., certain species of jellyfish (e.g., Irukandji jellyfish and box jellyfish), hydrozoa (e.g., Portuguese Man o'War), sea anemones, certain species of coral), lizards (e.g., Gila monster), monster, Mexican bearded lizard, certain species of Varanus (e.g., Komodo dragon, perentie, and lace monitor), mammals (e.g., Southern short-tailed shrew, platypus, European mole, Eurasian water shrew, Mediterranean water shrew, Northern short-tailed shrew, Elliot's short-tailed shrew, shrew, certain species of solenodon (e.g., Cuban solenodon, Haitian solenodon, slow loris), mollusks (e.g., certain species of cone snails), cephalopods (e.g., certain species of octopus (e.g., blue-ringed octopus, squid, and cuttlefish), amphibians (e.g., frogs such as poison dart frogs, blue stone frogs, and Greening's frogs), salamanders (e.g., fire salamanders, Iberian ribbed newts, etc.),It is derived from toxins in venom such as that from the venom of the genus Neanderthal.

[0138] In some embodiments, the toxin is derived from a plant or a fungus (eg, a mushroom).

[0139] In some embodiments, the toxin immunogen is derived from a toxin such as a cyanotoxin, a dinotoxin, a myotoxin, a cytotoxin (e.g., ricin, apitoxin, a mycotoxin (e.g., aflatoxin), an ochratoxin, a citrinin, an ergot alkaloid, a patulin, a fusarium, a fumonisin, a trichothecene, a cardiotoxin), a tetrodotoxin, a batrachotoxin, a botulinum toxin A, a tetanus toxin A, a diphtheria toxin, a dioxin, a muscarine, a bufotoxin, a sarin, a hematotoxin, a phototoxin, a necrotic toxin, a nephrotoxin, and a neurotoxin (e.g., a calciceptin, a cobrotoxin, a calcicludin, a fasciculin-I, a caryotoxin).

[0140] Immunogens from any number of microorganisms or cancers can be used in circular or linear polyribonucleotides. In some cases, the immunogen is associated with or expressed by one of the microorganisms disclosed above. In some embodiments, the immunogen is associated with or expressed by two or more of the microorganisms disclosed above. In some embodiments, the immunogen is associated with or expressed by one of the cancers disclosed above. In some embodiments, the immunogen is associated with or expressed by two or more of the cancers disclosed above. In some embodiments, the immunogen is derived from a toxin disclosed above. In some embodiments, the immunogen is derived from two or more toxins disclosed above.

[0141] The two or more microorganisms may be related or unrelated. In some cases, the two or more microorganisms are phylogenetically related. For example, the circular or linear polyribonucleotide of the present disclosure comprises or encodes two or more viruses, two or more members of a viral family, two or more members of a viral class, two or more members of a viral lineage, two or more members of a viral genus, two or more members of a viral species, or two or more immunogens from bacterial pathogens. In some embodiments, the two or more microorganisms are not phylogenetically related.

[0142] In some cases, the two or more microorganisms are phenotypically related. For example, a circular or linear polyribonucleotide of the present disclosure comprises or encodes an immunogen from two or more respiratory pathogens, two or more selection agents, two or more microorganisms associated with severe disease, two or more microorganisms associated with adverse outcomes in immunocompromised subjects, two or more microorganisms associated with adverse pregnancy-related outcomes, two or more microorganisms associated with hemorrhagic fevers.

[0143] The immunogens of the present disclosure may comprise wild-type sequences. When describing an immunogen, the term "wild-type" refers to a sequence (e.g., a nucleic acid sequence or an amino acid sequence) that is naturally occurring and is encoded by a genome (e.g., a viral genome). A species (e.g., a microbial species) may have one wild-type sequence, or more than one wild-type sequence (e.g., one standard wild-type sequence present in a reference microbial genome, and additional mutant wild-type sequences present that arise from mutation).

[0144] When referring to an immunogen, the terms "derivative" and "derived from" refer to a sequence (e.g., a nucleic acid sequence or an amino acid sequence) that differs from a wild-type sequence in one or more nucleic acids or amino acids, e.g., that contains one or more amino acid insertions, deletions, and / or substitutions compared to the wild-type sequence.

[0145] An immunogenic derivative sequence is a sequence that has at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type sequence, e.g., a wild-type nucleic acid, protein, immunogen, or epitope sequence.

[0146] In some embodiments, the immunogen contains one or more amino acid insertions, deletions, substitutions, or combinations thereof that affect the structure of the encoded protein. In some embodiments, the immunogen contains one or more amino acid insertions, deletions, substitutions, or combinations thereof that affect the function of the encoded protein. In some embodiments, the immunogen contains one or more amino acid insertions, deletions, substitutions, or combinations thereof that affect the expression or processing of the encoded protein by a cell.

[0147] In some embodiments, the immunogen comprises one or more nucleic acid insertions, deletions, substitutions, or a combination thereof that affect the structure of the encoded immunogenic nucleic acid.

[0148] The amino acid insertion, deletion, substitution, or combination thereof may introduce a site for post-translational modification (e.g., introduce a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or a sequence targeted for cleavage). In certain embodiments, the amino acid insertion, deletion, substitution, or combination thereof removes a site for post-translational modification (e.g., removes a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or a sequence targeted for cleavage). In certain embodiments, the amino acid insertion, deletion, substitution, or combination thereof modifies a site for post-translational modification (e.g., a glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation, or lipidation site, or modifies a site to alter the efficiency or characteristics of cleavage).

[0149] Amino acid substitutions can be conservative or non-conservative. A conservative amino acid substitution can be the substitution of one amino acid for another amino acid with similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution can be the substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R). A conservative amino acid change can be an amino acid change from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids may include Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A conservative amino acid change may be an amino acid change from one acidic amino acid to another acidic amino acid. Acidic amino acids may include Glu (E) and Asp (D). A conservative amino acid change may be an amino acid change from one basic amino acid to another basic amino acid. Basic amino acids may include His (H), Arg (R), and Lys (K). A conservative amino acid change may be an amino acid change from one polar amino acid to another polar amino acid. Polar amino acids may include Asn (N), Gln (Q), Ser (S), and Thr (T). A conservative amino acid change may be an amino acid change from one non-polar amino acid to another non-polar amino acid. Nonpolar amino acids may include Leu (L), Val (V), Ile (I), Met (M), Gly (G) and Ala (A). A conservative amino acid change may be an amino acid change from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids may include Phe (F), Tyr (Y) and Trp (W). A conservative amino acid change may be an amino acid change from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids may include Ala (A), Val (V), Leu (L) and Ile (I).In one embodiment, a conservative amino acid substitution is an amino acid change from one amino acid to another within one of the following groups: Group I: ala, pro, gly, gln, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0150] In an embodiment, an immunogenic or epitope derivative of the disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid deletions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0151] In an embodiment, an immunogenic or epitope derivative of the disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid substitutions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0152] In an embodiment, an immunogenic or epitope derivative of the disclosure comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid substitutions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0153] In some embodiments, the immunogenic or epitope derivatives of the disclosure have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-1 Contains 0, 2 to 15, 2 to 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, or 20 to 25 amino acid substitutions.

[0154] In certain embodiments, an immunogenic or epitope derivative of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0155] The one or more amino acid substitutions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The amino acid substitutions may be consecutive, non-consecutive, or a combination thereof.

[0156] In an embodiment, an immunogenic or epitope derivative of the disclosure comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 120, no more than 140, no more than 160, no more than 180, or no more than 200 amino acid deletions compared to a sequence disclosed herein (e.g., a wild-type sequence).

[0157] In certain embodiments, the immunogenic or epitope derivatives of the disclosure have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, The deletion may include 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, 20 to 25, 20 to 30, 30 to 50, 50 to 100, or 100 to 200 amino acids.

[0158] In certain embodiments, an immunogenic or epitope derivative of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid deletions compared to the wild-type sequence.

[0159] The one or more amino acid deletions can be at the N-terminus, C-terminus, or a combination thereof within the amino acid sequence. The amino acid deletions can be contiguous, non-contiguous, or a combination thereof.

[0160] In an embodiment, an immunogenic or epitope derivative of the disclosure comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acid insertions compared to the wild-type sequence.

[0161] In an embodiment, an immunogenic or epitope derivative of the disclosure comprises no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 11, no more than 12, no more than 13, no more than 14, no more than 15, no more than 16, no more than 17, no more than 18, no more than 19, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, or no more than 50 amino acid insertions compared to the wild-type sequence.

[0162] In some embodiments, the antigen or epitope derivatives of the present disclosure have a sequence similar to or different from the wild-type sequence, but with a sequence similar or different to the wild-type sequence, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2- 20, 2 to 30, 2 to 40, 3 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 15, 3 to 20, 3 to 30, 3 to 40, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 15, 5 to 20, 5 to 30, 5 to 40, 10 to 15, 15 to 20, or 20 to 25 amino acid insertions.

[0163] In certain embodiments, an antigen or epitope derivative of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid insertions compared to the wild-type sequence.

[0164] The one or more amino acid insertions may be at the N-terminus, C-terminus, or a combination thereof within the amino acid sequence. The amino acid insertions may be contiguous, non-contiguous, or a combination thereof.

[0165] In some embodiments, the immunogen is expressed by a circular or linear polyribonucleotide. In some embodiments, the immunogen is the product of rolling circle amplification of a circular or linear polyribonucleotide.

[0166] The immunogen can be produced in substantial quantities. Thus, the immunogen can be any proteinaceous molecule that can be produced. The immunogen can be a polypeptide that can be secreted from a cell or that can be localized in the cytoplasm, nucleus, or membrane fraction of a cell. In some embodiments, the polypeptide encoded by the circular or linear polyribonucleotide of the present disclosure comprises a fusion protein that comprises two or more immunogens disclosed herein. In some embodiments, the polypeptide encoded by the circular or linear polyribonucleotide of the present disclosure comprises an epitope. In some embodiments, the polypeptide encoded by the circular or linear polyribonucleotide of the present disclosure comprises a fusion protein that comprises two or more epitopes disclosed herein, for example, an artificial peptide sequence that comprises multiple predicted epitopes from one or more microorganisms of the present disclosure.

[0167] In some embodiments, the immunogen that can be expressed from a circular or linear polyribonucleotide is a membrane protein, for example, includes a polypeptide sequence that is commonly found as a membrane protein or that has been modified to be a membrane protein. Exemplary immunogens that can be expressed from the circular or linear polyribonucleotides disclosed herein in some embodiments include intracellular or cytoplasmic immunogens.

[0168] In some embodiments, the immunogen is less than about 40,000 amino acids in length, less than about 35,000 amino acids in length, less than about 30,000 amino acids in length, less than about 25,000 amino acids in length, less than about 20,000 amino acids in length, less than about 15,000 amino acids in length, less than about 10,000 amino acids in length, less than about 9,000 amino acids in length, less than about 8,000 amino acids in length, less than about 7,000 amino acids in length, less than about 6,000 amino acids in length, less than about 5,000 amino acids in length, less than about 4,000 amino acids in length, less than about 3,000 amino acids in length, less than about 2,500 amino acids in length, less than about 2,000 amino acids in length, less than about 1,500 amino acids in length, less than about 1,000 amino acids in length, less than about 900 amino acids in length, less than about 800 amino acids in length, less than about 700 amino acids in length , less than about 600 amino acids in length, less than about 500 amino acids in length, less than about 400 amino acids in length, less than about 300 amino acids in length, less than about 250 amino acids in length, less than about 200 amino acids in length, less than about 150 amino acids in length, less than about 140 amino acids in length, less than about 130 amino acids in length, less than about 120 amino acids in length, less than about 110 amino acids in length, less than about 100 amino acids in length, less than about 90 amino acids in length, less than about 80 amino acids in length, less than about 70 amino acids in length, less than about 60 amino acids in length, less than about 50 amino acids in length, less than about 40 amino acids in length, less than about 30 amino acids in length, less than about 25 amino acids in length, less than about 20 amino acids in length, less than about 15 amino acids in length, less than about 10 amino acids in length, less than about 5 amino acids in length, any amino acid length therebetween or less may be useful.

[0169] In some embodiments, the circular or linear polyribonucleotide comprises one or more immunogenic sequences and is configured to be persistently expressed in the cells of a subject in vivo. In some embodiments, the circular or linear polyribonucleotide is configured so that the expression of one or more expression sequences in the cells at a later time point is equal to or higher than that at an earlier time point. In such embodiments, the expression of one or more immunogenic sequences can be maintained at a relatively stable level or increased over time. The expression of the immunogenic sequence can be relatively stable over an extended period of time. The expression of the immunogenic sequence can be relatively stable transiently or for a limited period of time, for example, at most 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0170] In some embodiments, the circular or linear polyribonucleotide expresses one or more immunogens in a subject (e.g., transiently or chronically). In certain embodiments, expression of the immunogen lasts for at least about 1 hour to about 30 days, or for at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or more, or any time in between. In certain embodiments, onset of the immunogen occurs within about 30 minutes to about 7 days, or within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 ... "Any period not exceeding 30 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or any time in between."

[0171] Expression of the immunogen includes translating at least a region of the circular or linear polyribonucleotide provided herein. For example, the circular or linear polyribonucleotide can be translated in a subject to produce a polypeptide comprising one or more immunogens of the present disclosure, thereby stimulating the production of an adaptive immune response (e.g., antibody response and / or T cell response) in the subject. In some embodiments, the circular or linear polyribonucleotide of the present disclosure is translated to produce one or more immunogens in a human or animal subject, thereby stimulating the production of an adaptive immune response (e.g., antibody response and / or T cell response) in a human or animal subject.

[0172] In some embodiments, the methods for immunogen expression include translation of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the total length of a circular or linear polyribonucleotide into a polypeptide. In some embodiments, the methods for immunogen expression include translation of a circular or linear polyribonucleotide into a polypeptide of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids. In some embodiments, the methods for protein expression include translation of a circular or linear polyribonucleotide into a polypeptide of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, or about 1000 amino acids. In some embodiments, the methods include translation of a circular or linear polyribonucleotide into a continuous polypeptide as provided herein, a separate polypeptide as provided herein, or both.

[0173] In some embodiments, the methods for immunogen expression include modification of the translation product, folding, or other post-translational modifications, hi some embodiments, the methods for immunogen expression include in vivo post-translational modifications (e.g., via cellular machinery).

[0174] Polymerization In certain embodiments, the cyclic polyribonucleotide may code for a multimerization domain. For example, the cyclic polyribonucleotide may code for a first polypeptide that is an immunogen and a second polypeptide that is a multimerization domain. For example, the multimerization domain may be encoded in the same open reading frame as the immunogen and expressed as a fusion protein with the immunogen. In some embodiments, the cyclic polyribonucleotide may code for two or more immunogens, and optionally each immunogen may be fused to a multimerization domain. The multimerization domain may promote the formation of an immunogen complex (e.g., a complex that includes multiple immunogens).

[0175] Multimerization of the encoded immunogen may be beneficial for the induction of immune responses. Fusing an immunogen to one or more multimerization elements (e.g., dimerization, trimerization, tetramerization, and oligomerization elements) may lead to the formation of a multimeric immunogen complex (e.g., the formation of a multimeric immunogen complex after expression in an immunized subject). In some embodiments, the formation of a multimeric immunogen complex increases the immunogenicity of the immunogen. For example, the formation of a multimeric immunogen complex may increase the immunogenicity of the immunogen by mimicking an infection with an exogenous pathogen (e.g., a virus), where multiple potential immunogens are often located on the pathogen envelope (e.g., the hemagglutinin (HA) immunogen of influenza virus). In some embodiments, the multimerized complex comprises at least 2, 3, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 immunogens. In some embodiments, the immunogen complex comprises 2-10, 2-50, 2-100, 5-10, 5-15, 5-20, 5-50, 5-100, 10-20, 10-30, 10-40, 10-50, 10-60, 10-100, 20-50, or 20-100 immunogens. In some embodiments, the immunogen complex comprises 6 copies of the immunogen (e.g., the cyclic polyribonucleotide encodes an immunogen-foldon-immunogen fusion protein). In some embodiments, the immunogen complex comprises 24 copies of the immunogen (e.g., the circular polyribonucleotide encodes an immunogen-ferritin fusion protein), while in some embodiments, the immunogen complex comprises 60 copies of the immunogen (e.g., the circular polyribonucleotide encodes an immunogen-AaLS fusion protein or encodes an immunogen-β-annulus peptide).

[0176] When used in conjunction with a polypeptide immunogen of interest in the context of the present disclosure, such multimerization elements can be located at the N-terminus or C-terminus of the polypeptide of interest. At the nucleic acid level, the coding sequence for such multimerization elements is typically located 5' or 3' to the coding sequence of the polypeptide or protein of interest, in the same reading frame.

[0177] The multimerization domain may have 10 to 500 amino acid residues (e.g., 10 to 450, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 50 to 500, 100 to 500, 150 to 500, 200 to 500, 250 to 500, 300 to 500, 350 to 500, 400 to 500, and 450 to 500 residues). In some embodiments, the multimerization domain may comprise 20 to 2500 amino acid residues (e.g., 20 to 250, 20 to 225, 20 to 200, 20 to 175, 20 to 150, 20 to 150, 20 to 125, 20 to 100, 20 to 75, 20 to 50, 50 to 250, 75 to 250, 100 to 250, 125 to 250, 150 to 250, 175 to 250, 200 to 250, and 225 to 250 residues).

[0178] In some embodiments, an immunogen fused to a multimerization domain is at least 2-fold, 5-fold, or 10-fold more immunogenic (e.g., in a human subject) than the immunogen. In some embodiments, an immunogen fused to a multimerization domain is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500% more immunogenic (e.g., in a human subject) than an immunogen that is not fused to a multimerization domain.

[0179] Particular multimerization elements are oligomerization elements, tetramerization elements, trimerization elements or dimerization elements. Dimerization elements can be selected, for example, from dimerization elements / domains of heat shock proteins, immunoglobulin Fc domains and leucine zippers (dimerization domains of the basic region leucine zipper transcription factor class). Trimerization and tetramerization elements can be selected, for example, from engineered leucine zippers (engineered alpha-helical coiled-coil peptides that adopt a parallel trimer state), fibritin foldon domains from enterobacteriaceae phage T4, GCN4pll, CCN4-pLI and p53. In some embodiments, the cyclic polyribonucleotide comprises a T4 foldon domain. In particular embodiments, the T4 foldon domain has an amino acid sequence at least 95% identical to GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 29). In some embodiments, the T4 foldon has the amino acid sequence of SEQ ID NO: 29. In some embodiments, the multimerization domain is a β-annulus peptide (see Matsuura et al. (2010), Angew. CHEM. INT. ED., 49:9662-9665). In some embodiments, the β-annulus peptide has an amino acid sequence of INHVGGTGGAIMAPVAVTRQLVGS (SEQ ID NO: 30), optionally with or without a C-terminal serine residue, or has an amino acid sequence at least 95% identical to SEQ ID NO: 30. In some embodiments, the cyclic polyribonucleotide comprises an AaLS peptide. In particular embodiments, the AaLS peptide has an amino acid sequence at least 95% identical to TDILGKYVINYLNKLKKKEDIFKEFLKW (SEQ ID NO: 31). In some embodiments, the AaLS peptide has the amino acid sequence of SEQ ID NO: 31.

[0180] The oligomerization element may be selected from, for example, ferritin, surfactant D, paramyxovirus phosphoprotein oligomerization domains, complement inhibitor C4 binding protein (C4bp) oligomerization domains, viral infectivity factor (Vif) oligomerization domains, sterile alpha motif (SAM) domains, and von Willebrand factor type D domains.

[0181] Ferritin is a highly conserved protein that forms oligomers and is found in all animals, bacteria, and plants. Ferritin is a protein that spontaneously forms nanoparticles with 24 identical subunits. Ferritin-immunogen fusion constructs may form oligomeric aggregates or "clusters" of immunogens that may result in an enhanced immune response. In some embodiments, the cyclic polyribonucleotide comprises a ferritin domain. In some embodiments, the cyclic polyribonucleotide comprises [ka] The ferritin domain has the amino acid sequence:

[0182] Surfactant D protein (SPD) is a hydrophilic glycoprotein that spontaneously self-assembles to form oligomers. SPD-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogen that can result in an enhanced immune response.

[0183] The phosphoproteins of paramyxoviruses (negative-strand RNA viruses) function as transcriptional transactivators of the viral polymerase. Phosphoprotein oligomerization is critical for viral genome replication. Phosphoprotein-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogen that can result in an enhanced immune response.

[0184] The complement inhibitor C4 binding protein (C4bp) may also be used as a fusion partner to generate oligomeric immunogen aggregates. The C-terminal domain of C4bp (57 amino acid residues in humans and 54 amino acid residues in mice) is necessary and sufficient for oligomerization of C4bp or other polypeptides fused to it. C4bp-immunogen fusion constructs may form oligomeric aggregates or "clusters" of immunogens that may result in an enhanced immune response. The viral infectivity factor (Vif) multimerization domain has been shown to form oligomers both in vitro and in vivo. Vif oligomerization involves sequence mapping between residues 151-164 of the C-terminal domain and the 161 PPLP 164 motif (in human HIV-1: TPKKIKPPLP (SEQ ID NO: 33)). Vif-immunogen fusion constructs may form oligomeric aggregates or "clusters" of immunogens that may result in an enhanced immune response.

[0185] The sterile alpha motif (SAM) domain is a protein interaction module present in a wide variety of proteins involved in many biological processes. SAM domains, spanning approximately 70 residues, are found in a variety of eukaryotic organisms. SAM domains have been shown to homo- and hetero-oligomerize to form multiple self-associated oligomeric structures. SAM-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can lead to enhanced immune responses. von Willebrand factor (vWF) contains several type D domains: D1 and D2 are present within the N-terminal propeptide, while the remaining D domains are required for oligomerization. This domain is found in a variety of plasma proteins: complement factors B, C2, C3, and CR4; integrins (l-domain); collagen types VI, VII, XII, and XIV; and other extracellular proteins. vWF-immunogen fusion constructs can form oligomeric aggregates or "clusters" of immunogens that can lead to enhanced immune responses.

[0186] In some embodiments, the multimerization domain is a lumazine synthase domain. Lumazine synthase can assemble into a complex comprising 60 copies of the lumazine synthase domain, where each lumazine synthase domain can be fused to one or more immunogens. In some embodiments, the lumazine synthase domain comprises an amino acid sequence of any of SEQ ID NOs: 34-44 and 115, or an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 34-44 and 115. SEQ ID NO:34 [ka] SEQ ID NO:35 [ka] SEQ ID NO:36 [ka] SEQ ID NO:37 [ka] SEQ ID NO:115 [ka]

[0187] Providing one or more cysteine ​​substitutions in the lumazine synthase domain to introduce non-natural disulfide bonds stabilizes the lumazine synthase complex formed by self-assembled subunits.In some embodiments, non-natural disulfide bonds are introduced by L121C-K131C, L121CG-K131C, L121GC-K131C, K7C-R40C, I3C-L50C, I82C-K131CG, E5C-R52C, or E95C-A101C substitutions, or combinations thereof (such as I3C-L50C and I82C-K131CG; E5C-R52C and I82C-K131CG; or E95C-A101C and I82C-K131CG).Residue numbering is based on the lumazine synthase subunit shown as SEQ ID NO:34. Non-limiting examples include: SEQ ID NO:38 (L121C-K131C) [ka] SEQ ID NO: 39 (L121CG-K131C) [ka] SEQ ID NO: 40 (L121GC-K131C) [ka] SEQ ID NO: 41 (K7C-R40C) [ka] SEQ ID NO: 42 (I3C-L50C, I82C-K131CG) [ka] SEQ ID NO: 43 (E5C-R52C, I82C-K131CG) [ka] SEQ ID NO: 44 (E95C-A101C, I82C-K131CG) [ka]

[0188] Various methods for multimerizing polypeptides are described in WO 2020 / 061564, page 25, line 1 to page 26, line 20 (which is incorporated by reference herein).

[0189] In some embodiments, the multimerization domain is a riboflavin synthase domain.For example, the riboflavin synthase domain can have an amino acid sequence with at least 95% sequence identity with TDILGKYVINYLNKLKKKEDIFKEFLKW (SEQ ID NO: 116).In some embodiments, the riboflavin synthase domain can have the amino acid sequence of SEQ ID NO: 116.

[0190] In some embodiments, the cyclic polyribonucleotide may include one or more multimerization domains. For example, the cyclic polyribonucleotide may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 multimerization domains. In some embodiments, the cyclic polyribonucleotide includes two multimerization domains. The two or more multimerization domains may be adjacent to each other. Alternatively, the two or more multimerization domains may be separated by one or more other elements. For example, the two multimerization domains may be separated by an immunogen. In a detailed embodiment, the cyclic polyribonucleotide may include a ferritin domain and a T4 Foldon domain. The ferritin and T4 Foldon domains may be linked (e.g., by a Gly-Ser linker). In some embodiments, the ferritin domain linked to the T4 Foldon domain has the following amino acid sequence: [ka]

[0191] Suitable multimerization domains may be selected, for example, from the list of amino acid sequences according to SEQ ID NOs: 1116 to 1167 of WO 2017 / 081082, or fragments or variants of these sequences.

[0192] In some embodiments, the circular polyribonucleotide encodes an open reading frame (e.g., an open reading frame operably linked to an IRES) that includes the elements as set forth in Table 1 or Table 2 below, in the order in which they appear. For the embodiments set forth in Table 1 or Table 2, each immunogen optionally includes a secretory signal sequence. When an embodiment of Table 1 or Table 2 includes multiple immunogens, the immunogens may be the same or different (e.g., selected from any of the immunogens described herein). When an embodiment of Table 1 includes multiple multimerization domains, the multimerization domains may be the same or different (e.g., selected from any of the multimerization domains described herein). In some embodiments, the circular polyribonucleotide includes multiple open reading frames, where each open reading frame is set forth in Table 1 or Table 2.

[0193] [Table 1]

[0194] [Table 2]

[0195] Internal ribosome entry site In some embodiments, the circular polyribonucleotide described herein comprises one or more internal ribosome entry sites (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences, where each expression sequence optionally encodes an immunogen, such as an immunogen that comprises a multimerization domain). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence (e.g., the coding sequence that encodes an immunogen that comprises a multimerization domain).

[0196] Suitable IRES elements for inclusion in polyribonucleotides include RNA sequences capable of associating with eukaryotic ribosomes. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

[0197] In some embodiments, the IRES element is derived from DNA of organisms including, but not limited to, viruses, mammals, and Drosophila. Such viral DNA can be derived from picomavirus complementary DNA (cDNA), including, but not limited to, encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, the Drosophila DNA from which the IRES element is derived includes, but is not limited to, the antennapedia gene of Drosophila melanogaster.

[0198] In some embodiments, the IRES sequence is selected from the group consisting of Taura syndrome virus, Assassin bug virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Fuman poliovirus 1, Plautia stall enteric virus, Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua) picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus type 1, Black queen cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus (AEV), Acute bee paralysis virus, Hibiscus chlorotic mottle virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-l, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, salivirus, cosavirus, parechovirus, Drosophila hairless, S. cerevisiae TFIID, S.cerevisiae YAP1, human c-src, human FGF-1, simian picomavirus, turnip crinkle virus, Aichi virus, Kurohi virus, echovirus 11, an aptamer for eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2) IRES sequence. In yet another embodiment, the IRES is a Coxsackievirus B3 (CVB3) IRES sequence. In a further embodiment, the IRES is an encephalomyocarditis virus IRES sequence. In a further embodiment, the IRES is a Theiler's encephalomyelitis virus IRES sequence.

[0199] The IRES sequence may have a modified sequence compared to the wild-type IRES sequence. In some embodiments, when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified so that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence in which the terminal adenosine residue is modified to be a cytosine residue. In some embodiments, the modified CVB3 IRES is [ka] The nucleic acid sequence may be:

[0200] In some embodiments, the IRES sequence is an Enterovirus 71 (EV17) IRES. In some embodiments, the terminal guanosine residue of the EV17 IRES sequence is modified to become a cytosine residue. In some embodiments, the modified EV71 IRES is [ka] The nucleic acid sequence may be:

[0201] In some embodiments, the polyribonucleotide comprises at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expressed sequence. In some embodiments, an IRES flanks both sides of at least one (e.g., 2, 3, 4, 5 or more) expressed sequence. In some embodiments, the polyribonucleotide comprises one or more IRES sequences on one or both sides of each expressed sequence, leading to the separation of the resulting 0001 peptides and / or polypeptides. For example, the polyribonucleotide described herein can comprise a first IRES operably linked to a first expressed sequence (e.g., encoding a first immunogen, such as a first immunogen comprising a multimerization domain) and a second IRES operably linked to a second expressed sequence (e.g., encoding a second immunogen, such as a second immunogen comprising a multimerization domain).

[0202] In some embodiments, a polyribonucleotide described herein comprises an IRES (eg, an IRES operably linked to a coding region). For example, polyribonucleotides are Chen et al. MOL. CELL 81(20):4300-18,2021; Jopling et al. ONCOGENE 20:2664-70,2001; Baranick et al. PNAS 105(12):4733-38,2008; Lang et al. MOLECULAR BIOLOGY OF THE CELL 13(5):1792-1801,2002; Dorokhov et al. PNAS 99(8):5301-06,2002; Wang et al. NUCLEIC ACIDS RESEARCH 33(7):2248-58,2005; Petz et al. NUCLEIC ACIDS RESEARCH 35(8):2473-82,2007; Chen et al. SCIENCE 268:415-417, 1995; Fan et al. NATURE COMMUNICATION 13(1):3751-3765, 2022, and International Publication No. WO 2021 / 263124 (each of which is hereby incorporated by reference in its entirety).

[0203] Signal sequence In some embodiments, exemplary immunogens that may be expressed from the circular polyribonucleotides disclosed herein include secretory proteins, such as proteins (e.g., immunogens) that naturally include a signal sequence, or that do not normally encode a signal sequence but are modified to include one. In some embodiments, the immunogens encoded by the circular polyribonucleotides include a secretory signal. For example, the secretory signal may be a naturally encoded secretory signal in a secretory protein. In another example, the secretory signal may be a modified secretory signal in a secretory protein. In other embodiments, the immunogens encoded by the circular polyribonucleotides do not include a secretory signal.

[0204] In some embodiments, the signal sequence is selected from SecSP38 (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO:1); SecD4 (MWWLLLLLLLLWPMVWA; SEQ ID NO:2), gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO:3); INHC1 (MASRLTLLTLLLLLLAGDRASS; SEQ ID NO:4); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO:5); and IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO:6).

[0205] In some embodiments, the circular polyribonucleotide encodes multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the same immunogen. In some embodiments, at least one copy of the immunogen includes a signal sequence and at least one copy of the immunogen does not include a signal sequence. In some embodiments, the circular polyribonucleotide encodes multiple immunogens (e.g., multiple different immunogens or multiple immunogens with less than 100% sequence identity), where at least one of the multiple immunogens includes a signal sequence and at least one copy of the multiple immunogens does not include a signal sequence.

[0206] In some embodiments, the signal sequence is a wild-type signal sequence (e.g., when endogenously expressed) that is present on the N-terminus of the corresponding wild-type immunogen. In some embodiments, the signal sequence is heterologous to the immunogen (e.g., not present when the wild-type immunogen is endogenously expressed). The polyribonucleotide sequence encoding the immunogen may be modified to remove nucleotide sequences encoding the wild-type signal sequence and / or to add sequences encoding a heterologous signal sequence.

[0207] The circular polyribonucleotide may further comprise one or more adjuvants, each with or without a signal sequence. In some embodiments, the circular polyribonucleotide encodes at least one adjuvant and at least one immunogen. In some embodiments, at least one encoded adjuvant comprises a signal sequence and at least one encoded immunogen does not comprise a signal sequence. In some embodiments, at least one encoded adjuvant comprises a signal sequence and at least one encoded immunogen comprises a signal sequence. In some embodiments, at least one encoded adjuvant does not comprise a signal sequence and at least one encoded immunogen comprises a signal sequence. In some embodiments, neither the encoded adjuvant nor the encoded immunogen comprises a signal sequence.

[0208] In some embodiments, the signal sequence is a wild-type signal sequence present at the N-terminus of the corresponding wild-type adjuvant (e.g., when endogenously expressed). In some embodiments, the signal sequence is heterologous to the adjuvant (e.g., not present when the wild-type adjuvant is endogenously expressed). The polyribonucleotide sequence encoding the adjuvant may be modified such that nucleotide sequences encoding the wild-type signal sequence are removed and / or sequences encoding a heterologous signal sequence are added.

[0209] A polypeptide encoded by a polyribonucleotide (e.g., an immunogen or an adjuvant encoded by a polyribonucleotide) may include a signal sequence that directs the immunogen or adjuvant into the secretory pathway. In some embodiments, the signal sequence may direct the immunogen or adjuvant to be present in a particular organelle (e.g., the endoplasmic reticulum, the Golgi apparatus, or an endosome). In some embodiments, the signal sequence directs the immunogen or adjuvant to be secreted from the cell. In the case of a secreted protein, the signal sequence may be cleaved after secretion, resulting in the mature protein. In other embodiments, the signal sequence may become embedded in the membrane of the cell or a particular organelle, creating a transmembrane segment that anchors the protein to the membrane of the cell, the endoplasmic reticulum, or the Golgi apparatus. In certain embodiments, the signal sequence of a transmembrane protein is a short sequence at the N-terminus of the polypeptide. In other embodiments, the first transmembrane domain acts as a first signal sequence that targets the protein to the membrane.

[0210] In some embodiments, the polyribonucleotide-encoded adjuvant comprises a secretory signal sequence. In some embodiments, the polyribonucleotide-encoded immunogen comprises a secretory signal sequence, a transmembrane insertion signal sequence, or no signal sequence.

[0211] Adjustment element The circular polyribonucleotide includes one or more regulatory elements (eg, one or more sequences that regulate expression of an expression sequence within the circular polyribonucleotide).

[0212] A regulatory element may include a sequence located adjacent to an expression sequence that encodes an expression product. The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of the expressed product compared to the amount of the product expressed in the absence of the regulatory element. A regulatory element may be used to increase the expression of one or more immunogens and / or adjuvants encoded by the circular polyribonucleotide. Similarly, a regulatory element may be used to decrease the expression of one or more immunogens and / or adjuvants encoded by the circular polyribonucleotide. In some embodiments, a regulatory element may be used to increase the expression of an immunogen and / or adjuvant and another regulatory element may be used to decrease the expression of another immunogen and / or adjuvant for the same circular polyribonucleotide. Furthermore, a regulatory element may increase the amount of a product (e.g., an immunogen or an adjuvant) expressed for multiple expression sequences linked side by side. Thus, a regulatory element may promote the expression of one or more expression sequences (e.g., an immunogen or an adjuvant). Multiple regulatory elements can also be used, for example, to differentially regulate expression of different expression sequences.

[0213] In some embodiments, the regulatory element provided herein may include a selective translation sequence. As used herein, the term "selective translation sequence" refers to a nucleic acid sequence that selectively initiates or activates the translation of an expression sequence in a circular polyribonucleotide, such as a specific riboswitch aptazyme. The regulatory element may also include a selective degradation sequence. As used herein, the term "selective degradation sequence" refers to a nucleic acid sequence that initiates the degradation of a circular polyribonucleotide or an expression product of a circular polyribonucleotide. In some embodiments, the regulatory element is a translation modulator. A translation modulator may regulate the translation of an expression sequence in a circular polyribonucleotide. A translation modulator may be a translation enhancer or suppressor. In some embodiments, a translation initiation sequence may function as a regulatory element.

[0214] In some embodiments, the cyclic polyribonucleotide produces expression products in a stoichiometric ratio. Rolling circle translation produces expression products in a substantially equal ratio in succession. In some embodiments, the cyclic polyribonucleotide exhibits stoichiometric translation efficiency, resulting in expression products being produced in a substantially equal ratio. In some embodiments, the cyclic polyribonucleotide exhibits stoichiometric translation efficiency of multiple expression products (e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more expression sequences). In some embodiments, the cyclic polyribonucleotide produces expression products in substantially different ratios. For example, the translation efficiency of multiple expression products may exhibit a ratio of 1:10,000; 1:7000, 1:5000, 1:1000, 1:700, 1:500, 1:100, 1:50, 1:10, 1:5, 1:4, 1:3 or 1:2. In some embodiments, the ratio of multiple expression products may be modified using regulatory elements.

[0215] Further examples of regulatory elements are described in paragraphs

[0154] to

[0161] of International Publication No. 2019 / 118919, the entirety of which is incorporated herein by reference.

[0216] Cleavage domain A circular polyribonucleotide of the disclosure may include a cleavage domain (e.g., a stagger element or cleavage sequence).

[0217] The term "stagger element" refers to a moiety, such as a nucleotide sequence, that induces ribosome posing during translation. In some embodiments, a stagger element is a non-conserved sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence -D(V / I)ExNPGP, where x=any amino acid (SEQ ID NO:7). In some embodiments, a stagger element can include a chemical moiety, such as glycerol, a non-nucleic acid linking moiety, a chemical modification, a modified nucleic acid, or any combination thereof.

[0218] In some embodiments, the circular polyribonucleotide comprises at least one stagger element flanking the expressed sequence. In some embodiments, the circular polyribonucleotide comprises a stagger element flanking each expressed sequence. In some embodiments, the stagger element is present on one or both sides of each expressed sequence, leading to separation of the expressed products (e.g., immunogens and / or adjuvants). In some embodiments, the stagger element is part of one or more expressed sequences. In some embodiments, the circular polyribonucleotide comprises one or more expressed sequences (e.g., immunogens and / or adjuvants), each of which is separated from the subsequent expressed sequence (e.g., immunogens and / or adjuvants) on the circular polyribonucleotide by a stagger element. In some embodiments, the stagger element prevents the generation of a single polypeptide from (a) two rounds of translation of a single expressed sequence, or (b) one or more rounds of translation of two or more expressed sequences. In some embodiments, the stagger element is a separate sequence from the one or more expressed sequences. In some embodiments, the stagger element comprises a portion of an expressed sequence in the one or more expressed sequences.

[0219] Examples of stagger elements are described in paragraphs

[0172] to

[0175] of WO 2019 / 118919, which is hereby incorporated by reference in its entirety.

[0220] In some embodiments, multiple immunogens and / or adjuvants encoded by a cyclic ribonucleotide may be separated by an IRES between each immunogen (e.g., each immunogen is operably linked to a separate IRES). For example, a cyclic polyribonucleotide may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence. The IRES may be the same IRES among all immunogens. The IRES may be different among different immunogens.

[0221] In some embodiments, multiple immunogens and / or adjuvants may be separated by a 2A self-cleaving peptide. For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first immunogen, 2A, and a second immunogen.

[0222] In some embodiments, multiple immunogens and / or adjuvants may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first immunogen, a protease cleavage site (e.g., a furin cleavage site), and a second immunogen.

[0223] In some embodiments, the multiple immunogens and / or adjuvants may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first immunogen, 2A, a protease cleavage site (e.g., a furin cleavage site), and a second immunogen. A circular polyribonucleotide may also encode an IRES operably linked to an open reading frame encoding a first immunogen, a protease cleavage site (e.g., a furin cleavage site), 2A, and a second immunogen. The tandem 2A and furin cleavage site may be referred to as furin-2A (which includes furin-2A or 2A-furin in either orientation).

[0224] Furthermore, multiple immunogens and / or adjuvants encoded by cyclic ribonucleotides may be separated by both an IRES sequence and a 2A sequence. For example, there may be an IRES between one immunogen and / or adjuvant and a second immunogen and / or adjuvant, while there may be a 2A peptide between a second immunogen and / or adjuvant and a third immunogen and / or adjuvant. The specific selection of the IRES or 2A self-cleaving peptide may be used to control the expression level of the immunogen and / or adjuvant under the control of the IRES or 2A sequence. For example, depending on the selection of the IRES and / or 2A peptide, the expression on the polypeptide may be high or low.

[0225] To maintain rolling circle translation while avoiding the production of consecutive expression products (e.g., immunogens and / or adjuvants), ribosome posing during translation may be induced by including a stagger element. In some embodiments, the stagger element is at the 3' end of at least one of the one or more expressed sequences. The stagger element can be configured to stall the ribosome during rolling circle translation of the circular polyribonucleotide. The stagger element may include, but is not limited to, 2A-like or CHYSEL (SEQ ID NO: 8) (cis-acting hydrolase element) sequences. In some embodiments, the stagger element encodes a sequence having a C-terminal consensus sequence of X1X2X3EX5NPGP, where X1 is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid (SEQ ID NO: 9). In some embodiments, this sequence comprises a non-conserved sequence of amino acids with strong alpha-helical propensity followed by the consensus sequence -D(V / I)ExNPGP (SEQ ID NO:7), where x=any amino acid. Some non-limiting examples of stagger elements include GDVESNPGP (SEQ ID NO:10), GDIEENPGP (SEQ ID NO:11), VEPNPGP (SEQ ID NO:12), IETNPGP (SEQ ID NO:13), GDIESNPGP (SEQ ID NO:14), GDVELNPGP (SEQ ID NO:15), GDIETNPGP (SEQ ID NO:16), GDVENPGP (SEQ ID NO:17), GDVEENPGP (SEQ ID NO:18), GDVEQNPGP (SEQ ID NO:19), IESNPGP (SEQ ID NO:20), GDIELNPGP (SEQ ID NO:21), HDIETPGP (SEQ ID NO:22), HDVETNPGP (SEQ ID NO:23), HDVEMNPGP (SEQ ID NO:24), GDMESNPGP (SEQ ID NO:25), GDVETNPGP (SEQ ID NO:26), GDIEQNPGP (SEQ ID NO:27), and DSEFNPGP (SEQ ID NO:28).

[0226] In some embodiments, the stagger elements described herein cleave the expression product, such as between the G and P of the consensus sequences described herein. As one non-limiting example, the cyclic polyribonucleotide comprises at least one stagger element to cleave the expression product. In some embodiments, the cyclic polyribonucleotide comprises a stagger element adjacent to at least one expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element after each expressed sequence. In some embodiments, the cyclic polyribonucleotide comprises a stagger element present on one or both sides of each expressed sequence, thus leading to the translation of individual peptides and / or polypeptides from each expressed sequence.

[0227] In some embodiments, the stagger element comprises one or more modified or non-natural nucleotides that induce ribosome posing during translation. Non-natural nucleotides can include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Such examples are distinguished from naturally occurring DNA or RNA by changes in the backbone of the molecule. Exemplary modifications can include any modification to the sugar, nucleobase, internucleoside bond (e.g., to the linking phosphate / to the phosphodiester bond / to the phosphodiester backbone), and any combination thereof that can induce ribosome posing during translation. Some of the exemplary modifications provided herein are described elsewhere herein.

[0228] In some embodiments, the stagger element is present in other forms in the circular polyribonucleotide. For example, in some exemplary circular polyribonucleotides, the stagger element comprises a termination element of a first expressed sequence in the circular polyribonucleotide and a nucleotide spacer sequence that separates the termination element from a first translation initiation sequence of a subsequent expression of the first expressed sequence. In some examples, the first stagger element of the first expressed sequence is upstream (5') of the first translation initiation sequence of a subsequent expression of the first expressed sequence in the circular polyribonucleotide. In some cases, the first expressed sequence and the subsequent expressed sequence of the first expressed sequence are two separate expressed sequences in the circular polyribonucleotide. The distance between the first stagger element and the first translation initiation sequence may allow for continuous translation of the first expressed sequence and its subsequent expressed sequence. In some embodiments, the first stagger element comprises a termination element to separate the expression product of the first expressed sequence from the expression product of its subsequent expressed sequence, thereby creating discontinuous expression products. In some cases, a circular polyribonucleotide that includes a first stagger element upstream of a first translation initiation sequence of a subsequent sequence in a circular polyribonucleotide is translated continuously, while a corresponding circular polyribonucleotide that includes a stagger element of a second expressed sequence upstream of a second translation initiation sequence of a subsequent expressed sequence of a second expressed sequence is not translated continuously. In some cases, there is only one expressed sequence in a circular polyribonucleotide, and the first expressed sequence and the subsequent expressed sequence are the same expressed sequence. In some exemplary circular polyribonucleotides, the stagger element includes a first termination element of a first expressed sequence in a circular polyribonucleotide and a nucleotide spacer sequence that separates the termination element from a downstream translation initiation sequence. In some such examples, the first stagger element is upstream (5') of the first translation initiation sequence of the first expressed sequence in a circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation initiation sequence allows for continuous translation of the first expressed sequence and any subsequent expressed sequence.In some embodiments, the first stagger element separates the expression products of one round of the first expression sequence from the expression products of the next round of the first expression sequence, thereby creating discontinuous expression products. In some cases, a circular polyribonucleotide that includes a first stagger element upstream of a first translation initiation sequence of a first expression sequence in a circular polyribonucleotide is translated continuously, while a corresponding circular polyribonucleotide that includes a stagger element upstream of a second translation initiation sequence of a second expression sequence in a corresponding circular polyribonucleotide is not translated continuously. In some cases, the distance between the second stagger element and the second translation initiation sequence in the corresponding circular polyribonucleotide is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than the distance between the first stagger element and the first translation initiation in the circular polyribonucleotide. In some cases, the distance between the first stagger element and the first translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or more. In some embodiments, the distance between the second stagger element and the second translation start is at least 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, or greater than the distance between the first stagger element and the first translation start. In some embodiments, the circular polyribonucleotide comprises two or more expressed sequences.

[0229] In some embodiments, the circular polyribonucleotide comprises at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to an expression sequence. In some embodiments, the cleavage sequence is between two expression sequences. In some embodiments, the cleavage sequence is included in the expression sequence. In some embodiments, the circular polyribonucleotide comprises 2-10 cleavage sequences. In some embodiments, the circular polyribonucleotide comprises 2-5 cleavage sequences. In some embodiments, the multiple cleavage sequences are between multiple expression sequences; for example, the circular polyribonucleotide may comprise 3 expression sequences, 2 cleavage sequences, with a cleavage sequence between each expression sequence. In some embodiments, the circular polyribonucleotide comprises a cleavage sequence, such as those found in sacrificial circRNAs, cleavable circRNAs, or self-cleaving circRNAs. In some embodiments, the circular polyribonucleotide comprises more than one cleavage sequence, leading to the separation of the circular polyribonucleotide into multiple products (e.g., miRNAs, linear RNAs, smaller circular polyribonucleotides, etc.).

[0230] In some embodiments, the cleavage sequence comprises a ribozyme RNA sequence. Ribozymes (from ribonucleic acid enzymes, also known as RNA enzymes or catalytic RNAs) are RNA molecules that catalyze chemical reactions. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of ribosomes. Catalytic RNAs can be "evolved" by in vitro methods. Similar to the riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In some embodiments, catalytic RNAs or ribozymes can be placed within larger non-coding RNAs such that the ribozyme is present in multiple copies within the cell for chemical conversion of molecules from bulk volumes. In some embodiments, the aptamer and ribozyme can both be encoded in the same non-coding RNA.

[0231] In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, the polyribonucleotide may encode two or more immunogens (e.g., where two or more immunogens are encoded by a single open reading frame (ORF)). For example, two or more immunogens may be encoded by a single open reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORF further encodes a polypeptide linker, such that, for example, the expression product of the ORF encodes two or more immunogens, each separated by a sequence encoding a polypeptide linker (e.g., a linker of 5-200, 5-100, 5-50, 5-20, 50-100, or 50-200 amino acids). The polypeptide linker may include a cleavage site, for example, that is recognized and cleaved by a protease (e.g., an endogenous protease of the subject upon administration of the polyribonucleotide to the subject). In such an embodiment, a single expression product comprising the amino acid sequences of two or more immunogens is cleaved during expression, such that the two or more immunogens are separated after expression. Exemplary protease cleavage sites are known to those of skill in the art and include, for example, amino acid sequences that serve as protease cleavage sites recognized by metalloproteinases (e.g., matrix metalloproteinases (MMPs), such as any one or more of MMP1-28), a disintegrin and metalloproteinase (ADAM, such as any one or more of ADAM2, 7-12, 15, 17-23, 28-30, and 33), serine proteases (e.g., furin), urokinase-type plasminogen activator, matriptase, cysteine ​​proteases, aspartic acid proteases, or cathepsin proteases. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.

[0232] In some embodiments, the cyclic polyribonucleotide described herein is a sacrificial cyclic polyribonucleotide, a cleavable cyclic polyribonucleotide, or a self-cleaving cyclic polyribonucleotide. The cyclic polyribonucleotide can deliver cellular components, including, for example, RNA, lncRNA, lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some embodiments, the cyclic polyribonucleotide comprises an miRNA separated by (i) a self-cleavable element; (ii) a cleavage recruitment site; (iii) a degradable linker; (iv) a chemical linker; and / or (v) a spacer sequence. In some embodiments, the circRNA comprises an siRNA separated by (i) a self-cleavable element; (ii) a cleavage recruitment site (e.g., ADAR); (iii) a degradable linker (e.g., glycerol); (iv) a chemical linker; and / or (v) a spacer sequence. Non-limiting examples of self-cleavable elements include hammerhead splicing elements, hairpins, Hepatitis D virus (HDV), Varkud satellite (VS), and the glmS ribozyme.

[0233] Translation initiation sequence In some embodiments, the circular polyribonucleotide encodes an immunogen and includes a translation initiation sequence (e.g., a start codon). In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the translation initiation sequence includes a Kozak sequence. In some embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some embodiments, the circular polyribonucleotide includes a translation initiation sequence (e.g., a Kozak sequence) adjacent to an expression sequence. In some embodiments, the translation initiation sequence is a non-coding start codon. In some embodiments, a translation initiation sequence (e.g., a Kozak sequence) is present on one or both sides of each expression sequence, resulting in separation of the expression products. In some embodiments, the circular polyribonucleotide includes at least one translation initiation sequence adjacent to an expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the circular polyribonucleotide. In some embodiments, the translation initiation sequence is within a substantially single-stranded region of the circular polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs

[0163] to

[0165] of International Publication No. 2019 / 118919, the entire contents of which are incorporated herein by reference.

[0234] A circular polyribonucleotide may include two or more start codons, such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons. Translation may begin at the first start codon or may begin downstream of the first start codon.

[0235] In some embodiments, the circular polyribonucleotide may initiate at a codon that is not the first start codon (e.g., AUG). Translation of the circular polyribonucleotide may initiate at an alternative translation initiation sequence, such as those described in International Patent Publication WO 2019 / 118919A1, which is incorporated herein by reference in its entirety.

[0236] In some embodiments, translation is initiated by processing of eukaryotic initiation factor 4A (eIF4A) with RocaGlate (translation is suppressed by blocking 43S scanning, causing premature upstream translation initiation and reduced protein expression from transcripts with RocA-eIF4A target sequences, see, e.g., www.nature.com / articles / nature17978).

[0237] Untranslated Regions In some embodiments, the circular polyribonucleotide comprises an untranslated region (UTR). The UTR of a genomic region that comprises a gene may be transcribed but not translated. In some embodiments, the UTR may be included upstream of the translation initiation sequence of the expression sequence described herein. In some embodiments, the UTR may be included downstream of the expression sequence described herein. In some cases, one UTR for a first expression sequence is the same as or contiguous with or overlaps with another UTR for a second expression sequence.

[0238] Exemplary untranslated regions are described in paragraphs

[0197] to

[0201] of International Publication No. 2019 / 118919, the entirety of which is incorporated herein by reference.

[0239] In some embodiments, the cyclic polyribonucleotide comprises a polyA sequence. Exemplary polyA sequences are described in paragraphs

[0202] to

[0205] of WO 2019 / 118919, the entirety of which is incorporated herein by reference. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence.

[0240] In some embodiments, the cyclic polyribonucleotide comprises a UTR that contains one or more stretches of adenosines and uridines. These AU-rich signatures may increase the turnover rate of the expression product.

[0241] The introduction, removal, or modification of AU-rich elements (AREs) in the UTRs can be useful for modulating the stability or immunogenicity (e.g., the level of one or more markers of immune or inflammatory response) of a cyclic polyribonucleotide. When modifying a particular cyclic polyribonucleotide, one or more copies of an ARE may be introduced into the cyclic polyribonucleotide, and the copies of the ARE may modulate the translation and / or production of the expression product. Similarly, AREs can be identified and removed or modified into a cyclic polyribonucleotide to modulate the intracellular stability, which in turn affects the translation and production of the resulting protein.

[0242] It should be understood that any UTR from any gene may be incorporated into each flanking region of the circular polyribonucleotide.

[0243] In some embodiments, the circular polyribonucleotide lacks a 5'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 3'UTR and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a polyA sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination sequence and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a cap and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5'UTR, a 3'UTR, and an IRES and is capable of protein expression from its one or more expression sequences. In some embodiments, the circular polyribonucleotide comprises one or more of the following sequences: a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding an exogenous gene, a sequence encoding a therapeutic, a regulatory element (e.g., a translation modulator, e.g., a translation enhancer or suppressor), a translation initiation sequence, one or more regulatory nucleic acids (e.g., siRNA, lncRNA, shRNA) targeting an endogenous gene, and a sequence encoding a therapeutic mRNA or protein.

[0244] In some embodiments, the cyclic polyribonucleotide lacks a 5'UTR. In some embodiments, the cyclic polyribonucleotide lacks a 3'UTR. In some embodiments, the cyclic polyribonucleotide lacks a polyA sequence. In some embodiments, the cyclic polyribonucleotide lacks a termination sequence. In some embodiments, the cyclic polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks susceptibility to degradation by exonucleases. In some embodiments, the fact that the cyclic polyribonucleotide lacks susceptibility to degradation can mean that the cyclic polyribonucleotide is not degraded by exonucleases or is degraded to a limited extent in the presence of exonucleases (e.g., is equivalent or similar in the absence of exonucleases). In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, the cyclic polyribonucleotide has reduced degradation when exposed to exonucleases. In some embodiments, the circular polyribonucleotide lacks a linkage to a cap-binding protein. In some embodiments, the circular polyribonucleotide lacks a 5' cap.

[0245] Closing element In some embodiments, the polyribonucleotide described herein comprises at least one termination element. In some embodiments, the polyribonucleotide comprises a termination element operably linked to the expression sequence. In some embodiments, the polynucleotide lacks a termination element.

[0246] In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element. In some embodiments, the polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element, such that the polyribonucleotide is translated in a continuous manner. The removal of the termination element may result in rolling circle translation or continuous expression of the expression product.

[0247] In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, each of which may or may not have a termination element, resulting in the circular polyribonucleotide being translated in a continuous manner. The removal of the termination element may result in rolling circle translation or continuous expression of expression products (e.g., due to the absence of peptide or polypeptide, ribosome stalling or shedding). In such embodiments, rolling circle translation results in the expression of continuous expression products through each of the expression sequences. In some other embodiments, the termination element of the expression sequence may be part of a stagger element. In some embodiments, one or more of the expression sequences in the circular polyribonucleotide comprises a termination element. However, rolling circle translation or expression of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the circular polyribonucleotide is performed. In such cases, the ribosome may fall off the expression product when it encounters a termination element (e.g., a stop codon and terminates translation). In some embodiments, translation terminates while the ribosome (e.g., at least one subunit of the ribosome) remains in contact with the circular polyribonucleotide.

[0248] In some embodiments, the circular polyribonucleotide comprises a termination element at the end of one or more expressed sequences. In some embodiments, one or more expressed sequences comprise two or more termination elements in succession. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome completely disengages from the circular polyribonucleotide. In some such embodiments, production of subsequent expressed sequences (e.g., second, third, fourth, fifth, etc.) in the circular polyribonucleotide may require the ribosome to reassociate with the circular polyribonucleotide before translation begins. Generally, the termination element comprises an in-frame nucleotide triplet (e.g., UAA, UGA, UAG) that signals the termination of translation. In some embodiments, one or more termination elements in the circular polyribonucleotide may cause a frame shift, such as, but not limited to, off-frame or -1 and +1 shifted reading frame (e.g., cryptic stop), thereby terminating translation. Frameshifted termination elements include the nucleotide triplets TAA, TAG, and TGA, which appear in the second and third reading frames of the expressed sequence. Frameshifted termination elements can be important in preventing misreading of mRNA, which is often harmful to cells. In some embodiments, the termination element is a stop codon.

[0249] In some embodiments, the expressed sequence includes a polyA sequence (eg, at the 3' end of the expressed sequence, eg, 3' to the termination element). In some embodiments, the length of the polyA sequence is greater than 10 nucleotides in length. In one embodiment, the polyA sequence is greater than 15 nucleotides in length (e.g., greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides in length). In some embodiments, the polyA sequence is designed according to the description of polyA sequences at

[0202] to

[0204] of WO 2019 / 118919 A1, which is incorporated by reference in its entirety. In some embodiments, the expression sequence lacks a polyA sequence (e.g., at the 3' end of the expression sequence).

[0250] In some embodiments, the cyclic polyribonucleotide comprises a polyA, lacks a polyA, or has a modified polyA to modulate one or more properties of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide lacks a polyA or has a modified polyA to improve one or more functional properties (e.g., immunogenicity (e.g., levels of one or more markers of an immune or inflammatory response), half-life, and / or expression efficiency).

[0251] Further examples of termination elements are described in paragraphs

[0169] to

[0170] of WO 2019 / 118919, which is hereby incorporated by reference in its entirety.

[0252] Spacer sequence In some embodiments, the circular polyribonucleotides described herein comprise a spacer sequence. In some embodiments, the polyribonucleotides described herein comprise one or more spacer sequences. A spacer refers to any contiguous nucleotide sequence (e.g., one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. A spacer can be present between any of the nucleic acid elements described herein. A spacer can also be present within the nucleic acid elements described herein.

[0253] The spacer can be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides long. In some embodiments, each spacer region is at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides long. Each spacer region can be, for example, 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides long. The first spacer region, the second spacer region, or the first spacer region and the second spacer region can include a polyA sequence. The first spacer region, the second spacer region, or the first spacer region and the second spacer region can include a polyAC sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly-AG sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a poly-AT sequence. In some embodiments, the first spacer region, the second spacer region, or the first spacer region and the second spacer region comprise a random sequence.

[0254] In some embodiments, the spacer sequence may be, for example, at least 10 nucleotides long, at least 15 nucleotides long, or at least 30 nucleotides long. In some embodiments, the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides long. In some embodiments, the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides long. In some embodiments, the spacer sequence is 20-50 nucleotides long. In certain embodiments, the spacer sequence is 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.

[0255] The spacer sequence may be a polyA sequence, a polyAC sequence, a polyC sequence, or a polyU sequence.

[0256] In some embodiments, the spacer sequence may be polyAT, polyAC, polyAG, or a random sequence.

[0257] Exemplary spacer sequences are described in paragraphs

[0293] to

[0302] of WO 2019 / 118919, which is hereby incorporated by reference in its entirety.

[0258] qualification It is within the scope of the present disclosure that a circular polyribonucleotide may contain one or more substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly a parent polyribonucleotide.

[0259] In some embodiments, the circular polyribonucleotide comprises one or more post-transcriptional modifications (e.g., capping, truncation, polyadenylation, splicing, polyA sequences, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than 100 different nucleoside modifications identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-97). In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the polyribonucleotide comprises at least one nucleoside selected from the group described in paragraph

[0311] of International Patent Publication WO 2019 / 118919 A1, which is incorporated by reference in its entirety.

[0260] Cyclic polyribonucleotides may include any useful modification, for example, to the sugar, nucleobase, or internucleoside linkage (e.g., linking phosphate / phosphodiester linkage / phosphodiester backbone). One or more atoms of the pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and the internucleoside linkage. The modification may be of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof). Further modifications are described herein.

[0261] In some embodiments, the cyclic polyribonucleotide comprises at least one N(6) methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the m6A modification may reduce the immunogenicity of the cyclic polyribonucleotide (e.g., reduce the level of one or more markers of an immune or inflammatory response).

[0262] In some embodiments, the modification may include chemical or cell-induced modifications. For example, some non-limiting examples of intracellular RNA modifications are described by Lewis and Pan, "RNA modifications and structures cooperate to guide RNA-protein interactions" in Nat Reviews Mol Cell Biol, 2017, 18:202-10.

[0263] In some embodiments, chemical modifications to the ribonucleotides of the cyclic polyribonucleotides may enhance immune evasion. The cyclic polyribonucleotides may be synthesized and / or modified by methods well established in the art, such as those described in Current protocols in nucleic acid chemistry, Beaucage, SLet al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA (hereby incorporated by reference). Modifications include, for example, terminal modifications, such as 5'-terminal modifications (phosphorylation (mono-, di- and tri-), conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), base modifications (e.g., replacement with a stabilizing base, a destabilizing base, or a base that base pairs with an expanded repertoire of partners), removal of a base (abasic nucleotide), or conjugated base. Modified ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In some embodiments, base modifications can regulate the expression, immune response, stability, and intracellular localization of cyclic polyribonucleotides, to name a few functional effects. In some embodiments, modifications include biorthogonal nucleotides, such as unnatural bases. See, for example, Kimoto et al, Chem Commun (Camb), 2017, 53:12309, DOI:10.1039 / c7cc06661a (herein incorporated by reference).

[0264] In some embodiments, the sugar modification (e.g., at the 2' or 4' position) or sugar substitution of one or more ribonucleotides of the cyclic polyribonucleotide, as well as the backbone modification, may include modification or substitution of the phosphodiester bond. Specific examples of cyclic polyribonucleotides include cyclic polyribonucleotides that include modified backbones or non-natural internucleoside linkages, such as internucleoside modifications, including, but not limited to, modification or substitution of the phosphodiester bond. Cyclic polyribonucleotides with modified backbones include, in particular, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides. In certain embodiments, the cyclic polyribonucleotide will include ribonucleotides that have a phosphorus atom in the internucleoside backbone.

[0265] Modified cyclic polyribonucleotide backbones may include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with inverted polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. In some embodiments, the cyclic polyribonucleotides may be negatively or positively charged.

[0266] Modified nucleotides that can be incorporated into cyclic polyribonucleotides can be modified with respect to the internucleoside bond (e.g., the phosphate backbone). In the present specification, in the context of polynucleotide backbones, the terms "phosphate" and "phosphodiester" are used interchangeably. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides can include extensive replacement of unmodified phosphate moieties with alternative internucleoside linkages as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Dithiophosphates have both non-linked oxygens replaced by sulfur. Phosphate linkers can also be modified by replacement of the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates).

[0267] The a-thio substituted phosphate moieties are provided to provide stability to RNA and DNA polymers via non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have enhanced nuclease resistance and subsequent extended half-life in cellular environments. Phosphorothioates linked to circular polyribonucleotides are expected to reduce innate immune responses via weaker binding / activation of cellular innate immune molecules.

[0268] In specific embodiments, the modified nucleoside comprises an α-thionucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0269] Other internucleoside linkages that may be used in accordance with the present disclosure are described herein, including internucleoside linkages that do not contain phosphorous atoms.

[0270] In some embodiments, the cyclic polyribonucleotide may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into the cyclic polyribonucleotide, such as bifunctional modifications. Cytotoxic nucleosides may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Further examples include fludarabine phosphate, N4-behenoyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl-1-β-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidate).

[0271] A cyclic polyribonucleotide may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally occurring nucleotides, purines or pyrimidines, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in a cyclic polyribonucleotide or in a given predetermined sequence region thereof. In some embodiments, a cyclic polyribonucleotide includes pseudouridine. In some embodiments, a cyclic polyribonucleotide includes inosine, which may contribute to the immune system characterizing cyclic polyribonucleotides as endogenous RNA and viral RNA. Incorporation of inosine may also mediate improved RNA stability / reduced degradation. See, for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

[0272] In some embodiments, all nucleotides in a cyclic polyribonucleotide (or a given sequence region thereof) are modified. In some embodiments, modifications may include m6A, which may enhance expression; inosine, which may attenuate immune response; pseudouridine, which may enhance RNA stability or translational read-through (staggered elements), m5C, which may enhance stability; and 2,2,7-trimethylguanosine, which aids in intracellular translocation (e.g., nuclear localization).

[0273] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) may be present at various positions in the cyclic polyribonucleotide. One skilled in the art will appreciate that nucleotide analogs or other modifications may be placed at any position in the cyclic polyribonucleotide without substantially reducing the function of the cyclic polyribonucleotide. Modifications may also be non-coding region modifications. Cyclic polyribonucleotides may be present in an amount ranging from about 1% to about 100% (either based on total nucleotide content, or on one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any percentage therebetween (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-95% , 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%) of modified nucleotides.

[0274] Method of preparation The present disclosure provides methods for making circular polyribonucleotides, including, for example, recombinant techniques or chemical synthesis. For example, the DNA molecules used to make the RNA circle can include DNA sequences of naturally occurring nucleic acid sequences, modified versions thereof, or DNA sequences that code for synthetic polypeptides not normally found in nature (e.g., chimeric molecules or fusion proteins). DNA and RNA molecules can be modified using a variety of techniques, including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme digestion of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification or mutagenesis of selected regions of nucleic acid sequences, synthesis of mixtures of oligonucleotides and ligation of mixtures to "build" mixtures of nucleic acid molecules, and combinations thereof.

[0275] Circular polyribonucleotides may be prepared by any available technique, including but not limited to chemical synthesis and enzymatic synthesis. In some embodiments, linear primary constructs or linear RNAs may be circularized or concatemerized to create circRNAs as described herein. The circularization or concatemerization mechanism may occur through methods such as chemical, enzymatic, splint ligation, or ribozyme catalysis. The newly formed 5'-3' linkage may be an intramolecular or intermolecular linkage. For example, splint ligases such as SplintR® ligase may be used for splint ligation. According to this method, a single-stranded polynucleotide (splint), such as a single-stranded DNA or RNA, may be designed to hybridize with both ends of a linear polyribonucleotide, such that the two ends are placed side-by-side upon hybridization with the single-stranded splint. The splint ligase may catalyze the ligation of the two ends of the linear polyribonucleotide placed side-by-side in this manner, resulting in a circRNA. In some embodiments, a DNA or RNA ligase may be used in the synthesis of a circular polynucleotide. As a non-limiting example, the ligase may be a circ ligase or a circular ligase.

[0276] In another example, either the 5' or 3' end of the linear polyribonucleotide can encode a ligase ribozyme sequence, such that upon in vitro transcription, the resulting linear circRNA will contain an active ribozyme sequence capable of ligating the 5' end of the linear polyribonucleotide to the 3' end of the linear polyribonucleotide. The ligase ribozyme may be derived from a group I intron, hepatitis D virus, a hairpin ribozyme, or may be selected by SELEX (enrichment of exogenous sequences).

[0277] In another example, a linear polyribonucleotide may be cyclized or concatemerized by using at least one non-nucleic acid moiety. For example, the at least one non-nucleic acid moiety may react with a region or feature near the 5' end or near the 3' end of the linear polyribonucleotide to cyclize or concatemerize the linear polyribonucleotide. In another example, the at least one non-nucleic acid moiety may be located at or near the 5' end or the 3' end of the linear polyribonucleotide or may be linked to it. The non-nucleic acid moiety may be homogeneous or heterogeneous. As a non-limiting example, the non-nucleic acid moiety may be a linking chain, such as a hydrophobic linking chain, an ionic linking chain, a biodegradable linking chain, or a cleavable linking chain. As another non-limiting example, the non-nucleic acid moiety may be a ligation moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, such as an aptamer or a non-nucleic acid linker as described herein.

[0278] In another example, the linear polyribonucleotide may be circularized or concatemerized by self-splicing. In some embodiments, the linear polyribonucleotide contains a loop E sequence to self-ligate. In another embodiment, the linear polyribonucleotide may contain a self-cyclizing intron, such as a 5' and 3' slice junction, or a self-cyclizing catalytic intron, such as a group I, group II, or group III intron. Non-limiting examples of group I intron self-splicing sequences include the self-splicing permuted intron-exon sequence from the T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of Tetrahymena, the cyanobacterium Anabaena pre-tRNA-Leu gene, or the Tetrahymena pre-rRNA.

[0279] In some embodiments, the polyribonucleotide may include a catalytic intron fragment, such as a 3' half fragment of a group I catalytic intron and a 5' half fragment of a group I catalytic intron. The first and second annealing regions may be located within the catalytic intron fragment. The group I catalytic intron is a self-splicing ribozyme that catalyzes its own cleavage from mRNA, tRNA, and rRNA precursors by a two-metal ion phosphoryl transfer mechanism. Importantly, the RNA itself self-catalyzes intron removal without the need for exogenous enzymes such as ligases.

[0280] In some embodiments, the 3' half fragment of the group I catalytic intron and the 5' half fragment of the group I catalytic intron are from a cyanobacterial Anabaena pre-tRNA-Leu gene or a Tetrahymena pre-rRNA.

[0281] In some embodiments, the 3' half fragment of the group I catalytic intron and the 5' half fragment of the group I catalytic intron are from a cyanobacterium Anabaena pre-tRNA-Leu gene, the 3' exon fragment comprises a first annealing region, and the 5' exon fragment comprises a second annealing region. The first annealing region may comprise, for example, 5 to 50, e.g., 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides, and the second annealing region may comprise, for example, 5 to 50, e.g., 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides.

[0282] In some embodiments, the 3' half fragment of the group I catalytic intron and the 5' half fragment of the group I catalytic intron are from Tetrahymena pre-rRNA, and the 3' half fragment of the group I catalytic intron comprises a first annealing region and the 5' exon fragment comprises a second annealing region. In some embodiments, the 3' exon comprises a first annealing region and the 5' half fragment of the group I catalytic intron comprises a second annealing region. The first annealing region may comprise, for example, 6 to 50, e.g., 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, 6 to 50, e.g., 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0283] In some embodiments, the 3' half fragment of the group I catalytic intron and the 5' half fragment of the group I catalytic intron are from the cyanobacterium Anabaena pre-tRNA-Leu gene, Tetrahymena pre-rRNA, or T4 phage td gene.

[0284] In some embodiments, the 3' half fragment of the group I catalytic intron and the 5' half fragment of the group I catalytic intron are from the T4 phage td gene. The 3' exon fragment may comprise a first annealing region, and the 5' half fragment of the group I catalytic intron may comprise a second annealing region. The first annealing region may comprise, for example, 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, 2 to 16, e.g., 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

[0285] In some embodiments, the 3' half fragment of the Group I catalytic intron is the 5' end of the linear polynucleotide.

[0286] In some embodiments, the 5' half-fragment of the Group I catalytic intron is the 3' end of a linear polyribonucleotide.

[0287] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0288] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0289] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:97 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:98.

[0290] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0291] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0292] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:99 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:100.

[0293] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0294] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0295] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:101 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:102.

[0296] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0297] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0298] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:103 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:104.

[0299] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0300] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0301] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:105 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:106.

[0302] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0303] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0304] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:107 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:108.

[0305] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0306] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0307] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:109 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:110.

[0308] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0309] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0310] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:111 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:112.

[0311] In some embodiments, the 3' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0312] In some embodiments, the 5' half fragment of the Group I catalytic intron comprises: [ka] The sequence has at least 80% (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity with the sequence of

[0313] In some embodiments, the 3' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:113 and the 5' half fragment of the Group I catalytic intron has the sequence of SEQ ID NO:114.

[0314] In another example, a linear polyribonucleotide may be cyclized or concatemerized by a non-nucleic acid moiety that creates an attractive force between atoms, molecular surfaces, at, near, or connected to the 5' and 3' ends of the linear polyribonucleotide. One or more linear polyribonucleotides may be cyclized or concatemerized by intermolecular or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der Waals forces, and London dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, agnostic bonds, dipole bonds, conjugation, hyperconjugation, and antibonds.

[0315] In another example, a linear polyribonucleotide may contain ribozyme RNA sequences near the 5' and 3' ends. The ribozyme RNA sequences may be covalently linked to a peptide when the sequences are exposed to the remainder of the ribozyme. Peptides covalently linked to ribozyme RNA sequences near the 5' and 3' ends may associate with each other, thereby causing circularization or concatemerization of the linear polyribonucleotide. In another example, peptides covalently linked to ribozyme RNA near the 5' and 3' ends may cause circularization or concatemerization of a linear primary construct or linear mRNA after being subjected to ligation using various methods known in the art, including but not limited to protein ligation. A non-limiting list of examples of ribozymes or methods of incorporating or covalently linking peptides for use in the linear primary constructs or linear polyribonucleotides of the invention is provided in U.S. Patent Application Publication No. 20030082768, the contents of which are incorporated by reference in their entirety herein.

[0316] In yet another example, chemical cyclization methods may be used to generate circular polyribonucleotides, including, but not limited to, click chemistry (e.g., alkyne and azide-based methods, or click reactive bases), olefin metathesis, phosphoramidate ligation, hemiaminal-imine crosslinking, base modification, and any combination thereof.

[0317] In another example, circular polyribonucleotides can be made by using a deoxyribonucleotide template that is transcribed in a cell-free system (e.g., by in vitro transcription) to make linear RNA, which can produce splicing-competent polyribonucleotides that can self-splice to make circular polyribonucleotides.

[0318] In some embodiments, the disclosure provides methods of making a circular polyribonucleotide (e.g., in a cell-free system) by providing a linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide; thereby making a circular polyribonucleotide.

[0319] In some embodiments, the disclosure provides methods of making a circular polyribonucleotide by providing deoxyribonucleotides encoding a linear polyribonucleotide; transcribing the deoxyribonucleotides in a cell-free system to make a linear polyribonucleotide; optionally purifying the splicing-compatible linear polyribonucleotide; and self-splicing the linear polyribonucleotide under conditions suitable for splicing of the 3' and 5' splice sites of the linear polyribonucleotide, thereby making a circular polyribonucleotide.

[0320] In some embodiments, the present disclosure provides a method for producing a circular polyribonucleotide by providing a deoxyribonucleotide encoding a linear polyribonucleotide; transcribing the deoxyribonucleotide in a cell-free system to produce a linear polyribonucleotide, the transcription being carried out in solution under conditions suitable for splicing the 3' and 5' splice sites of the linear polyribonucleotide, thereby producing a circular polyribonucleotide. In some embodiments, the linear polyribonucleotide comprises a 5' split-intron and a 3' split-intron (e.g., a self-splicing construct for producing a circular polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5' annealing region and a 3' annealing region.

[0321] Suitable conditions for in vitro transcription and / or self-splicing can include any conditions (e.g., solutions or buffers, such as aqueous buffers or solutions) that mimic physiological conditions in one or more respects. In some embodiments, suitable conditions include 0.1-100 mM Mg2+ ions or salts thereof (e.g., 1-100 mM, 1-50 mM, 1-20 mM, 5-50 mM, 5-20 mM, or 5-15 mM). In some embodiments, suitable conditions include 1-1000 mM K+ ions or salts thereof, such as KCl (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include 1-1000 mM Cl- ion or a salt thereof such as KCl (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include 0.1-100 mM Mn2+ ion or a salt thereof such as MnCl2 (e.g., 0.1-100 mM, 0.1-50 mM, 0.1-20 mM, 0.1-10 mM, 0.1-5 mM, 0.1-2 mM, 0.5-50 mM, 0.5-20 mM, 0.5-15 mM, 0.5-5 mM, 0.5-2 mM, or 0.1-10 mM). In some embodiments, suitable conditions include dithiothreitol (DTT) (e.g., 1 to 1000 μM, 1 to 500 μM, 1 to 200 μM, 50 to 500 μM, 100 to 500 μM, 100 to 300 μM, 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 20 mM, 0.1 to 10 mM, 0.1 to 5 mM, 0.1 to 2 mM, 0.5 to 50 mM, 0.5 to 20 mM, 0.5 to 15 mM, 0.5 to 5 mM, 0.5 to 2 mM, or 0.1 to 10 mM). In some embodiments, suitable conditions include 0.1 mM to 100 mM ribonucleoside triphosphate (NTP) (e.g., 0.1 to 100 mM, 0.1 to 50 mM, 0.1 to 10 mM, 1 to 100 mM, 1 to 50 mM, or 1 to 10 mM). In some embodiments, suitable conditions include a pH of 4 to 10 (e.g., a pH of 5 to 9, a pH of 6 to 9, or a pH of 6.5 to 8.5).In some embodiments, suitable conditions include a temperature between 4°C and 50°C (e.g., between 10°C and 40°C, between 15°C and 40°C, between 20°C and 40°C, or between 30°C and 40°C).

[0322] In some embodiments, the linear polyribonucleotide is generated from a deoxyribonucleic acid, such as a deoxyribonucleic acid described herein, such as a DNA vector, a linearized DNA vector, or a cDNA. In some embodiments, the linear polyribonucleotide is transcribed from a deoxyribonucleic acid by transcription in a cell-free system (e.g., in vitro transcription).

[0323] In another example, the circular polyribonucleotide may be made in a cell, for example, a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is provided with an exogenous polyribonucleotide (e.g., a linear polyribonucleotide described herein or a DNA molecule encoding the transcription of a linear polyribonucleotide described herein). The linear polyribonucleotide may be transcribed in the cell from an exogenous DNA molecule provided to the cell. The linear polyribonucleotide may be transcribed in the cell from an exogenous recombinant DNA molecule transiently provided to the cell. In some embodiments, the exogenous DNA molecule is not integrated into the genome of the cell. In some embodiments, the linear polyribonucleotide is transcribed in the cell from a recombinant DNA molecule integrated into the genome of the cell.

[0324] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell comprising the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. For example, prokaryotic cells comprising the polyribonucleotides described herein include, but are not limited to, Escherichia coli, halophilic archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g., Synechococcus elongatus, Spirulina spp. (Arthrospira spp., and Synechocystis spp.), Streptomyces, Actinomycetes (e.g., Nonomuraea, Kitasatospora, or Thermobifida), Bacillus spp., and the like. The prokaryotic cells may be selected from the group consisting of Bacillus spp. (e.g., Bacillus subtilis, Bacillus anthracis, Bacillus cereus), Betaproteobacteria (e.g., Burkholderia), Alphaproteobacteria (e.g., Agrobacterium), Pseudomonas (e.g., Pseudomonas putida), and Enterobacteria. The prokaryotic cells may be grown in a culture medium. The prokaryotic cells may be placed in a bioreactor.

[0325] The cell may be a eukaryotic cell. In some embodiments, the eukaryotic cell is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic organism is a unicellular fungal cell, such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp., Torulaspora spp., and Pichia spp.). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. The unicellular animal cell may be a cell isolated from a multicellular animal and grown in culture. In some embodiments, the unicellular animal cell may be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. The unicellular plant cell may be a cell isolated from a multicellular plant and grown in culture, or a daughter cell thereof. In some embodiments, the unicellular plant cell may be dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In some embodiments, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algae cell, such as a unicellular green alga, a diatom, a euglenid, or a dinoflagellate.Non-limiting examples of unicellular eukaryotic algae of interest include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon botryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cell.

[0326] In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryotic organism. For example, the multicellular eukaryotic organism may be selected from the group consisting of a vertebrate, an invertebrate, a multicellular fungus, a multicellular alga, and a multicellular plant. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryotic organism is an invertebrate. In some embodiments, the eukaryotic organism is a multicellular fungus. In some embodiments, the eukaryotic organism is a multicellular plant. In embodiments, the eukaryotic cell is a human cell or a non-human mammalian cell, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., bovine, including cow, buffalo, bison, sheep, goat, and muskox; pig; camelid, including camel, llama, and alpaca; deer, antelope; and equine, including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare) cell. In embodiments, the eukaryotic cell is a cell of an avian, e.g., a member of the avian taxonomic groups Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, geese), Paleaognathae (e.g., ostrich, emu), Columbiformes (e.g., pigeon, dove), or Psittaciformes (e.g., parrot). In embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, worm, or mollusc. In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm (which may be dicotyledonous or monocotyledonous) or gymnosperm (e.g., conifers, cycads, ephedra, ginkgo), ferns, horsetails, club mosses, etc. In embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular alga.

[0327] The eukaryotic cells may be grown in a culture medium. The eukaryotic cells may be contained in a bioreactor.

[0328] Examples of bioreactors include, without limitation, stirred tank (e.g., well-mixed) and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibrating mixers, fluidized bed reactors, and membrane bioreactors. The operation mode of the bioreactor can be a batch process or a continuous process. A bioreactor is continuous when the reagent and product streams are continuously fed and withdrawn from the system. A batch bioreactor has a continuous recycle stream, but the reagent feed or product withdrawal can be non-continuous. Some methods of the present disclosure relate to large-scale production of cyclic polyribonucleotides. For large-scale production methods, the method may be carried out in volumes of 1 liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, or more). In some embodiments, the method may be carried out in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L. In some embodiments, the bioreactor is capable of producing at least 1 g of circular RNA. In some embodiments, a bioreactor may produce 1-200 g of circular RNA (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, 50-200 g of circular RNA). In some embodiments, production is measured per liter (e.g., 1-200 g per liter), per batch or reaction (e.g., 1-200 g per batch or reaction), or per unit of time (e.g., 1-200 g per hour or day). In some embodiments, two or more bioreactors may be utilized in series to increase production capacity (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 bioreactors may be utilized in series).

[0329] Methods for making the circular polyribonucleotides described herein are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press (2002); in Zhao, SYNTHETIC BIOLOGY: TOOLS AND APPLICATIONS, (First Edition), Academic Press (2013); and Egli & Herdewijn, CHEMISTRY AND BIOLOGY OF ARTIFICIAL NUCLEIC ACIDS, (First Edition), Wiley-VCH (2012).

[0330] Various methods of synthesizing circular polyribonucleotides have also been described elsewhere (see, e.g., U.S. Pat. No. 6,210,931, U.S. Pat. No. 5,773,244, U.S. Pat. No. 5,766,903, U.S. Pat. No. 5,712,128, U.S. Pat. No. 5,426,180, U.S. Patent Publication No. 20100137407, WO 1992001813, WO 2010084371, and Petkovic et al., Nucleic Acids Res. 43:2454-65 (2015); the contents of each of which are incorporated by reference in their entirety herein).

[0331] In some embodiments, the circular polyribonucleotides are purified, e.g., free ribonucleic acid, linear or nicked RNA, DNA, proteins, etc. are removed. In some embodiments, the circular polyribonucleotides may be purified by any known method commonly used in the art. Non-limiting examples of purification methods include column chromatography, gel excision, size exclusion, etc.

[0332] immunization In some embodiments, the method of the disclosure includes immunizing a subject with an immunogenic composition comprising a cyclic polyribonucleotide as disclosed herein. In some embodiments, the immunogen is expressed from the cyclic polyribonucleotide. In some embodiments, the immunization induces an immune response in the subject against the immunogen expressed from the cyclic polyribonucleotide. In some embodiments, the immunization induces an immune response in the subject (e.g., induces the production of antibodies that bind to the immunogen expressed from the cyclic polyribonucleotide). In some embodiments, the immunization is for treating or preventing a disease, disorder, or condition in the subject (e.g., a human subject). In some embodiments, the immunization is for producing antibodies in the subject (e.g., producing antibodies for purification, as in the case of non-human mammals). In some embodiments, the immunogenic composition comprises the cyclic polyribonucleotide and a diluent, carrier, first adjuvant, or a combination thereof in a single composition. In some embodiments, the subject is further immunized with a second adjuvant. In some embodiments, the subject is further immunized with a second immunogenic composition.

[0333] The subject is immunized with one or more immunogenic compositions comprising any number of cyclic polyribonucleotides. The subject is, for example, immunized with one or more immunogenic compositions comprising at least one cyclic polyribonucleotide. The subject is, for example, immunized with one or more immunogenic compositions comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20 different cyclic polyribonucleotides, or more different cyclic polyribonucleotides. In some embodiments, the subject is immunized with one or more immunogenic compositions comprising at most one cyclic polyribonucleotide. In some embodiments, the subject is immunized with one or more immunogenic compositions comprising about one cyclic polyribonucleotide. In some embodiments, a subject is immunized with one or more immunogenic compositions comprising about 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-15, or 15-20 different cyclic polyribonucleotides. Different cyclic polyribonucleotides have different sequences from each other. For example, they can contain or code for different immunogens, overlapping immunogens, similar immunogens, or the same immunogen (e.g., with the same or different regulatory elements, initiation sequences, promoters, termination elements, or other elements of the present disclosure). When a subject is immunized with one or more immunogenic compositions that contain two or more different cyclic polyribonucleotides, the two or more different cyclic polyribonucleotides can be in the same or different immunogenic compositions and immunized at the same or different time points. The immunogenic compositions that contain two or more different cyclic polyribonucleotides can be administered to the same anatomical location or different anatomical locations.

[0334] In some embodiments, the immunogenic composition comprises a cyclic polyribonucleotide and a diluent, a carrier, a first adjuvant, or a combination thereof. In a detailed embodiment, the immunogenic composition comprises a cyclic polyribonucleotide as described herein and a carrier or a diluent that does not contain any carrier. In some embodiments, the immunogenic composition comprising a cyclic polyribonucleotide together with a diluent that does not contain any carrier is used for naked delivery of the cyclic polyribonucleotide to a subject. In another detailed embodiment, the immunogenic composition comprises a cyclic polyribonucleotide as described herein and a first adjuvant.

[0335] In certain embodiments, the subject is further administered a second adjuvant. The adjuvant enhances the innate immune response, which in turn enhances the adaptive immune response of the subject. The adjuvant can be any adjuvant as discussed below. In certain embodiments, the adjuvant is formulated with the cyclic polyribonucleotide as part of the immunogenic composition. In certain embodiments, the adjuvant is not part of the immunogenic composition comprising the cyclic polyribonucleotide. In certain embodiments, the adjuvant is administered separately from the immunogenic composition comprising the cyclic polyribonucleotide. In this aspect, the adjuvant is co-administered (e.g., administered at the same time) or at different times with the immunogenic composition comprising the cyclic polyribonucleotide to the subject. For example, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minutes or hours in between, after the immunogenic composition comprising a cyclic polyribonucleotide. In some embodiments, the adjuvant is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minutes or hours in between, before the immunogenic composition comprising the cyclic polyribonucleotide. For example, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any days in between, after the immunogenic composition comprising the cyclic polyribonucleotide. In some embodiments, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, prior to the immunogenic composition comprising a cyclic polyribonucleotide.The adjuvant may be administered in the same anatomical location as the immunogenic composition comprising the cyclic polyribonucleotide or in a different anatomical location.

[0336] In some embodiments, the subject is further immunized with a second agent, for example, a vaccine that is not a cyclic polyribonucleotide (as described below). This vaccine is co-administered (for example, administered simultaneously) with the immunogenic composition comprising cyclic polyribonucleotide to the subject, or is administered at a different time. For example, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours after the immunogenic composition comprising cyclic polyribonucleotide, or any minutes or hours therebetween. In some embodiments, the vaccine is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minutes or hours in between, before the immunogenic composition comprising cyclic polyribonucleotide. For example, the vaccine is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any days in between, after the immunogenic composition comprising cyclic polyribonucleotide. In some embodiments, the vaccine is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any number of days in between, prior to the immunogenic composition comprising a cyclic polyribonucleotide.

[0337] A subject may be immunized with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combinations thereof any number of times suitable to achieve the desired response. For example, a prime-boost immunization strategy can be utilized to elicit systemic and / or mucosal immunity. A subject may be immunized with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combinations thereof of the present disclosure, for example, at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or at least 15 times or more.

[0338] In some embodiments, a subject may be immunized with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine) of the disclosure, or a combination thereof, no more than two times, no more than three times, no more than four times, no more than five times, no more than six times, no more than seven times, no more than eight times, no more than nine times, no more than ten times, no more than fifteen times, or no more than twenty times, or less.

[0339] In some embodiments, a subject may be immunized about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine) of the disclosure, or a combination thereof.

[0340] In some embodiments, a subject may be immunized once with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combination thereof of the present disclosure. In some embodiments, a subject may be immunized twice with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combination thereof of the present disclosure. In some embodiments, a subject may be immunized three times with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combination thereof of the present disclosure. In some embodiments, a subject may be immunized four times with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combination thereof of the present disclosure. In some embodiments, a subject may be immunized five times with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combination thereof of the present disclosure. In some embodiments, a subject may be immunized seven times with an immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combination thereof of the present disclosure.

[0341] A suitable time interval between two or more immunizations can be selected. The time interval can be applied to multiple immunizations with the same immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combinations thereof, for example, the same immunogenic composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or combinations thereof, can be administered in the same amount or in different amounts, by the same immunization route, or by different immunization routes. The time interval can be applied to multiple immunizations with different immunogenic compositions, adjuvants, or vaccines (e.g., protein subunit vaccine), or combinations thereof, for example, different immunogenic compositions, adjuvants, or vaccines (e.g., protein subunit vaccine), or combinations thereof, can be administered in the same amount or in different amounts, by the same immunization route, or by different immunization routes. The time interval can be applied to immunizations with different agents, for example, a first immunogenic composition comprising a first cyclic polyribonucleotide and a second immunogenic composition comprising a second cyclic polyribonucleotide. The time interval can be applied to immunization with different agents, for example, a first immunogenic composition comprising a first cyclic polyribonucleotide and a second immunogenic composition comprising a protein immunogen (e.g., a protein subunit). In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 48, or 72 hours pass between two immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 21, 24, 28, or 30 days pass between two immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, or 8 weeks pass between two immunizations, In some embodiments, about 1, 2, 3, 4, 5, 6, 7, or 8 months pass between two immunizations.

[0342] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 24, at least 36, or at least 72 hours or more elapse between the two immunizations. In some embodiments, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 24, at most 36, or at most 72 hours or less elapse between the two immunizations.

[0343] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26 at least 27, at least 28, at least 29, or at least 30 days or more elapse between the two immunizations. In some embodiments, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 32, at most 34, or at most 36 days or less elapse between the two immunizations.

[0344] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 weeks or more elapse between two immunizations, in some embodiments, not more than 2, not more than 3, not more than 4, not more than 5, not more than 6, not more than 7, not more than 8 weeks elapse between two immunizations.

[0345] In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 months or more elapse between the two immunizations, in some embodiments, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8 months, at most 9 months, at most 10 months, at most 11 months, or at most 12 months or less elapse between the two immunizations.

[0346] In some embodiments, the method includes pre-administering an agent to the subject to improve the immunogenic response to a cyclic polyribonucleotide comprising a sequence encoding an immunogen. In some embodiments, the agent is an immunogen (e.g., a protein immunogen) as disclosed herein. For example, the method includes administering the protein immunogen 1-7 days prior to administering the cyclic polyribonucleotide comprising a sequence encoding the protein immunogen. In some embodiments, the protein immunogen is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administering the cyclic polyribonucleotide comprising a sequence encoding the protein immunogen. The protein immunogen may be administered as a protein preparation, encoded on a plasmid (pDNA), presented as a virus-like particle (VLP), formulated in a lipid nanoparticle, etc.

[0347] In some embodiments, the method includes administering an agent to the subject to improve the immunogenic response to a cyclic polyribonucleotide comprising a sequence encoding an immunogen after the cyclic polyribonucleotide comprising a sequence encoding an immunogen is administered to the subject. In some embodiments, the agent is an immunogen (e.g., a protein immunogen) as disclosed herein. In some embodiments, the cyclic polyribonucleotide comprises a sequence encoding a protein immunogen. For example, the method includes administering a protein immunogen within one year (e.g., within 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, and 1 month) of administering a cyclic polyribonucleotide comprising a sequence encoding an immunogen to the subject. In some embodiments, the method includes administering any one of the cyclic polyribonucleotides described herein or any one of the immunogenic compositions described herein and a protein subunit to the subject.

[0348] In some embodiments, the protein immunogen has the same amino acid sequence as the immunogen encoded by the cyclic polyribonucleotide. For example, the polypeptide immunogen can correspond to (e.g., share 90%, 95%, 96%, 97%, 98%, or 100% amino acid sequence identity with) the polypeptide immunogen encoded by the sequence of the cyclic polyribonucleotide. In some embodiments, the protein immunogen has a different amino acid sequence from the amino acid sequence of the immunogen encoded by the cyclic polyribonucleotide. For example, the polypeptide immunogen can share less than 90% (e.g., 80%, 70%, 30%, 20%, or 10%) amino acid sequence identity with the polypeptide immunogen encoded by the sequence of the cyclic polyribonucleotide.

[0349] A subject may be immunized with an immunogenic composition, adjuvant, or vaccine (e.g., a protein subunit vaccine), or combinations thereof, at any suitable number of anatomical sites. Multiple anatomical sites may be administered the same immunogenic composition, adjuvant, vaccine (e.g., a protein subunit vaccine), or combinations thereof, different anatomical sites may be administered different immunogenic compositions comprising the same or different cyclic polyribonucleotides, adjuvants, vaccines (e.g., a protein subunit vaccine), or combinations thereof, different anatomical sites may be administered different immunogenic compositions comprising the same or different cyclic polyribonucleotides, adjuvants, vaccines (e.g., a protein subunit vaccine), or combinations thereof, or any combination thereof. For example, an immunogenic composition comprising cyclic polyribonucleotides may be administered at two different anatomical sites, and / or an immunogenic composition comprising cyclic polyribonucleotides may be administered at one anatomical site, and an adjuvant may be administered at a different anatomical site.

[0350] Immunization in any two or more anatomical routes may be via the same immunization route (e.g., intramuscular) or by two or more immunization routes. In some embodiments, the immunogenic composition comprising the cyclic polyribonucleotide, adjuvant, or vaccine (e.g., protein subunit vaccine) of the present disclosure, or a combination thereof, is immunized into at least one, at least two, at least three, at least four, at least five, or at least six anatomical sites of the subject. In some embodiments, the immunogenic composition comprising the cyclic polyribonucleotide, adjuvant, or vaccine (e.g., protein subunit vaccine) of the present disclosure, or a combination thereof, is immunized into at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten anatomical sites of the subject, or fewer sites. In some embodiments, the immunogenic composition comprising the cyclic polyribonucleotide of the present disclosure, or an adjuvant, is immunized into at least one, two, three, four, five, six, seven, eight, nine, or ten anatomical sites of the subject.

[0351] Immunization can be by any suitable route. Non-limiting examples of immunization routes include, for example, injection and infusion, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular, intralesional, intraventricular, intracisternal, or intraparenchymal. In some cases, immunization can be by inhalation. Two or more immunizations can be by the same route or by different routes.

[0352] The subject of the present disclosure can be administered any suitable amount of cyclic polyribonucleotide.For example, the subject can be immunized with at least about 1 ng, at least about 10 ng, at least about 100 ng, at least about 1 μg, at least about 10 μg, at least about, at least about 100 μg, at least about 1 mg, at least about 10 mg, at least about 100 mg, or at least about 1 g of cyclic polyribonucleotide.In some embodiments, the subject can be immunized with at most about 1 ng, at most about 10 ng, at most about 100 ng, at most about 1 μg, at most about 10 μg, at most about, at most about 100 μg, at most about 1 mg, at most about 10 mg, at most about 100 mg, or at most about 1 g of cyclic polyribonucleotide. In some embodiments, a subject may be immunized with about 1 ng, about 10 ng, about 100 ng, about 1 μg, about 10 μg, about, about 100 μg, about 1 mg, about 10 mg, about 100 mg, or about 1 g of cyclic polyribonucleotide.

[0353] In some embodiments, the method further comprises evaluating the subject for an antibody response to the immunogen. In some embodiments, the evaluation is before and / or after administration of a circular polyribonucleotide comprising a sequence encoding the immunogen.

[0354] Antibody production and purification Immunization of a subject with a polyribonucleotide described herein (e.g., a polyribonucleotide encoding an immunogen comprising a multimerization domain) can induce the production of antibodies (e.g., generate antibodies) in the subject that bind to the immunogen expressed from the cyclic polyribonucleotide. In some embodiments, immunization is aimed at producing antibodies in the subject (e.g., a human or non-human animal), which are quantified or purified from the subject (e.g., for diagnostic or therapeutic use). Thus, the cyclic polyribonucleotides of the invention can be used in methods for producing polyclonal or monoclonal antibodies (e.g., polyclonal or monoclonal antibodies).

[0355] For example, the disclosure provides for administering a cyclic polyribonucleotide described herein (e.g., encoding an immunogen comprising a multimerization domain) to a non-human animal (e.g., a non-human mammal such as a goat, pig, rabbit, rat, mouse, llama, camel, horse, donkey, or cow (dairy cow)). The cyclic polyribonucleotide may be administered according to any composition, formulation, route or administration, amount, or dose administration regimen described herein (e.g., optionally with an adjuvant administered in the same composition or as part of the dose administration regimen). In some embodiments, the non-human animal has a humanized immune system (e.g., a cow with a humanized immune system).

[0356] From a subject immunized with cyclic polyribonucleotides, plasma containing polyclonal antibodies produced from an immunogenic composition comprising cyclic polyribonucleotides as disclosed herein can be collected. Such polyclonal antibodies can be quantified (e.g., for diagnostic purposes in human subjects) or purified (e.g., for use in therapeutic methods or for the development of monoclonal antibodies). Plasma can be collected by methods known to those skilled in the art, for example, by plasmapheresis. Plasma can be collected from the same subject one or more times, for example, multiple times after each given period of time from immunization, multiple times after immunization, multiple times during immunization, or any combination thereof.

[0357] According to the methods described herein, antibodies, or fragments thereof (e.g., polyclonal antibodies, such as human or humanized polyclonal antibodies) that specifically bind to an immunogen comprising a multimerization domain can be produced. The antibodies, or fragments thereof, may be purified from blood (e.g., from plasma or serum) by methods known to those of skill in the art.

[0358] Polyclonal antibodies can be purified from plasma using techniques well known to those skilled in the art. For example, adjust the pH of plasma to 4.8 (e.g., by adding 20% ​​acetic acid dropwise), fractionate with caprylic acid at a caprylic acid / total protein ratio of 1.0, and then clarify by centrifugation (e.g., 10,000g for 20 minutes at room temperature). The supernatant containing polyclonal antibodies (e.g., IgG polyclonal antibodies) is neutralized to pH 7.5 with 1M Tris, filtered through a 0.22 μM filter, and affinity purified with an anti-human immunoglobulin specific column (e.g., an anti-human IgG light chain specific column). The polyclonal antibodies are further purified by passing through an affinity column that specifically binds impurities, e.g., non-human antibodies from non-human animals. The polyclonal antibody is stored in a suitable buffer, for example, a sterile filtered buffer consisting of 10 mM monosodium glutamate, 262 mM D-sorbitol, and Tween® (0.05 mg / ml), pH 5.5. The quantity and concentration of the purified polyclonal antibody is determined. HPLC size exclusion chromatography is performed to determine whether aggregates or multimers are present. In some embodiments, human polyclonal antibodies are purified from a non-human animal with a humanized immune system according to Beigel, JH et al., LANCET INFECT. DIS., 18:410-18 (2018), including Supplementary appendix, which is incorporated herein by reference in its entirety.

[0359] The present disclosure also provides methods of producing antibodies in a human subject, e.g., for therapeutic treatment and / or diagnosis. For example, the present disclosure provides methods of quantifying anti-immunogen antibody levels in a subject after administration of a cyclic polyribonucleotide or immunogenic composition described herein. Quantification may be performed by methods known in the art (e.g., performing an antibody titer), e.g., by obtaining a blood sample from the subject and quantifying anti-immunogen antibody levels using standard techniques such as enzyme-linked immunosorbent assay (ELISA). Antibodies may also be purified by methods known to those of skill in the art.

[0360] Adjuvants Adjuvants enhance the immune response (humoral and / or cellular) elicited in a subject receiving the adjuvant and / or an immunogenic composition comprising the adjuvant. In some embodiments, the adjuvant is administered to the subject as disclosed herein. In some embodiments, the adjuvant is used in the methods described herein to generate an immune response as described herein. In particular embodiments, the adjuvant is used to enhance the subject's immune response to the immunogen expressed from the cyclic polyribonucleotide. In some embodiments, the adjuvant and the polyribonucleotide are co-administered in separate compositions. In some embodiments, the adjuvant is mixed or formulated with the polyribonucleotide in a single composition and administered to the subject. In some embodiments, the adjuvant and the cyclic polyribonucleotide are co-administered in separate compositions. In some embodiments, the adjuvant is mixed or formulated with the cyclic polyribonucleotide in a single composition to obtain the immunogenic composition administered to the subject.

[0361] The adjuvant may be a component of the cyclic polyribonucleotide (e.g., a polyribonucleotide sequence), may be a polypeptide adjuvant encoded by the expressed sequence of the polyribonucleotide, or may be a molecule not encoded by the polyribonucleotide (e.g., a small molecule, a polypeptide, or a nucleic acid molecule). The adjuvant may be formulated in the same pharmaceutical composition with the polyribonucleotide. The adjuvant may be administered separately in combination with the polyribonucleotide (e.g., as a separate pharmaceutical composition).

[0362] In some embodiments, the adjuvant is encoded by a cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide encodes two or more adjuvants. For example, the cyclic polyribonucleotide encodes between 2 and 100 adjuvants. In some embodiments, the cyclic polyribonucleotide encodes between 2 and 10 adjuvants. In some embodiments, the cyclic polyribonucleotide encodes two adjuvants. One or more of the adjuvants encoded by the cyclic polyribonucleotide may include an N-terminal signal sequence, for example, to direct the expressed polypeptide adjuvant to the secretory pathway. In some embodiments, the polyribonucleotide encodes three adjuvants. In some embodiments, the polyribonucleotide encodes four adjuvants. In some embodiments, the polyribonucleotide encodes five adjuvants. In some embodiments, the adjuvant is encoded by the same polyribonucleotide that encodes one or more immunogens. The adjuvant and the immunogen may be co-delivered on the same polyribonucleotide. In some embodiments, the polyribonucleotide-encoded adjuvant is a sequence (e.g., a polyribonucleotide sequence) that is an innate immune system stimulator. The innate immune system stimulator sequence may comprise at least 5, at least 10, at least 20, at least 50, at least 100, or at least 500 ribonucleotides. The innate immune system stimulator sequence may comprise 5-1000, 10-500, 20-500, 10-100, 20-100, 20-50, 100-500, 500-1000, or 10-1000 ribonucleotides. For example, the innate immune system stimulator sequence may be selected from a GU-rich motif, an AU-rich motif, a structured region comprising dsRNA, or an aptamer.

[0363] The adjuvant may be a TH1 adjuvant and / or a TH2 adjuvant. Additional adjuvants contemplated by the present disclosure include, but are not limited to, one or more of the following:

[0364] Mineral-containing compositions. Mineral-containing compositions suitable for use as adjuvants in the present disclosure include mineral salts, such as aluminum salts and calcium salts. The present disclosure includes mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, etc., or mixtures of different mineral compounds in any suitable form (e.g., gel, crystalline, amorphous, etc.). Calcium salts include calcium phosphate (e.g., "CAP"). Aluminum salts include hydroxides, phosphates, sulfates, etc.

[0365] Oil-based emulsion compositions. Oil-based emulsion compositions suitable for use as adjuvants in the present disclosure include squalene-water emulsions such as MF59 (5% squalene, 0.5% Tween® 80 and 0.5% Span®, formulated into submicron particles using a microfluidizer), AS03 (α-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion), Montanide formulations (e.g., Montanide ISA 51, Montanide ISA 720), incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), and incomplete Freund's adjuvant (IFA).

[0366] Small molecules suitable for use as adjuvants in the present disclosure include imiquimod or 847, resiquimod or R848, and gardquimod.

[0367] Polymeric nanoparticles. Polymeric nanoparticles suitable for use as adjuvants in the present disclosure include poly(a-hydroxy acids), polyhydroxybutyric acids, polylactones (including polycaprolactones), polydioxanones, polyvalerolactones, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine derivative polycarbonates, polyvinyl-pyrrolidinones or polyester-amides, and combinations thereof.

[0368] Saponins (i.e., glycosides, polycyclic aglycones attached to one or more sugar side chains). Saponin formulations suitable for use as adjuvants in the present disclosure include purified preparations, such as QS21, and lipid formulations, such as ISCOMs and ISCOM matrices. QS21 is sold under the trade name STIMULON™. Saponin formulations may also include a sterol, such as cholesterol. A combination of saponin and cholesterol can be used to form unique particles called immune stimulating complexes (ISCOMs). ISCOMs also typically include a phospholipid, such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in the ISCOMs. Preferably, the ISCOMs include one or more of QuilA, QHA, and QHC. Optionally, the ISCOMs may lack additional detergents.

[0369] Lipopolysaccharides. Suitable adjuvants for use in the present disclosure include non-toxic derivatives of enterobacterial lipopolysaccharide (LPS). Such derivatives include monophosphoryl lipid A (MPLA), glucopyranosyl lipid A (GLA) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of three de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives, e.g., RC-529.

[0370] Liposomes. Liposomes suitable for use as adjuvants in the present disclosure include virosomes and CAF01.

[0371] Lipid Nanoparticles. Adjuvants suitable for use in the present disclosure include lipid nanoparticles (LNPs) and components thereof.

[0372] Lipopeptides (i.e., compounds that contain one or more fatty acid residues and two or more amino acid residues). Lipopeptides suitable for use as adjuvants in the present disclosure include Pam2 (Pam2CSK4) and Pam3 (Pam3CSK4).

[0373] Glycolipids. Glycolipids suitable for use as adjuvants in the present disclosure include Cord Factor (trehalose dimycolate).

[0374] Peptides and peptidoglycans (synthetic or purified) derived from Gram-negative or Gram-positive bacteria, such as MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine), are suitable for use as adjuvants in the present disclosure.

[0375] Carbohydrates (carbohydrate-containing) or polysaccharides suitable for use as adjuvants include dextran (eg, branched microbial polysaccharides), dextran sulfate, lentinan, zymosan, beta-glucan, deltin, mannan, and chitin.

[0376] RNA-based adjuvants. RNA-based adjuvants suitable for use in the present disclosure are poly IC, poly IC:LC, hairpin RNA with or without 5' triphosphate, viral sequences, poly U-containing sequences, dsRNA natural or synthetic RNA sequences (e.g., poly I:C), and nucleic acid analogs (e.g., cyclic GMP-AMP or other cyclic dinucleotides, e.g., cyclic di-GMP, immunostimulatory base analogs, e.g., C8-substituted and N7,C8-disubstituted guanine ribonucleotides). In some embodiments, the adjuvant is a linear polyribonucleotide counterpart of the cyclic polyribonucleotides described herein.

[0377] DNA-Based Adjuvants DNA-based adjuvants suitable for use in the present disclosure include CpGs (e.g., CpG1018), dsDNA, and natural or synthetic immunostimulatory DNA sequences.

[0378] Proteins or Peptides. Proteins and peptides suitable for use as adjuvants in the present disclosure include flagellin fusion proteins, MBL (mannose-binding lectin), cytokines, and chemokines.

[0379] Viral particles.Viral particles suitable for use as adjuvants include virosomes (phospholipid cell membrane bilayers).

[0380] Adjuvants for use in the present disclosure may be bacterially derived, such as flagellin, LPS, or bacterial toxins (e.g., enterotoxins (proteins), e.g., heat-labile toxins or cholera toxins). Adjuvants for use in the present disclosure may be hybrid molecules, such as CpG conjugated to imiquimod. Adjuvants for use in the present disclosure may be fungal or oomycete microbe-associated molecular patterns (MAMPs), such as chitin or beta-glucan. In some embodiments, the adjuvant is an inorganic nanoparticle, such as gold nanorods or silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSNs)). In some embodiments, the adjuvant is a multicomponent adjuvant or adjuvant system such as AS01 (AS01B), AS03, AS04 (MLP5 + alum), alum (a mixture of aluminum hydroxide and magnesium hydroxide), aluminum hydroxide, magnesium hydroxide, CFA (Complete Freund's adjuvant: IFA + peptiglycan + trehalose dimycolate), CAF01 (a two-component cationic liposomal vehicle (dimethyldioctadecylammonium (DDA)) stabilized with a glycolipid immunomodulator (trehalose 6,6-dibehenate (TDB), which may be a synthetic variant of cord factor located in the mycobacterial cell wall).

[0381] Cytokines. The adjuvant may be partial or full length DNA encoding a cytokine, such as a proinflammatory cytokine (e.g., GM-CSF, IL-1α, IL-1β, TGF-β, TNF-α, TNF-β), a Th-1 inducing cytokine (e.g., IFN-γ, IL-2, IL-12, IL-15, IL-18), or a Th-2 inducing cytokine (e.g., IL-4, IL-5, IL-6, IL-10, IL-13).

[0382] Chemokines. The adjuvant may be a partial or full-length DNA or RNA (e.g., circular polyribonucleotide or mRNA) encoding a chemokine, such as MCP-1, MIP-1α, MIP-1β, RANTES, or TCA-3.

[0383] The adjuvant may be partial or full-length DNA encoding a costimulatory molecule, such as CD80, CD86, CD40-L, CD70, or CD27.

[0384] The adjuvant may be an innate immune system stimulator (partial, full-length, or mutated), such as TLR4, TLR3, TLR3, TLR9, TLR7, TLR8, TLR7, RIG-I / DDX58, or MDA-5 / IFIH1; or partial or full-length DNA or RNA (e.g., circular polyribonucleotide or mRNA) encoding a constitutively active (ca) innate immune stimulator, such as caTLR4, caTLR3, caTLR3, caTLR9, caTLR7, caTLR8, caTLR7, caRIG-I / DDX58, or caMDA-5 / IFIH1.

[0385] The adjuvant may be partial or full-length DNA or RNA (e.g., circular polyribonucleotide or mRNA) encoding an adaptor or signaling molecule, such as STING (e.g., caSTING), TRIF, TRAM, MyD88, IPS1, ASC, MAVS, MAPKs, IKK-α, IKK complex, TBK1, β-catenin, and caspase-1.

[0386] The adjuvant may be partial or full-length DNA or RNA (e.g., circular polyribonucleotide or mRNA) encoding a transcription activator, such as a transcription activator (e.g., AP1, NF-κB, IRF3, IRF7, IRF1, or IRF5) capable of upregulating an immune response. The adjuvant may be partial or full-length DNA encoding a cytokine receptor, such as IL-2β, IFN-γ, or IL-6.

[0387] The adjuvant may be partial or full-length DNA or RNA (eg, circular polyribonucleotide or mRNA) encoding a bacterial component, such as flagellin or MBL.

[0388] The adjuvant may be partial or full-length DNA or RNA (eg, circular polyribonucleotide or mRNA) encoding any component of the innate immune system.

[0389] In some embodiments, a subject is administered a cyclic polyribonucleotide encoding one or more immunogens in combination with an adjuvant (e.g., an adjuvant that is a molecular entity separate from the cyclic polyribonucleotide or an adjuvant encoded by a separate polyribonucleotide). As used throughout this description, the term "in combination with" includes any two compositions administered as part of a therapeutic regimen. This may include, for example, a polyribonucleotide and an adjuvant formulated as a single pharmaceutical composition. This may also include, for example, a polyribonucleotide and an adjuvant administered to a subject as separate compositions according to a defined therapeutic or dosing regimen. The adjuvant may be administered to a subject before, at substantially the same time, or after administration of the polyribonucleotide. The adjuvant may be administered within 1 day, 2 days, 5 days, 10 days, 20 days, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months before or after administration of the polyribonucleotide. The adjuvant may be administered by the same route of administration as the polyribonucleotide (eg, intradermally, intramuscularly, subcutaneously, intravenously, intraperitoneally, topically, or orally) or by a different route.

[0390] delivery The cyclic polyribonucleotides described herein may be included in a pharmaceutical composition with or without a carrier.

[0391] The pharmaceutical compositions described herein may be formulated to include carriers such as, for example, pharmaceutical and / or polymeric carriers, e.g., liposomes, and delivered to a subject in need thereof (e.g., human or non-human agricultural or livestock animals, e.g., cows, dogs, cats, horses, poultry) by known methods. Such methods include, but are not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, particle bombardment ("gene gun"), fugene, direct sonic loading, cell compression, optical transfection, protoplast fusion, imparefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. Methods of delivery are also described, for example, in Gori et al., Delivery and Specificity of CRISPR / Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. July 2015, 26(7):443-451. doi:10.1089 / hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80.

[0392] In some embodiments, the cyclic polyribonucleotides may be delivered in a "naked" delivery formulation, which delivers the cyclic polyribonucleotides to cells without the aid of a carrier and without covalent modification of the cyclic polyribonucleotide or partial or complete encapsulation of the cyclic polyribonucleotide.

[0393] A naked delivery formulation is a formulation free from a carrier, where the cyclic polyribonucleotide is not covalently modified to bind to a moiety that aids in delivery to a cell, and the cyclic polyribonucleotide is not partially or completely encapsulated. In some embodiments, the cyclic polyribonucleotide is not covalently bound to a moiety, such as a protein, small molecule, particle, polymer, or biopolymer, that aids in delivery to a cell. In some embodiments, the cyclic polyribonucleotide may be delivered in a delivery formulation with protamine or a protamine salt (e.g., protamine sulfate).

[0394] A polyribonucleotide without a covalent modification that is attached to a moiety that aids in delivery to a cell may not include modified phosphate groups. For example, a polyribonucleotide without a covalent modification that is attached to a moiety that aids in delivery to a cell may not include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.

[0395] In some embodiments, the naked delivery formulation may not include a transfection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier. For example, naked delivery formulations include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydride-modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxypropyl)-1,1-dimethylethyl]-2-hydroxypropyl ... 2,3-Dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HC1), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

[0396] The naked delivery formulation may include a non-carrier excipient. In some embodiments, the non-carrier excipient may include an inactive ingredient that does not exhibit an active cell permeabilizing effect. In some embodiments, the non-carrier excipient may include a buffer, for example, PBS. In some embodiments, the non-carrier excipient may be a solvent, a non-aqueous solvent, a diluent, a suspension aid, a surfactant, an isotonicity agent, a thickener, an emulsifier, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersant, a granulating agent, a disintegrant, a binder, a buffer, a lubricant, or an oil.

[0397] In some embodiments, the naked delivery formulation may include a diluent, such as a parenterally acceptable diluent. The diluent (e.g., parenterally acceptable diluent) may be a liquid diluent or a solid diluent. In some embodiments, the diluent (e.g., parenterally acceptable diluent) may be an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers include 2-(N-morpholino)ethanesulfonic acid (MES), bis-tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), Tris, Tricine, Gly-Gly, Bicine, or phosphoric acid. Examples of isotonicity agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

[0398] In some embodiments, the formulation includes a cell-penetrating agent. In some embodiments, the formulation is a topical formulation and includes a cell-penetrating agent. The cell-penetrating agent can include organic compounds, such as alcohols having one or more hydroxyl functional groups. In some cases, the cell-penetrating agent can include an alcohol, such as, but not limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic alcohols, and alicyclic alcohols. The cell-penetrating agent can include one or more of methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl alcohol, special denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene glycol (PEG)-400, ethoxylated fatty acid, or hydroxyethyl cellulose. In certain embodiments, the cell-penetrating agent can include ethanol. The cell permeation agent can include any cell permeation agent, in any amount or in any formulation, as described in WO 2020 / 180751 or WO 2020 / 180752, which are hereby incorporated by reference in their entireties.

[0399] In some embodiments, the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the drug substance as disclosed, or the pharmaceutical formulation as disclosed herein is included in a parenteral nucleic acid delivery system. The parent nucleic acid delivery system may include the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the drug substance as disclosed, or the pharmaceutical formulation as disclosed herein, and a parenterally acceptable diluent. In some embodiments, the pharmaceutical preparation as disclosed herein, the pharmaceutical composition as disclosed herein, the drug substance as disclosed, or the pharmaceutical formulation as disclosed herein in the parenteral nucleic acid delivery system does not include any carrier.

[0400] The present disclosure is further directed to a host or host cell comprising the cyclic polyribonucleotides described herein. In some embodiments, the host or host cell is a vertebrate, mammal (e.g., human), or other organism or cell.

[0401] In some embodiments, the cyclic polyribonucleotide has a reduced or no undesirable response by the host's immune system compared to the response induced by a reference compound, for example, a linear polynucleotide corresponding to the described cyclic polyribonucleotide. In embodiments, the cyclic polyribonucleotide is non-immunogenic in the host. Some immune responses include, but are not limited to, humoral immune responses (e.g., production of antibodies specific to the immunogen) and cell-mediated immune responses (e.g., lymphocyte proliferation).

[0402] In some embodiments, a host or host cell is contacted with (e.g., delivered or administered to) a cyclic polyribonucleotide. In some embodiments, the host is a mammal, such as a human. The amount of cyclic or linear polyribonucleotide, expression product, or both in the host can be measured at any time after administration. In certain embodiments, a time course of host growth in culture is measured. If growth is enhanced or reduced in the presence of cyclic or linear polyribonucleotide, the cyclic polyribonucleotide or expression product, or both, is identified as effective for enhancing or reducing host growth.

[0403] Methods for delivering a circular polyribonucleotide molecule as described herein to a cell, tissue, or subject include administering to the cell, tissue, or subject a pharmaceutical composition, drug substance, or pharmaceutical formulation as described herein.

[0404] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an ungulate cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is an immune cell. In some embodiments, the tissue is connective tissue, muscle tissue, nervous tissue, or epithelial tissue. In some embodiments, the tissue is an organ (e.g., liver, lung, spleen, kidney, etc.).

[0405] In some embodiments, the method of delivery is an in vivo method. For example, the method of delivery of cyclic polyribonucleotide as described herein comprises parenterally administering to a subject in need thereof a pharmaceutical composition, drug substance or pharmaceutical formulation as described herein. As another example, the method of delivery of cyclic polyribonucleotide to a cell or tissue of a subject comprises parenterally administering to a cell or tissue a pharmaceutical composition, drug substance or pharmaceutical formulation as described herein. In some embodiments, the cyclic polyribonucleotide is in an amount effective to induce a biological response in the subject. In some embodiments, the cyclic polyribonucleotide is in an amount effective to have a biological effect on a cell or tissue in the subject. In some embodiments, the pharmaceutical composition, drug substance or pharmaceutical formulation as described herein comprises a carrier. In some embodiments, the pharmaceutical composition, drug substance or pharmaceutical formulation as described herein comprises a diluent and does not comprise any carrier.

[0406] In some embodiments, the pharmaceutical composition, drug substance, or pharmaceutical formulation is administered parenterally. In some embodiments, the pharmaceutical composition, drug substance, or pharmaceutical formulation is administered intravenously, intraarterially, intraperitoneally, intradermally, intracranially, intrathecally, intralymphatically, subcutaneously, or intramuscularly. In some embodiments, the parenteral administration is intravenous, intramuscular, ophthalmic, subcutaneous, intradermal, or topical.

[0407] In some embodiments, a pharmaceutical composition, drug substance, or pharmaceutical formulation as described herein is administered intramuscularly. In some embodiments, a pharmaceutical composition, drug substance, or pharmaceutical formulation as described herein is administered subcutaneously. In some embodiments, a pharmaceutical composition, drug substance, or pharmaceutical formulation as described herein is administered topically. In some embodiments, a pharmaceutical composition, drug substance, or pharmaceutical formulation is administered intratracheally.

[0408] In some embodiments, the pharmaceutical composition, drug substance, or pharmaceutical formulation is administered by injection. Administration can be systemic or local. In some embodiments, any of the delivery methods as described herein are performed with a carrier. In some embodiments, any of the delivery methods as described herein are performed without the aid of a carrier or cell permeation agent.

[0409] In some embodiments, the cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide is detected in the cell, tissue, or subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days after the administering step. In some embodiments, the presence of the cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide is assessed in the cell, tissue, or subject before the administering step. In some embodiments, the presence of the cyclic polyribonucleotide or a product translated from the cyclic polyribonucleotide is assessed in the cell, tissue, or subject after the administering step.

[0410] formulation In some embodiments of the present disclosure, the polyribonucleotide (e.g., cyclic polyribonucleotide) or a preparation thereof prepared by the methods described herein may be formulated into a composition, e.g., a composition for delivery to a cell, a plant, an invertebrate, a non-human vertebrate, or a human subject, e.g., an agricultural composition, a veterinary composition, or a pharmaceutical composition. In some embodiments, the polyribonucleotide is formulated into a pharmaceutical composition. In some embodiments, the composition comprises a polyribonucleotide and a diluent, a carrier, an adjuvant, or a combination thereof. In particular embodiments, the composition comprises a polyribonucleotide as described herein and a carrier or a diluent without any carrier. In some embodiments, a composition comprising a polyribonucleotide with a diluent without any carrier is used for naked delivery of the polyribonucleotide (e.g., cyclic polyribonucleotide) to a subject.

[0411] The pharmaceutical composition may optionally include one or more additional active substances, for example, therapeutically and / or prophylactically active substances. The pharmaceutical composition may optionally include inactive substances that act as excipients or vehicles for the compositions described herein (e.g., compositions that include cyclic polyribonucleotides, any one of the inactive ingredients listed in the inactive ingredient database approved by the U.S. Food and Drug Administration (FDA), etc.). The pharmaceutical composition of the present invention may be sterile and / or pyrogen-free. For general considerations in the manufacture of formulations and / or pharmaceuticals, see, for example, Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-limiting examples of inert substances include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersion liquids, suspending aids, surface active agents, isotonic agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersing agents, granulating agents, disintegrating agents, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof.

[0412] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and those skilled in the art of veterinary pharmacology can design and / or perform such modifications with no more than routine experimentation, if any. Subjects to which the pharmaceutical compositions are contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially valuable mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially valuable birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0413] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients and then, as necessary and / or desired, portioning, shaping, and / or packaging the product.

[0414] In some embodiments, a reference standard for the amount of linear polyribonucleotide molecules present in a preparation is 1 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 200 ng / ml ml, 300ng / ml, 400ng / ml, 500ng / ml, 600ng / ml, 1μg / ml, 10μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, 300μg / ml, 400μg / ml, 500μg / ml, 600μg / ml, 700μg / ml, 800μg / ml, 900μg / ml, 1mg / ml, 1.5mg / ml, or 2mg / ml.

[0415] In some embodiments, a reference standard for the amount of circular polyribonucleotide molecules present in a preparation is at least 30% (w / w), 40% (w / w), 50% (w / w), 60% (w / w), 70% (w / w), 80% (w / w), 85% (w / w), 90% (w / w), 91% (w / w), 92% (w / w), 93% (w / w), 94% (w / w), 95% (w / w), 96% (w / w), 97% (w / w), 98% (w / w), 99% (w / w), 100% (w / w), 101% (w / w), 102% (w / w), 103% (w / w), 104% (w / w), 105% (w / w), 106% (w / w), 107% (w / w), 108% (w / w), 109% (w / w), 110% (w / w), 111% (w / w), 112% (w / w), 113% (w / w), 114% (w / w), 115% (w / w), 116% (w / w), 117% (w / w), 118% (w / w), 119% (w / w), 120% (w / w), 121% (w / w), 122% (w / w), 123% (w / w), 124% (w / w), 125% (w / w), 126% (w / w), 127% (w / w), 128% (w / w), 129% (w / w), 130% (w / w), 131% (w / w), 132% (w / w), 133 %(w / w), 95%(w / w), 96%(w / w), 97%(w / w), 98%(w / w), 99%(w / w), 99.1%(w / w), 99.2%(w / w), 99.3%(w / w), 99.4%(w / w), 99.5%(w / w), 99.6%(w / w), 99.7%(w / w), 99.8%(w / w), 99.9%(w / w), or 100%(w / w) of the molecule.

[0416] In some embodiments, a reference standard for the amount of linear polyribonucleotide molecules present in a preparation is that linear polyribonucleotide molecules represent no more than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) of the total ribonucleotide molecules in the pharmaceutical preparation.

[0417] In some embodiments, a reference standard for the amount of nicked polyribonucleotide molecules present in a preparation is nicked polyribonucleotide molecules that are less than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), or 15% (w / w) of the total ribonucleotide molecules in the pharmaceutical preparation.

[0418] In some embodiments, the reference standard for the amount of nicked and linear polyribonucleotide molecules present in the preparation is less than 0.5% (w / w), 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), 30% (w / w), 40% (w / w), 50% (w / w) of the total ribonucleotide molecules in the pharmaceutical preparation. In some embodiments, the pharmaceutical preparation is an intermediate pharmaceutical preparation of a final circular polyribonucleotide drug product. In some embodiments, the pharmaceutical preparation is a drug substance or active pharmaceutical ingredient (API). In some embodiments, the pharmaceutical preparation is a drug product for administration to a subject.

[0419] In some embodiments, the preparation of circular polyribonucleotides is further processed (before, during or after reduction of linear RNA) to substantially remove DNA, protein contaminants (e.g., cellular proteins such as host cell proteins or protein processing impurities), endotoxins, mononucleotide molecules, and / or processing-related impurities.

[0420] In some embodiments, the pharmaceutical formulations disclosed herein may include (i) a compound disclosed herein (e.g., a cyclic polyribonucleotide); (ii) a buffering agent; (iii) a non-ionic surfactant; (iv) a tonicity agent; and / or (v) a stabilizer. In some embodiments, the pharmaceutical formulations disclosed herein are stable liquid pharmaceutical formulations. In some embodiments, the pharmaceutical formulations disclosed herein include protamine or a protamine salt (e.g., protamine sulfate).

[0421] The present disclosure provides an immunogenic composition comprising the cyclic polyribonucleotide described herein. The immunogenic composition of the present disclosure may comprise a diluent or carrier, an adjuvant, or any combination thereof. The immunogenic composition of the present disclosure may also comprise one or more immunomodulatory agents, for example, one or more adjuvants. The adjuvant may comprise a TH1 adjuvant and / or a TH2 adjuvant, which are further discussed below. In some embodiments, the immunogenic composition comprises a diluent without any carrier and is used for naked delivery of cyclic polyribonucleotide to a subject.

[0422] The immunogenic compositions of the present disclosure are used to generate an immune response in a subject. The immune response is preferably protective and preferably includes an antibody response (usually including IgG) and / or a cell-mediated immune response. For example, a subject is immunized with an immunogenic composition comprising a cyclic polyribonucleotide of the present disclosure to induce an immune response. In another example, a subject is immunized with an immunogenic composition comprising a linear polyribonucleotide comprising an immunogen to stimulate the production of antibodies that bind to the immunogen. By generating an immune response in a subject through these uses and methods, the subject may be protected against various diseases and / or infections, for example, against bacterial and / or viral diseases as discussed above. In certain embodiments, the immunogenic composition is a vaccine composition. A vaccine according to the present disclosure may be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., to treat infection), but will typically be prophylactic. In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal, preferably a mammal, such as a human. In one embodiment, the subject is a human. In other embodiments, the subject is a non-human mammal, e.g., selected from cows (e.g., dairy and beef cattle), sheep, goats, pigs, horses, dogs, or cats. In other embodiments, the subject is an avian, e.g., a hen or rooster, a turkey, or a parrot. In some embodiments, the animal is not a mouse or a rabbit or a cow. In certain embodiments, if the immunogenic composition is for prophylactic use, the human is a child (e.g., a toddler or infant) or a teenager. In another embodiment, if the immunogenic composition is for therapeutic use, the human is a teenager or an adult. Immunogenic compositions intended for pediatric use may also be administered to adults, e.g., to assess safety, dosage, immunogenicity, etc.

[0423] Both children and adults may be treated with the immunogenic compositions prepared according to the present disclosure. The human subject may be less than 1 year old, less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. In certain embodiments, the human subject to which the immunogenic composition is administered is elderly (e.g., ≧50 years old, ≧60 years old, and ≧65 years old), young adults (e.g., ≦5 years old), hospitalized patients, healthcare workers, armed service and military personnel, pregnant women, chronically ill, or immunocompromised patients. The immunogenic compositions are not suitable for these groups alone, but may be used more generally within the population.

[0424] In some embodiments, the subject is further immunized with an adjuvant. In some embodiments, the subject is further immunized with a vaccine.

[0425] Preservatives The compositions or pharmaceutical compositions provided herein may include material for single administration or may include material for multiple administration (e.g., "multi-dose" kits). Polyribonucleotides may be present in either linear or cyclic form. The compositions or pharmaceutical compositions may include one or more preservatives, such as thiomersal or 2-phenoxyethanol. Preservatives may be used to prevent microbial contamination during use. Suitable preservatives include benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium, sorbic acid, Onamer M, or other agents known to those skilled in the art. In ophthalmic products, for example, such preservatives may be used at levels of 0.004% to 0.02%. In the compositions described herein, preservatives, such as benzalkonium chloride, may be used at levels of 0.001% to less than 0.01%, for example, 0.001% to 0.008%, preferably about 0.005% by weight.

[0426] Polyribonucleotides may be sensitive to ribonucleases (RNases), which may be present in large amounts in the surrounding environment. The compositions provided herein may contain reagents that inhibit ribonuclease activity, thereby protecting polyribonucleotides from degradation. In some cases, the compositions or pharmaceutical compositions contain any ribonuclease inhibitor known to those skilled in the art. Alternatively or additionally, the polyribonucleotides in the compositions provided herein, as well as the cell permeation agent and / or the pharma- ceutically acceptable diluent or carrier, vehicle, excipient, or other reagent, may be prepared in a ribonuclease-free environment. The compositions may be formulated in a ribonuclease-free environment.

[0427] In some cases, the compositions provided herein may be sterile.The compositions may be formulated as sterile solutions or suspensions in suitable media as known in the art.The compositions may be sterilized by conventional sterilization techniques, for example, the compositions may be sterile filtered.

[0428] salt In some cases, the compositions or pharmaceutical compositions provided herein include one or more salts. To control osmolality, physiological salts such as sodium salts can be included in the compositions provided herein. Other salts can include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and / or magnesium chloride, and the like. In some cases, the compositions are formulated with one or more pharma- ceutically acceptable salts. The one or more pharma-ceutically acceptable salts can include those of inorganic ions, such as, for example, sodium, potassium, calcium, and magnesium ions. Such salts can include salts of inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid, or maleic acid. Polyribonucleotides can exist in either linear or cyclic form.

[0429] Buffer / pH The compositions or pharmaceutical compositions provided herein may include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (e.g., with aluminum hydroxide adjuvant), or citrate buffer, etc. The buffers are in some cases within the range of 5-20 mM.

[0430] The compositions or pharmaceutical compositions provided herein can have a pH between about 5.0 and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about 7.5, or between about 7.0 and about 7.8. The compositions or pharmaceutical compositions can have a pH of about 7. Polyribonucleotides can exist in either linear or circular form.

[0431] Detergents / Surfactants The compositions or pharmaceutical compositions provided herein may contain, depending on the intended route of administration, one or more detergents and / or surfactants, such as polyoxyethylene sorbitan ester surfactants (commonly referred to as "Tween®"), such as polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade name DOWFAX™; octoxynols, which may vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, such as octoxynol-9 (Triton® X-100, or t-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL®); CA-630 / NP-40; phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol (known as Brij® surfactants), such as triethylene glycol monolauryl ether (Brij® 30); and sorbitan esters (commonly known as “SPAN®”) such as sorbitan trioleate (Span® 85) and sorbitan monolaurate, octoxynol (such as octoxynol-9 (Triton® X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB"), or sodium deoxycholate. One or more detergents and / or surfactants may be included only in trace amounts. In some examples, the composition may contain less than 1 mg / ml each of octoxynol-10 and polysorbate 80. Non-ionic surfactants may be used herein. Surfactants may be classified by their "HLB" (hydrophilic / lipophilic balance). In some examples, the surfactant has an HLB of at least 10, at least 15, and / or at least 16.The polyribonucleotide may be included in a linear or circular form.

[0432] Diluent In some embodiments, the immunogenic compositions of the present disclosure comprise a cyclic polyribonucleotide and a diluent.

[0433] The diluent may be a non-carrier excipient. The non-carrier excipient serves as a vehicle or medium for the composition, such as the cyclic polyribonucleotide described herein. Non-limiting examples of non-carrier excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonicity agents, thickening agents, emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, dispersants, granulating agents, disintegrants, binders, buffers (e.g., phosphate buffered saline (PBS)), lubricants, oils, and mixtures thereof. The non-carrier excipient may be any of the inactive ingredients listed in the Inactive Ingredient Database that are approved by the United States Food and Drug Administration (FDA) and do not exhibit cell-penetrating effects. The non-carrier excipient may be any inactive ingredient suitable for administration to non-human animals, for example, suitable for veterinary use. Modifications of compositions suitable for administration to humans to make the compositions suitable for administration to a variety of animals are well understood and a veterinary pharmacologist of ordinary skill can design and / or make such modifications with only routine experimentation, if any.

[0434] In some embodiments, the cyclic polyribonucleotides can be delivered as a naked delivery formulation, such as one that includes a diluent. Naked delivery formulations deliver the cyclic polyribonucleotides to cells without a carrier and without modification or partial or complete encapsulation of the cyclic polyribonucleotides, capped polyribonucleotides, or complexes thereof.

[0435] Naked delivery formulations are formulations that do not contain carriers, where the cyclic polyribonucleotide does not have a covalent modification that is attached to a moiety that aids in delivery to cells, or does not have partial or complete encapsulation of the cyclic polyribonucleotide. In some embodiments, the cyclic polyribonucleotide that does not have a covalent modification that is attached to a moiety that aids in delivery to cells is a polyribonucleotide that is not covalently attached to a protein, small molecule, particle, polymer, or biopolymer. The cyclic polyribonucleotide that does not have a covalent modification that is attached to a moiety that aids in delivery to cells does not contain modified phosphate groups. For example, the cyclic polyribonucleotide that does not have a covalent modification that is attached to a moiety that aids in delivery to cells does not contain phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, phosphorodiamidates, alkyl or aryl phosphonates, or phosphotriesters.

[0436] In some embodiments, the naked delivery formulation does not include any or all of a transfection reagent, a cationic carrier, a carbohydrate carrier, a nanoparticle carrier, or a protein carrier. In certain embodiments, the naked delivery formulation is selected from the group consisting of phytoglycogen octenyl succinate, phytoglycogen β-dextrin, anhydrous modified phytoglycogen β-dextrin, lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3 -(2-Hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N,N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

[0437] In some embodiments, the naked delivery formulation comprises a non-carrier excipient. In some embodiments, the non-carrier excipient comprises an inactive ingredient that does not exhibit a cell-penetrating effect. In some embodiments, the non-carrier excipient comprises a buffer, such as PBS. In some embodiments, the non-carrier excipient is a solvent, a non-aqueous solvent, a diluent, a suspending aid, a surfactant, an isotonicity agent, a thickener, an emulsifier, a preservative, a polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersant, a granulating agent, a disintegrant, a binder, a buffer, a lubricant, or an oil.

[0438] In some embodiments, the naked delivery formulation comprises a diluent. The diluent can be a liquid diluent or a solid diluent. In some embodiments, the diluent is an RNA solubilizing agent, a buffer, or an isotonic agent. Examples of RNA solubilizing agents include water, ethanol, methanol, acetone, formamide, and 2-propanol. Examples of buffers include 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of isotonicity agents include glycerin, mannitol, polyethylene glycol, propylene glycol, trehalose, or sucrose.

[0439] Carrier In certain embodiments, the immunogenic composition of the present disclosure comprises a cyclic polyribonucleotide and a carrier.

[0440] In certain embodiments, the immunogenic composition comprises a circular polyribonucleotide described herein in a vesicle or other membrane-based carrier.

[0441] In other embodiments, the immunogenic composition comprises cyclic polyribonucleotides in or through cells, vesicles, or other membrane-based carriers. In one embodiment, the immunogenic composition comprises cyclic polyribonucleotides in liposomes or other similar vesicles. Liposomes are spherical vesicular structures composed of a mono- or multi-membrane lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, and can deliver both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes and transporting their load across biological membranes and the blood-brain barrier (BBB) ​​(see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for a review).

[0442] Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods for the preparation of multilamellar vesicle lipids are known in the art (see, for example, U.S. Pat. No. 6,693,086, the teachings of which are incorporated herein by reference for their related preparation of multilamellar vesicle lipids). When lipid membranes are mixed with aqueous solution, vesicle formation can occur spontaneously, but it can also be promoted by applying force in the form of shaking, by using homogenizers, sonicators, or extrusion devices (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155 / 2011 / 469679 for review). Extruded lipids can be prepared by extrusion through reduced size filters as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference.

[0443] In certain embodiments, the immunogenic composition of the present disclosure comprises a cyclic polyribonucleotide and a lipid nanoparticle, e.g., a lipid nanoparticle, as described herein. Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for the cyclic polyribonucleotide molecules described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets, improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, the two components increase drug encapsulation efficiency, promote surface modification, and prevent leakage of water-soluble drugs. For review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.

[0444] Further non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), protein carriers (e.g., proteins covalently bound to cyclic polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phytoglycogen octenyl succinate, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin. Non-limiting examples of cationic carriers include lipofectamine, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)-2-hydroxypropyl]-2-hydroxypropyl-2-hydroxypropyl, and the like. N,N-diisopropyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), 3B-[N-(N\N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HC1), diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC). Non-limiting examples of protein carriers include human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin.

[0445] Exosomes can also be used as drug delivery vehicles for the circular RNA compositions or preparations described herein. For review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; https: / / doi.org / 10.1016 / j.apsb.2016.02.001.

[0446] Ex vivo differentiated red blood cells can also be used as carriers for the circular RNA compositions or preparations described herein. See, for example, WO 2015 / 073587; WO 2017 / 123646; WO 2017 / 123644; WO 2018 / 102740; WO 2016 / 183482; WO 2015 / 153102; WO 2018 / 151829; WO 2018 / 009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28):10131-136; U.S. Pat. No. 9,644,180; Huang et al. 2017. Nature Communications 8:423; Shi et al. 2014. Proc Natl Acad Sci USA. See USA.111(28):10131-136.

[0447] For example, fusosome compositions such as those described in WO 2018 / 208728 may also be used as carriers for delivering the circular polyribonucleotide molecules described herein.

[0448] Virosomes and virus-like particles (VLPs) may also be used as carriers to deliver the circular polyribonucleotide molecules described herein to target cells.

[0449] Plant nanovesicles and plant messenger packs (PMPs), for example as described in International Patent Publication Nos. WO 2011 / 097480, WO 2013 / 070324, WO 2017 / 004526, or WO 2020041784, may also be used as carriers for delivering the circular RNA compositions or preparations described herein.

[0450] Microbubbles can also be used as carriers for delivering the circular polyribonucleotide molecules described herein.See, for example, US Patent No. 7,115,583;Beeri, R. et al., Circulation.2002 Oct 1;106(14):1756-59;Bez, M. et al., Nat Protoc.2019 Apr;14(4):1015-26;Hernot, S. et al., Adv Drug Deliv Rev.2008 Jun 30;60(10):1153-66;Rychak, JJ et al., Adv Drug Deliv Rev.2014 Jun;72:82-93.In some embodiments, the microbubbles are albumin-coated perfluorocarbon microbubbles.

[0451] A carrier comprising a cyclic polyribonucleotide as described herein may comprise a plurality of particles. The particles may have a median article size of 30-700 nanometers (e.g., 30-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 100-500, 50-500, or 200-700 nanometers). The size of the particles may be optimized to favor deposition of the payload comprising cyclic polyribonucleotide into cells. Deposition of cyclic polyribonucleotide into specific cell types may favor different particle sizes. For example, the particle size may be optimized for deposition of cyclic polyribonucleotide into antigen-presenting cells. The particle size may be optimized for deposition of cyclic polyribonucleotide into dendritic cells. Additionally, the particle size may be optimized for deposition of cyclic polyribonucleotide into draining lymph node cells.

[0452] Lipid Nanoparticles The compositions, methods, and delivery systems provided by the present disclosure may use any suitable carrier or delivery modality, including, in certain embodiments, lipid nanoparticles (LNPs) as described herein. The lipid nanoparticles, in certain embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or amphoteric lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers as described in Table 5 of International Publication No. WO2019217941, which is incorporated herein by reference in its entirety); and one or more sterols (e.g., cholesterol).

[0453] Lipids (e.g., lipid nanoparticles) that can be used in nanoparticle formation include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference - for example, a lipid-containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941. The lipid nanoparticle can include additional elements, such as a polymer, for example, a polymer described in Table 5 of WO2019217941, which is incorporated by reference.

[0454] In certain embodiments, the conjugated lipid, if present, is PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecyl)glycerol), PEG-glyceryl-2,3-diamino-2-(trimethylsilyl)-1,1-dimethylethyl)-2,2-dimethylethyl)-1,1-dimethylethyl)-2,3-dimethylethyl ... The polyoxyalkylene glycols may include one or more of the following: N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, N-(carbonyloxypropyl-l-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those listed in Table 2 of WO2019051289 (incorporated by reference), as well as combinations of the above.

[0455] In some embodiments, sterols that may be incorporated into the lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009 / 127060 or U.S. Patent Publication No. 2010 / 0130588, which are incorporated by reference. Further exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated by reference herein.

[0456] In some embodiments, the lipid particles include an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amounts of these components can be independently varied to achieve desired properties. For example, in some embodiments, the lipid nanoparticles include an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid (in other embodiments, it can be 20-70% (mol), 30-60% (mol), or 40-50% (mol); about 50 mol% to about 90 mol% of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid, a conjugated lipid in an amount of about 0.5 mol% to about 20 mol% of the total lipid, and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid. The ratio of total lipid to nucleic acid can be varied as needed. For example, the ratio of total lipid to nucleic acid (mass or weight) can be about 10:1 to about 30:1.

[0457] In certain embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipid and nucleic acid can be adjusted to obtain a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid nanoparticle formulation can range from about 5 mg / ml to about 30 mg / mL.

[0458] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of the compositions described herein, e.g., the nucleic acids described herein (e.g., RNA (e.g., circular polyribonucleotides, linear polyribonucleotides)), include the following: [ka] In certain embodiments, an LNP comprising formula (i) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0459] [ka] In certain embodiments, an LNP comprising formula (ii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0460] [ka] In certain embodiments, an LNP comprising formula (iii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0461] [ka] In certain embodiments, an LNP comprising formula (v) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0462] [ka] In certain embodiments, an LNP comprising formula (vi) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0463] [ka] In certain embodiments, an LNP comprising formula (viii) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0464] [ka] In certain embodiments, an LNP comprising formula (ix) is used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0465] [ka] During the ceremony, X 1 But, O, NR 1 or a direct bond, X 2 is C2-5 alkylene, and X 3 is C(=O) or a direct bond, and R 1 is H or Me, and R 3 is C1-3 alkyl, R 2 is C1-3 alkyl, or R 2 The nitrogen atom to which it is attached and X 2 together with 1 to 3 carbon atoms of X to form a 4-, 5-, or ...

Claims

1. A circular polyribonucleotide comprising an open reading frame comprising a sequence encoding an immunogen and a sequence encoding a multimerization domain.

2. The open reading frame comprises, in 5' to 3' order: (a) a first sequence encoding an immunogen and a second sequence encoding a multimerization domain; (b) a first sequence encoding an immunogen, a second sequence encoding a multimerization domain, and a third sequence encoding an immunogen; (c) a first sequence encoding an immunogen, a second sequence encoding a multimerization domain, a third sequence encoding an immunogen, and a fourth sequence encoding a multimerization domain; (d) a first sequence encoding an immunogen, a second sequence encoding a multimerization domain, and a third sequence encoding a multimerization domain; (e) a first sequence encoding a multimerization domain and a second sequence encoding an immunogen; (f) a first sequence encoding a multimerization domain, a second sequence encoding an immunogen, and a third sequence encoding a multimerization domain; (g) a first sequence encoding a multimerization domain, a second sequence encoding an immunogen, a third sequence encoding a multimerization domain, and a fourth sequence encoding an immunogen; or (h) a first sequence encoding a multimerization domain, a second sequence encoding a multimerization domain, and a third sequence encoding an immunogen. The cyclic polyribonucleotide of claim 1, comprising:

3. the or each multimerization domain comprises: (a) T4 Foldon domain; (b) ferritin domain; (c) β-annulus peptide; (d) an AaLS peptide; or (e) Lumazine synthase domain The cyclic polyribonucleotide of claim 1, comprising:

4. The open reading frame comprises, in 5' to 3' order: (a) a first sequence encoding an immunogen and a second sequence encoding a T4 Foldon domain; (b) a first sequence encoding an immunogen and a second sequence encoding a ferritin domain; (c) a first sequence encoding an immunogen and a second sequence encoding a β-annulus peptide; (d) a first sequence encoding an immunogen and a second sequence encoding an AaLS peptide; (e) a first sequence encoding an immunogen, a second sequence encoding a T4 Foldon domain, and a third sequence encoding an immunogen; (f) a first sequence encoding an immunogen, a second sequence encoding a T4 Foldon domain, and a third sequence encoding a ferritin domain; or (g) a first sequence encoding an immunogen, a second sequence encoding a ferritin domain, and a third sequence encoding a T4 Foldon domain. The cyclic polyribonucleotide of claim 1, comprising:

5. 2. The circular polyribonucleotide of claim 1, wherein each immunogen is independently operably linked to a secretory signal sequence.

6. The circular polyribonucleotide of claim 1, wherein the open reading frame is operably linked to an IRES.

7. 7. The circular polyribonucleotide of claim 6, further comprising a second open reading frame encoding a second polypeptide operably linked to a second IRES.

8. 8. The cyclic polyribonucleotide of claim 7, wherein the second polypeptide is a polypeptide immunogen.

9. 8. The cyclic polyribonucleotide of claim 7, wherein the second polypeptide is a polypeptide adjuvant.

10. 2. The circular polyribonucleotide of claim 1, further comprising a non-coding ribonucleic acid sequence that is an innate immune system stimulator.

11. 11. The cyclic polyribonucleotide of claim 10, wherein the innate immune system stimulator is selected from a GU-rich motif, an AU-rich motif, a structured region comprising dsRNA, or an aptamer.

12. An immunogenic composition comprising the cyclic polyribonucleotide of claim 1 and a pharmaceutically acceptable excipient.

13. The immunogenic composition of claim 12, further comprising a second cyclic polyribonucleotide.

14. The immunogenic composition of claim 13 , wherein the second circular polyribonucleotide comprises an open reading frame encoding an immunogen.

15. 14. The immunogenic composition of claim 13, wherein the second cyclic polyribonucleotide comprises an open reading frame encoding a polypeptide adjuvant.

16. The immunogenic composition of claim 13, wherein the second circular polyribonucleotide comprises a non-coding ribonucleic acid sequence that is an innate immune system stimulator.

17. 17. A composition comprising a cyclic polyribonucleotide according to any one of claims 1 to 11 or an immunogenic composition according to any one of claims 12 to 16 for use in a method of inducing an immune response to an immunogen in a subject, said method comprising administering said composition to said subject.

18. 17. A composition comprising a cyclic polyribonucleotide according to any one of claims 1 to 11 or an immunogenic composition according to any one of claims 12 to 16 for use in a method of treating or preventing a disease, condition or disorder in a subject, said method comprising administering said composition to said subject.

19. The composition for use according to claim 17, wherein the composition is administered to the subject in combination with an adjuvant.

20. A composition for use as described in claim 17, characterized in that the composition is administered to the subject in combination with a polypeptide immunogen.

21. The composition for use according to claim 18, wherein the composition is administered to the subject in combination with an adjuvant.

22. A composition for use as described in claim 18, characterized in that the composition is administered to the subject in combination with a polypeptide immunogen.

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