Method of purifying circular RNA

Abstract

Embodiments of the invention include a method of purifying circular RNA (circRNA) from a solution. The method an include steps of (a) incubating the solution at about 55°C, (b) cellulose chromatography, (c) desalting and purifying RNA reactions with magnetic carboxylic acid beads, (d) ethanol precipitation to remove double stranded RNA byproducts and concentrated RNA, (e) poly-A polymerase treatment, (f) desalting and purifying RNA reactions with magnetic carboxylic acid beads to remove poly A reaction components, (g) RNase R treatment, and (h) desalting and purifying the solution to yield purified circRNA. In aspects, the method removes, among other contaminates, in vitro transcribed circular RNA from linear and doubled stranded RNA.

Description

METHOD OF PURIFYING CIRCULAR RNARELATED APPLICATIONS

[0001] The present application claims priority to U. S. Patent Application No.63 / 717,213 filed November 6, 2025, the contents of which are incorporated by reference.TECHNICAL FIELD

[0002] The present disclosure relates generally to laboratory methods, and more specifically, to methods of purifying circular RNA for pharmaceutical use such as, for example, vaccines.BACKGROUND

[0003] RNA has emerged as an innovative candidate for a variety of pharmaceutical uses, but efficient purification continues to be a challenge. This is due, in part, to various types and combinations of unwanted contaminants in the sample that need to be separated from the desired RNA species in order to obtain a pure RNA sample. Such contaminants are generally components and by-products of any upstream process, such as RNA production. If in vitro transcription is used to produce large RNAs, after successful transcription, the sample generally contains the desired RNA species, unwanted RNA species, proteins, DN A or fragments thereof, pyrophosphate and free nucleotides.

[0004] Circular RNA (circRNA) is an endogenous noncoding RNA with a covalently closed cyclic structure. The covalently-closed structure of circRNAs offers a significant stability advantage over linear mRNA for therapeutic applications. Without 5' or 3' ends, circular RNAs are more resistant to exonuclease degradation than linear RNA. This increased stability may promote pharmacokinetic accumulation in various cell or tissue types. This characteristic could mean that lower doses are required for therapeutic impact, which would reduce manufacturing costs and potentially improving patient safety. In addition, without a 5' end, circRNA does not require a capping agent, which accounts for about 40 percent of the spend on mRNA raw materials, making circRNAconsiderably less expensive to manufacture than linear mRNA.

[0005] The circular structure may also prolong product shelf life compared to current mRNA vaccines and relieve stringent storage and shipping conditions. Research has shown that LNP-encapsulated circRNA vaccines can be preserved for at least four weeks at 4°C and two weeks at room temperature with little loss in expression. By comparison, linear mRNA resulted in lower levels of antigens produced at these temperatures.

[0006] One challenge associated with circular RNA is that it lacks the 5' end necessary for cap-dependent translation. However, circRNA can be modified to enable protein translation through an internal ribosome entry site or by incorporating m6A modifications upstream of the open reading frame. These modifications enable engineered circRNAs to express proteins similarly to modified mRNAs but for a longer duration.

[0007] The protein-coding ability of circular RNAs, coupled with the stability, storage and pharmacokinetic improvements compared to linear mRNA, suggest that circRNA may be the next revolution in nucleic acid therapeutics.

[0008] Another challenge with circRNA relates to purification. Impure circRNA may induce strong immune responses, adding safety risks for patients. Traditional purification methods (e.g., RNA-Seq) do not distinguish circRNAs from linear RNAs. Further, such methods are impractical or unsuitable for producing pharmaceutical circRNA. For example, conventional methods use DNase treatment followed by precipitation of the longer mRNA with lithium chloride. Such methods do not provide RNA of high purity, as contaminants such as DNA and protein remain present. Further, these methods use organic solvents and typically require many steps that involve manual sample handling at different conditions, including overnight incubation.

[0009] As a result, there is a need for improved methods to purify circRNA. The present invention includes methods of purifying circRNA with high recovery and purity.The product can be used in pharmaceutical applications such as, for example, a vaccine or component of a vaccine.SUMMARY

[0010] The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description. A full appreciation of the various aspects of the embodiments disclosed herein can be gained by taking into consideration the entire specification, claims, drawings, and abstract as a whole.

[0011] The invention includes a method of purifying circular RNA (circRNA) from a solution. In aspects, the method includes steps of (a) incubating the solution at about 55°C, (b) desalting and purifying RNA reactions with magnetic carboxylic acid beads, (c) cellulose chromatography, (d) ethanol precipitation to remove double stranded RNA byproducts and concentrated RNA, (e) poly-A polymerase treatment, (f) desalting and purifying RNA reactions with magnetic carboxylic acid beads to remove poly A reaction components, (g) RNase R treatment, and (h) desalting and purifying the solution to yield purified circRNA.

[0012] In aspects, the crude (i.e., starting) solution includes in vitro transcribed circular from linear and doubled stranded RNA contaminates.

[0013] In aspects, the method results in circular RNA that is >80% circular. In aspects, the method results in circular RNA contains no detectable double-stranded RNA (dsRNA).

[0014] In embodiments, the circRNA is a vaccine or component of a vaccine.

[0015] Embodiments include a method of purifying circRNA. The method can be implemented after RNA production by in vitro transcription. In aspects, the method includes the following steps:(a) circularization reaction: we incubate the circular RNA at 55°C in a buffered reaction containing 2mM GTP. This enhances the fraction of circRNA in our samples by ~10% (b) Desalt and purify RNA reactions with magnetic carboxylic acid beads to remove IVT and circularization reaction components(c) Cellulose chromatography followed by ethanol precipitation to remove double stranded RNA byproducts and concentrated RNA.(d) Poly-A polymerase treatment: Poly A treatment creates a single stranded 3’ end in order to enhance the efficiency of the subsequent RNase treatment(e) Desalt and purify RNA reactions with magnetic carboxylic acid beads to remove poly A reaction components(f) RNase R treatment: RNase R selectively degrades the linear RNA in the sample to produce circular RNA at >80% purity.(g) Desalt and purify RNA reactions Monarch RNA clean up kit to remove RNase reaction components and elute in pure water

[0016] Embodiments also include a method of producing a plasmid, the method can include steps of:a) obtaining a plasmid of interest, andb) linearizing the plasmid with one or more restriction enzymes.In aspects, the step of linearizing the plasmid entails incubation overnight (e.g., from six to twelve hours) at about 37°C.

[0017] In aspects, the method also includes a step of c) performing in vitro-transcription to generate RNA and subsequently treating with DNAse I to remove plasmid DNA.

[0018] In aspects, the transcribed RNA is circular RNA (circRNA).

[0019] Embodiments also include a method of purifying the circRNA. The method can include of:d) incubating the solution containing the circRNA at about 55°C,e) exposing the solution to cellulose chromatography to remove double stranded RNA,f) ethanol precipitation to concentrated RNA,g) desalting and purifying the solution with magnetic carboxylic acid beads to remove excess salts and / or adjust pH,h) poly-A polymerase treatment to make contaminant RNA more accessible for RNase treatment,i) desalting and the solution with magnetic carboxylic acid beads to remove poly A reaction components,j) RNase R treatment to remove linear RNA, andk) desalting and purifying the solution to yield purified circRNA.

[0020] Embodiments also include a method of producing and purifying circular RNA (circRNA) in a solution. The method can include steps of:a) obtaining a plasmid of interest,b) linearizing the plasmid with one or more restriction enzymes.c) conducting in vitro-transcription to generate RNA from the plasmid DNA, d) treat with DNAse I to remove plasmid DNA,e) incubating the solution containing the RNA at about 55°C,f) exposing the solution to cellulose chromatography to remove double stranded RNA,g) ethanol precipitation to concentrated RNA,h) desalting and purifying the solution with magnetic carboxylic acid beads to remove excess salts and / or adjust pH,i) poly-A polymerase treatment to make contaminant RNA more accessible for RNase treatment,j) desalting and the solution with magnetic carboxylic acid beads to remove poly A reaction components,k) RNase R treatment to remove linear RNA, andl) desalting and purifying the solution to yield purified circRNA.

[0021] In aspects, 2mM GTP is added to the solution before the step of incubation at about 55°C.

[0022] The contaminants can include plasmid DNA, linear RNA and doubled stranded RNA.

[0023] In aspects, the method yields more than 80% circular RNA.

[0024] In aspects, the method yields less than 0.1% double-stranded RNA.

[0025] The methods described herein can be used in vaccine production.Accordingly, the circRNA can include one or more antigenic sequences.

[0026] In aspects, the vaccine is an mRNA vaccine. In aspects, the mRNA vaccine is a circular mRNA vaccine. In aspects, the vaccine is a vaccine against a bacteria, a virus, a parasite or a cancer.

[0027] In aspects, the method removes 75%, 80%, 85%, 90%, 95%, 98%, 99% or more of contaminates (e.g. double stranded RNA). I, aspects, the method yields RNA that is 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher circular RNA.Definitions

[0028] Reference in this specification to "one embodiment / aspect" or "an embodiment / aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the disclosure. The use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places in the specification are not necessarily all referring to the same embodiment / aspect, nor are separate or alternative embodiments / aspects mutually exclusive of other embodiments / aspects. Moreover, various features are described which may beexhibited by some embodiments / aspects and not by others. Similarly, various requirements are described which may be requirements for some embodiments / aspects but not other embodiments / aspects. Embodiment and aspect can in certain instances be used interchangeably.

[0029] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure. It will be appreciated that the same thing can be said in more than one way.

[0030] Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. Nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and is not intended to further limit the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to various embodiments given in this specification.

[0031] The term “isolated” refers to a peptide or protein free from at least some of the components with which it naturally occurs.

[0032] The term “peptides,” “polypeptides” and “proteins” are used interchangeably and are defined herein as chains of amino acids (typically L-amino acids) in which the carbonyl group of one amino acid is linked to the amino group of a second amino acid by an amide linkage. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminal andincreasing in the direction of the carboxy terminal of the peptide. Thus, when one amino acid is said to “follow” another, that amino acid is positioned closer to the carboxy terminal of the peptide than the “preceding” amino acid.

[0033] The term “residue” refers to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond or an amide bond mimetic. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e. amino acid mimetics).Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

[0034] The term “antigen” refers to a molecule which can induce an immune response in an animal, preferably a mammal and most preferably a human. It induces the formation of an antibody. The term includes immunogens.

[0035] The term “vaccine” refers to the ability to stimulate an immune response in an organism (i.e., to produce specific immune cells that activate, proliferate, differentiate, and ultimately produce immune effector-specific antibodies or sensitized lymphocytes, and to be reactive).

[0036] The term “epitope” or “determinant” refers to the antibody binding site on an antigen.

[0037] The term “antibody” refers to a molecule produced by animals in response to antigen which has the particular property of combining specifically with the antigen which induced its formation.

[0038] The term “neutralizing antibody” refers to an antibody that blocks viral infection of a cell.

[0039] The term “neutralizing antigenic epitope” or “neutralizing epitope” refers to an epitope that elicits a neutralizing antibody.

[0040] The phrases “specifically binds to a peptide” or “specifically immunoreactive with”, when referring to an antibody, refers to a binding reaction which is determinative of the presence of the peptide, or an antibody to the peptide, in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular peptide and do not bind in a significant amount to other proteins present in the sample. Specific binding to a peptide under such conditions requires an antibody that is selected for its specificity for a particular protein or a particular epitope. In some embodiments, antibodies bind to a protein of one subtype or clade of influenza and not another, for example, antibodies bind to hemagglutinin from H5N1 subtype of influenza and not to H3N2. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solution or solid phase immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0041] The term “conservative variations” or “conservative modified variations” of a particular sequence refers to amino acids encoded by nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given peptide. Such nucleic acid variations are silent variations, which are one species of conservatively modified variations. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques.Accordingly, each silent variation of a nucleic acid which encodes a peptide is implicit in any described amino acid sequence. Further, one of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Serine (S), Threonine (T);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0042] Two polypeptides are said to be “identical” if the sequence of amino acid residues in the two sequences is the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman 1981 Adv Appl Math 2:482-489, by the homology alignment algorithm of Needleman and Wunsch 1970 J Mol Biol 48:443-453, by the search for similarity method of Pearson and Lipman 1988 Proc Natl Acad Sci USA 85:2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

[0043] The term “substantial identity” means that a polypeptide comprises a sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, or 85% sequence identity,preferably 90%, more preferably 95% or more, compared to a reference sequence. Another indication that polypeptide sequences are substantially identical is if one peptide is immunologically reactive with antibodies raised against the disclosed peptide. Thus, the peptides of the invention include peptides immunologically reactive with antibodies raised against the disclosed immunogenic peptides.

[0044] The term "substantial homology" or "substantial similarity," when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the aligned sequences. Preferably, the homology is overfull-length sequence, or an open reading frame thereof, or another suitable fragment which is at least 15 nucleotides in length. Examples of suitable fragments are described herein.

[0045] The terms "sequence identity" "percent sequence identity" or "percent identical" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired. Similarly, "percent sequence identity" may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. Suitably, a fragment is at least about 8 amino acids in length, and may be up to about 700 amino acids. Examples of suitable fragments are described herein.

[0046] The term "substantial homology" or "substantial similarity," when referring to amino acids or fragments thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95 to99% of the aligned sequences. Preferably, the homology is over full-length sequence, or a protein thereof, e.g., a cap protein, a rep protein, or a fragment thereof which is at least 8 amino acids, or more desirably, at least 15 amino acids in length. Examples of suitable fragments are described herein.

[0047] By the term "highly conserved" is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.

[0048] Generally, when referring to "identity", "homology", or "similarity" between two different adeno-associated viruses, "identity", "homology" or "similarity" is determined in reference to "aligned" sequences. "Aligned" sequences or "alignments" refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence. In the examples, AAV alignments are performed using the published AAV2 or AAV1 sequences as a reference point. However, one of skill in the art can readily select another AAV sequence as a reference. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Examples of such programs include, "Clustal W", "CAP Sequence Assembly", "MAP", and "MEME", which are accessible through Web Servers on the internet.

[0049] The term “artificial nucleic acid” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a nucleic acid molecule, e.g. a DNA or an RNA that does not occur naturally. In other words, an artificial nucleic acid may be understood as a non-natural nucleic acid molecule. Such nucleic acid molecule may be non-natural due to its individual sequence (which does not occur naturally, e.g. G / C content modified coding sequence, UTRs) and / or due to other modifications, e.g. structural modifications of nucleotides which do not occur naturally. An artificial nucleic acid may be a DNA molecule, an RNA molecule ora hybrid-molecule comprising DNA and RNA portions. Typically, artificial nucleicacids may be designed and / or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). In this context an artificial sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild type sequence by at least one nucleotide.

[0050] The term “wild type” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a sequence occurring in nature. Further, the term “artificial nucleic acid” is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of essentially identical molecules. Accordingly, it may relate to a plurality of essentially identical molecules contained in an aliquot or a sample.

[0051] The term “circular RNA” or “circRNA” refers to a type of single-stranded RNA which, unlike linear RNA, comprises a covalently closed continuous loop. clrcRNAs occur naturally in mammalian cells, and play important roles in various biological processes. clrcRNAs innately possess greater stability and resistance to intra- and extracellular RNAses than mRNAs, making them attractive candidates for delivery of key payloads where long-lasting expression is necessary. Recently, there has been an interest in using recombinant circRNAs to express a protein of interest, in vitro or in vivo. Introduction of an internal ribosome entry sequence (IRES) into a circular RNA allows translation of a protein encoded by a circRNA. However, IRES elements that exist in nature may or may not support translation from engineered circular RNAs, as IRES elements are often evolved in the context of linear RNA genomes.

[0052] The term “internal ribosome entry sequence” or “IRES” refers to an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis. Initiation of eukaryotic translation nearly always occurs at and is dependent on the 5' cap of mRNA molecules, where the translation initiation complex forms and ribosomes engage the mRNA. IRES elements, however allow ribosomes to engage the mRNA and begin translation independently of the 5' cap.

[0053] An "infectious" virus or viral particle is one that comprises a polynucleotide component that it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an "infectious" virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, "infectivity" refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as "transduction." The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and / or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA).

[0054] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0055] A "gene" refers to a polynucleotide containing at least one open reading framethat is capable of encoding a particular protein after being transcribed and translated.

[0056] The term "recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0057] The term "control element" or "control sequence" refers to a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.

[0058] The term "operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0059] The term "expression vector" refers to a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they areoperably linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0060] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.

[0061] The terms "genetic alteration" and "genetic modification" (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in this term.

[0062] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered(genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.

[0063] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.

[0064] The term “nanoparticle” refers to a particle of matter, generally between about 1 and 100 nanometers (nm) in diameter. As used herein, a nanoparticle can refer to a liposome, virus, viral vector or other viral particle.

[0065] The term “liposome” refers to a spherical vesicle having at least one lipid bilayer (i.e. an aqueous solution core surrounded by a hydrophobic membrane).Liposomes can be prepared by disrupting biological membranes (such as by sonication). Liposomes are formed when phospholipids and their derivatives are dispersed in water. Upon dispersion in water the phospholipids form closed vesicles called “liposomes,” which are characterized by lipid bilayers encapsulating an aqueous core. Various liposomes have been used as carriers for entrapped therapeutic agents, such as drugs, enzymes and genetic sequences for use in medical science, in pharmaceutical science and in biochemistry. Specific uses include delivery of nutrients and pharmaceutical drugs, such as lipid nanoparticles in mRNA vaccines and DNA vaccines. Liposomes can be modified by the incorporation of polyethylene glycol orother hydrophilic polymers (e.g., a PEG liposome where one or more of the constituent lipids is modified by attachment of PEG). Liposomes can also be modified to target particular cell types by incorporating targeting factors (e.g., “targeting ligands”) for particular cell types. Examples include asialoglycoprotein, folate, transferrin, antibodies, etc.

[0066] The term “exosome” refers to a membrane-bound extracellular vesicles that are produced in the endosomal compartment of most eukaryotic cells. In multicellular organisms, exosomes and other EVs are found in biological fluids including saliva, blood, urine and cerebrospinal fluid. Exosomes are similar to liposomes in terms of consisting of bilayered phospholipids, but the biogenesis of exosomes ensures their biocompatibility and low toxicity. It also significantly complicates pharmaceutical development, production and safety profiling (immunogenicity, and potential biological impurities).

[0067] The term “an effective amount” refers to the amount of the defined component sufficient to achieve the desired therapeutic result. In an embodiment, that result can be effective cancer treatment.

[0068] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0069] The terms "individual," "host," "subject," and "patient" are used interchangeablyherein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0070] The term “adaptive immune response” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to an antigen-specific response of the immune system. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is usually maintained in the body by “memory cells” (B-cells). In the context of the invention, the antigen (e.g. Bunyavirales peptide, protein, polyprotein) is provided by the artificial nucleic acid coding sequence encoding at least one antigenic peptide, protein or polyprotein of the invention.

[0071] The term “adaptive immune system” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a highly adaptable system typically regulating the adaptive immune response by providing the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered. The system is highly adaptable because of somatic hyper mutation (a process of accelerated somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because the gene rearrangement leads to an irreversible change in the DNA of each cell, all of the progeny (offspring) of such a cell will then inherit genes encoding the same receptor specificity, including the Memory B cells and Memory T cells that are the keys to induce long-lived specific immunity.

[0072] The term “antigen” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a substancewhich may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. In the context of the present invention, an antigen, e.g. a Bunyavirales antigen, may be the product of translation of a provided inventive artificial nucleic acid of the, preferably of the mRNA as specified herein. Also fragments, variants and derivatives of peptides, proteins, or polyproteins of a virus of the order Bunyavirales comprising at least one epitope are understood as antigens in the context of the invention.

[0073] The terms “cellular immunity” or “cellular immune response” or “cellular T-cell responses” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In more general terms, cellular immunity is not based on antibodies, but on the activation of cells of the immune system. Typically, a cellular immune response may be characterized e.g. by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g. specific immune cells like dendritic cells or other cells, displaying epitopes of foreign antigens on their surface. In the context of the invention, the antigen (e.g. Bunyavirales peptide, protein, polyprotein) is provided by the artificial nucleic acid coding sequence encoding at least one antigenic peptide, protein or polyprotein of the invention.

[0074] The term “derived from” as used throughout the present specification in the context of a nucleic acid, i.e. for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, more preferably at least 85%, 86%, 87%, 88%, 89% even more preferably at least 90%, 91%, 92%, 93%, 94%, even more preferably at least 95%,96%, 97%, and particularly preferably at least 98%, 99% sequence identity with the nucleic acid from which it is derived. The skilled person is aware that sequence identity is typically calculated for the same types of nucleic acids, i.e. for DNA sequences or for RNA sequences. Thus, it is understood, if a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the thymidines (T) by uracils (U) throughout the sequence). Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined. Preferably, a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g. in order to increase RNA stability even further and / or to prolong and / or increase protein production. It goes without saying that such modifications are preferred, which do not impair RNA stability, e.g. in comparison to the nucleic acid from which it is derived.

[0075] The term “epitope” (also called “antigen determinant” in the art) as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to T cell epitopes and B cell epitopes. T cell epitopes or parts of the antigenic peptides or proteins may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 to about 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens (e.g. Bunyavirales antigens), preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 aminoacids, which may be recognized by antibodies, i.e. in their native form. Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides. In this context antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.

[0076] The term “immune response” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response), or a combination thereof.

[0077] The term “immune system” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a system of the organism that may protect the organisms from infection. If a pathogen succeeds in passing a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts typically contains so called humoral and cellular components.

[0078] The term “innate immune system” (also known as non-specific or unspecific immune system) will be recognized and understood by the person of ordinary skill in theart, and is for example intended to refer to a system typically comprising the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system may recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system may be, e.g. activated by ligands of Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-1 like receptor, an immunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral agent.

[0079] The terms “isolate” or “isolate of a virus” as used herein, will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a specific isolated virus of a certain virus species. In the context of the invention, a natural Bunyavirales isolate is an instance of a particular natural virus or of a particular genetic strain (or variant). Isolates can be identical or slightly different in consensus or individual sequence from each other.

[0080] The term “peptide” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a polymer of amino acid monomers, linked by peptide bonds. It typically contains less than 50 amino acid monomers. Nevertheless, the term peptide is not a disclaimer for molecules having more than 50 amino acid monomers.

[0081] The terms “pharmaceutically effective amount” or “effective amount” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to an amount of a compound (e.g. the artificial nucleic acid of the invention) that is sufficient to induce a pharmaceutical effect, such as, in the context of the invention, an immune response (e.g. against an antigenic peptide, protein, polyprotein as defined herein).

[0082] The term “strain” or “strain of a virus” is a group of viruses that are genetically distinct from other groups of the same species. Accordingly, a “strain” is a variant of a given virus (species) that is recognizable because it possesses some unique phenotypic characteristics that remain stable under natural conditions. In the context of the invention, the terms “variant” of a virus and “strain” of a virus are used interchangeably.

[0083] The term “stabilized nucleic acid molecule” or “stabilized RNA” refer to is a nucleic acid molecule, preferably an RNA molecule that is modified such, that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digest, such as by an exo- or endonuclease degradation, than the nucleic acid molecule without the modification. Preferably, a stabilized nucleic acid molecule, e.g. stabilized RNA, in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell. The stabilization effect may also be exerted outside of cells, e.g. in a buffer solution etc., for example, in a manufacturing process for a pharmaceutical composition comprising the stabilized nucleic acid molecule.

[0084] The term “variant” as used herein in the context of a nucleic acid sequence will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a variant of nucleic acid sequences which forms the basis of a nucleic acid sequence. For example, a variant nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and / or substitutions compared to the nucleic acid sequence from which the variant is derived. Preferably, a variant of a nucleic acid sequence is at least 40%, preferably at least 50%, more preferably at least60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the nucleic acid sequence the variant is derived from. Preferably, the variant is a functional variant. A “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.

[0085] The term “variant” as used herein in the context of proteins or peptides will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a proteins or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and / or deleted amino acid(s). Preferably, these fragments and / or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleuine) by isoleuine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra). A'variant” of a protein orpeptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Preferably, a variant of a protein comprises a functional variant of the protein, which means that the variant exerts the same effect or functionality as the protein it is derived from.

[0086] The term “immunodominant antigen” refers to an antigen that elicits in at least one stage of the disease production of one or more types of antibodies (e.g., IgG, IgA, IgE, IgM, etc.) in at least 40%, more typically at least 70%, and most typically at least 90% of a population exposed to the antigen, and wherein, when compared to other antigens of the same pathogen, the average binding affinity and / or average quantity of the antibodies produced in the patient in at least one stage of the disease is at least in the upper fertile, more typically upper quartile, and most typically upper quintile. Most typically, the average binding affinity and / or average quantity of the antibodies is reflected in the signal intensity and signal intensity can therefore be used as a surrogate marker for average binding affinity and / or average quantity of the antibodies. In further aspects, preferred immunodominant antigens are also characterized by a response in the test group that is considered statistically significant when compared with control signal intensity, wherein the significance level p is preferably equal or less than 0.1, more preferably equal or less than 0.05, and most preferably equal or less than 0.01.

[0087] The term “plasmid prep” refers to a technique to isolate and purify plasmid DNA from bacterial cells for downstream applications like cloning, sequencing, or transfection. The process involves culturing bacteria, lysing the cells to release their contents, and then using methods like alkaline lysis and purification kits to separate the plasmid DNA from other cellular components, such as genomic DNA and proteins. The scale of the preparation can be adjusted, from small-scale minipreps to larger maxipreps, depending on the amount of DNA needed

[0088] The term “maxiprep” refers to a laboratory technique for purifying a large amount of plasmid DNA from bacterial cultures, yielding hundreds of micrograms tomilligrams of DNA. It is used for applications requiring large quantities of pure plasmid DNA, such as large-scale cloning, protein production, and viral vector production.Maxiprep kits simplify this process using methods like alkaline-SDS lysis and silica column-based purification, often completed in about an hour.

[0089] The term “in vitro-transcription” or “IVT” refers to a technique used to synthesize RNA from a DNA template outside of a living cell. It works by using a DNA template containing a promoter sequence and a specific RNA polymerase to produce RNA molecules, which can be used for applications like producing mRNA for vaccines or studying RNA structure and function. Key components for the process include a DNA template, a specific RNA polymerase (like T7, T3, or SP6), nucleotides, and the necessary buffer conditions.BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The accompanying drawings illustrate aspects of the present invention. In such drawings:

[0091] FIG. 1 is a flow chart that shows the steps in purifying circular RNA according to embodiments.

[0092] FIG. 2A is an agarose gel (and bar graph) that shows the relative purity of circular RNA after each purification step.

[0093] FIG. 2B is a table that shows the relative percentage of circular, linear and intron RNA.

[0094] FIG. 3A is a dot blot that shows the removal of double stranded RNA.

[0095] FIG. 3B is a graphical depiction of the total amount of double stranded RNA from the dot blot.DETAILED DESCRIPTION

[0096] The particular configurations discussed in the following description are nonlimiting examples that can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.

[0097] Conventional methods of purifying circRNA are impractical and generally unsuited for producing circRNA for pharmaceutical use. In embodiments, the methods described herein have high reproducibility and are suitable for purification of pharmaceutical-grade RNA on a commercial scale for implementation in an industrial process.Circular RNAs

[0098] Circular RNAs (circRNAs) are a class of single-stranded RNAs with covalently linked head-to-tail topology. In the decades since its initial discovery, their biogenesis, regulation, and function have rapidly disclosed, permitting a better understanding and adoption of them as new tools for medical applications. With the development of biotechnology and molecular medicine, artificial circRNAs have been engineered as a novel class of vaccines for disease treatment and prevention. Unlike the linear mRNA vaccine which applications were limited by its instability, inefficiency, and innate immunogenicity, circRNA vaccine which incorporate internal ribosome entry sites (IRESs) and open reading frame (ORF) provides an improved approach to RNA-based vaccination with safety, stability, simplicity of manufacture, and scalability. However, circRNA vaccines are at an early stage, and their optimization, delivery and applications require further development and evaluation.Circular mRNA Purification

[0099] Embodiments include a purification method to purify in vitro transcribed circular from linear and doubled stranded RNA contaminates. In aspects, the method results in circular RNA that is >80% circular and contains no detectable dsRNA.

[0100] The present invention includes methods of purifying mRNA, and particularlycircRNA. In aspects, the method uses RNA produced by in vitro transcription. FIG. 1 is a flowchart 100 that describes the steps in a method.

[0101] The first step 105 entails a circularization reaction. The circRNA is incubated at about 55°C in a buffered reaction containing 2mM GTP. This enhances the fraction of circRNA in samples by approximately 10%.

[0102] Thereafter, cellulose chromatography followed by ethanol precipitation is used to remove double stranded RNA byproducts and concentrated RNA 110.

[0103] The next step 115 entails desalting and purifying RNA reactions with magnetic carboxylic acid beads to remove IVT and circularization reaction components.

[0104] The circRNA is then subjected to poly-A polymerase treatment 120. Poly A treatment creates a single stranded 3’ end to enhance the efficiency of the subsequent RNase treatment

[0105] The next step 125 entails desalting and purifying RNA reactions with magnetic carboxylic acid beads to remove poly A reaction components. Thereafter, RNase R treatment 130 is used to selectively degrade the linear RNA in the sample to produce circular RNA at >80% purity.

[0106] The final step 135 includes desalting and purifying RNA reactions Monarch RNA clean up kit to remove RNase reaction components and elute in pure waterMethods of Use

[0107] In embodiments the methods / assays described herein are used to develop / produce a vaccine. In aspects, the vaccine elicits an immune response against a bacteria. Examples of bacteria include, for example, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani, Corynebacterium diphtheriae,Pseudomonas aeruginosa, Streptococcus agalactiae, Chlamydia trachomatis, Chlamydia pneumoniae, Helicobacter pylori, Escherichia coli, Bacillus anthracis, Yersinia pestis, Staphylococcus epidermis, Clostridium perfringens or Clostridium botulinums, Legionella pneumophila, Coxiella burnetiid, Brucella, (e.g., B. abortus, B.canis, B.melitensis, B.neotomae, B.ovis, B.suis, B.pinnipediae), Francisella, (e.g., F.novicida, F.philomiragia, F.tularensis). Neisseria gonorrhoeae, Treponema pallidum, Haemophilus ducreyi, Enterococcus faecalis or Enterococcus faeciumi, Staphylococcus saprophyticus, Yersinia enterocolitica, Mycobacterium tuberculosis, Rickettsia, Listeria monocytogenes, Vibrio cholerae, Salmonella typhi, Borrelia burgdorferi, Porphyromonas gingivalis and Kiebsiella.

[0108] In another embodiment the methods / assays described herein are used to develop / produce a vaccine. In aspects, the vaccine elicits an immune response against a virus. Examples of viruses include, for example, Orthomyxovirus, Paramyxoviridae viruses, Poxviridae, Picornavirus, enterovirus (e.g., EV71, coxsackie A or B), Bunyavirus, Orthobunyavirus (e.g., California encephalitis virus, a Phlebovirus, such as Rift Valley Fever virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever virus), Heparnavirus (e.g., hepatitis A virus), Filovirus, Togavirus (e.g., Rubivirus, an Alphavirus, or an Arterivirus. rubella virus, Flavivirus (e.g., Tick-borne encephalitis (TBE) virus, Dengue ( types 1, 2, 3 or 4) virus, Yellow Fever virus, Japanese encephalitis virus, Kyasanur Forest Virus, West Nile, Encephalitis virus (e.g, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus), Pestivirus (e.g., Bovine viral diarrhea, “BVDV” Classical swine fever “CSFV” or Border disease), Hepadnavirus (e.g., Hepatitis B virus, hepatitis C virus, delta hepatitis virus hepatitis E virus, or hepatitis G virus, Rhabdovirus (e.g. a Rabies virus and Vesiculovirus), Caliciviridae (e.g., Norovirus and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus), Coronavirus: (e.g., SARS coronavirus, avian infectious bronchitis (IBV), Mouse hepatitis virus (MEV), and Porcine transmissible gastroenteritis virus (TGEV), Retrovirus (e.g., Oncovirus, Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus, Reovirus: (e.g., Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus) Parvovirus, Herpesvirus: (e.g., Herpes Simplex Viruses (HSV) (e.g. H SV types 1 and 2),Varicella-zoster Virus (VZV), Epstein-Barr virus (EBV, Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8), Papovaviruses and Adenovirus.

[0109] In another embodiment the methods / assays described herein are used to develop / produce a vaccine. In aspects, the vaccine elicits an immune response against a fungus. Fungal immunogens can be derived from Dermatophytres, 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 mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton favifornie; or from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, 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 stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastontyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia, Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi; the less common are Brachiola spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces 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., Conidiobolus spp., Rhizopus spp., Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Rhizoctoniaspp, Paecilomyces spp, Pithomyces spp. and Cladosporium ssp.

[0110] In embodiments, the methods / assays described herein to develop / produce a vaccine against a parasite. The parasite can be from the Plasmodium genus, such as P.falciparum, P.vivax, P.malariae or P. ovale. Thus, the invention can be used for immunizing against malaria. In aspects, the immunogen elicits an immune response against a parasite from the Caligidae family (e.g., from the Lepeophtheirus and Caligus genera (sea lice) such as Lepeophtheirus salmonis or Caligus rogercresseyi).

[0111] In embodiments, the assays described herein to develop / produce a vaccine against cancer. The immunogen can be a tumor antigen selected from: (a) cancertestis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21 / Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c) overexpressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2 / neu (associated with, e.g., breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein,MUC-1 (associated with, e g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte differentiation antigens such as MART-1 / Melan A, gp1OO, MCIR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein-1 / TRPI and tyrosinase related protein-2 / TRP2 (associated with, e.g., melanoma); (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate cancer; (1) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, p15, 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, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, C A 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophilin C-associated protein), TAAL6, TAG-72, TLP, TPS, and the like.

[0112] Among other advantages, it should be recognized that contemplated compositions and methods presented herein allow for preparation of vaccines and diagnostic compositions comprising a plurality of antigens with known and predetermined affinity. As individual immune systems are known to exhibit significant variation with respect to antigen recognition, methods and compositions contemplated herein will allow statistically supported antigen identification to identify immunodominant antigens. Consequently, multiple targets can be used to elicit an immune response and / or detect a prior exposure, even where one or more of the targets may be evasive for detection or provide only a weak response.

[0113] In one embodiment, the circular RNA vaccine containing the desired transgene and cell-specific promoter for use in the target ocular cells as detailed above is optionally assessed for contamination by conventional methods and then formulated into a pharmaceutical composition. Such formulation can involve the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly one suitable for administration. For injection, the carrier will typically be a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline.

[0114] In certain embodiments of the methods described herein, the pharmaceutical composition described above is administered to the subject by injection. Other forms of administration that may be useful in the methods described herein include, for example, direct delivery to a desired organ (e.g., the eye), oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.

[0115] The composition can be delivered in a volume of from about 0.1 pL to about 1 mL, including all numbers within the range, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 pL. In another embodiment, the volume is about 70 pL. In another embodiment, the volume is about 100 pL. In another embodiment, the volume is about 125 pL. In another embodiment, the volume is about 150 pL. In another embodiment, the volume is about 175 pL. In yet another embodiment, the volume is about 200 pL. In another embodiment, the volume is about 250 pL. In another embodiment, the volume is about 300 pL. In another embodiment, the volume is about 450 pL. In another embodiment, the volume is about 500 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 750 pL. In another embodiment, the volume is about 850 pL. In another embodiment, the volume is about 1000 pL. An effective concentration of a recombinant adeno-associated virus carrying a nucleic acid sequence encoding the desired transgene under the control of the cell-specific promoter sequence desirably ranges from about 107and 1013vector genomes per milliliter (vg / mL) (also calledgenome copies / mL (GC / mL)). The rAAV infectious units are measured as described in S. K. McLaughlin et al, 1988 J. Virol., 62:1963, which is incorporated herein by reference. Preferably, the concentration in the retina is from about 1.5 x 109vg / mL to about 1.5 x 1012vg / mL, and more preferably from about 1.5 x 109vg / mL to about 1.5 x 1011vg / mL. In one embodiment, the effective concentration is about 1.4 x 108vg / mL. In one embodiment, the effective concentration is about 3.5 x 1010vg / mL. In another embodiment, the effective concentration is about 5.6 x 1011vg / mL. In another embodiment, the effective concentration is about 5.3 x 1012vg / mL. In yet another embodiment, the effective concentration is about 1.5 x 1012vg / mL. In another embodiment, the effective concentration is about 1.5 x 1013vg / mL. In one embodiment, the effective dosage (total genome copies delivered) is from about 107to 1013vector genomes. It is desirable that the lowest effective concentration of virus be utilized in order to reduce the risk of undesirable effects, such as toxicity, retinal dysplasia and detachment. Still other dosages and administration volumes in these ranges may be selected by the attending physician, taking into account the physical state of the subject, preferably human, being treated, the age of the subject, the particular ocular disorder and the degree to which the disorder, if progressive, has developed. For extra-ocular delivery, the dosage will be increased according to the scale-up from the retina.Intravenous delivery, for example may require doses on the order of 1.5 x 1013vg / kg.

[0116] Multiple doses can be administered to an individual in need thereof. Where multiple doses are administered over a period of time, an active agent is administered once a month to about once a year, from about once a year to once every 2 years, from about once every 2 years to once every 5 years, or from about once every 5 years to about once every 10 years, over a period of time. For example, a subject is administered over a period of from about 3 months to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, from about 10 years to about 20 years, or more than 20 years. The actual frequency of administration, and the actual duration of treatment, depends on various factors.

[0117] As an example, a subject at risk of exposure to an infection (or who issusceptible to cancer) can be immunized by administering an initial dose of a vaccine; and administering at least a second dose (a subsequent dose). Where two or more subsequent doses are administered, the subsequent dose(s) can be separated in time from each other by at least one month, at least 3 to 6 months, at least 6 months to 1 year, at least 1 year to 5 years, at least 5 years to 10 years, at least 10 years to 20 years, or more than 20 years.

[0118] Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0119] According to another aspect, a method for forming the above-described pharmaceutical composition is provided. The method includes introducing an expression vector for expressing a heterologous gene product into a cell to form a genetically modified cell and placing the genetically modified cell in a pharmaceutically acceptable carrier.

[0120] In embodiments, a purification method disclosed herein reduces the amount of contaminants in a circRNA solution. In aspects, the amount of contaminates is reduced by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In other aspects, the amount of contaminates is reduced by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%,about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.EXAMPLES

[0121] The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples are intended to be a mere subset of all possible contexts in which the components of the formulation may be combined. Thus, these examples should not be construed to limit any of the embodiments described in the present specification, including those pertaining to the type and amounts of components of the formulation and / or methods and uses thereofExample 1Purification of circular RNA

[0122] This example details steps of SOP-EMX-01 In vitro transcription and purification of circular RNA (circRNA) from plasmid DNA. This protocol produces about 80% circRNA and removes dsRNA (IVT byproduct). The expected yield is around 10 -20% of the IVT RNA.Materials:Maxiprep plasmid DNARestriction enzymes and 10x Cutsmart (NEB)DNA clean and concentrate kit (Zymo)NEB HiScribe IVT kit (NEB)Murine RNase inhibitor (NEB)RQ1 DNase (Promega)T4 RNA ligase buffer (NEB)100mM GTP (Invitrogen, NEB, or Millipore Sigma)Monarch RNA clean-up kit (500 ug) (NEB)Dynabead carboxylic acid beads and magnet (Thermo Fisher)E-Gel Ex 2% agarose gel (Thermo Fisher)ssRNA ladder, RNA loading dye (NEB)FormamideRNase R 20 U / uL (Lucigen)10x RNase R buffer (Lucigen)Poly-A polymerase (NEB)10x poly-A buffer (NEB)ATP (NEB)Cellulose (Sigma Aldrich)Ethanol3M Sodium acetate pH 5.22X Cellulose purification buffer (100mL):2 mL of 1M HEPES pH 7.540 pL of 0.5M EDTA pH 8.08.33 mL 3M NaCI32 mL 100% EtOH57.63 mL H2OPlasmid linearization

[0123] Plasmid linearization is performed with NEB restriction enzymes. pDNA should be obtained by maxiprep. Use 2 enzymes if possible. Start linearizing 5 pg of DNA as follows:

[0124] Incubate overnight at 37 °C and proceed to DNA clean up using the DNA clean and concentrator (Zymo®). Elute with >25pL of H2O. A yield of about 80 - 85% of DNAinput is expected after.In vitro transcription and circularization

[0125] Amounts for a single reaction are shown below. Reactions can be scaled up to increase yield. Expected yield is 180pg / 20pL reaction.• Incubate at 37 °C for 2 hours.• Add 0.4pL of RQ1 DNase per 20pL reaction. Incubate at 37 °C for 30 min.• Add 70pL H2O, 8pL of 10x T4 RNA ligase buffer, and 2pL of 10OmM GTP to each 20pL IVT reaction (100pL total per reaction). Incubate at 55 °C for 10 min.• Purify and desalt the sample using Dynabead™ carboxylic acid beads.Cellulose purification

[0126] For every 4 mg of RNA, resuspend 1 g of cellulose in 5 mL of 1x cellulose purification buffer. Mix well by vortexing. For large scale (20 mg) we use 50 mL falcon tubes, with 5g of cellulose per tube, resuspend in 25m L of 1x buffer.• Spin @ 1750 x g for 1 min. discard supernatant• Resuspend pellet in 5 mL of 1x purification buffer. Spin @ 1750 x g. Discard supernatant.• Dilute 4 mg of RNA in 2.5 mL of 1x purification buffer. Resuspend cellulose pelletwith the RNA mixture• Leave on horizontal speed shaker for 30 min. Can use any method that agitates the cellulose mixture and prevents settling.• Add cellulose solution to large, filtered spin column. Spin @ 2000 x g for 2 min.Collect supernatant• Add 10% of your total volume of 3M NaAc pH 5.2 (i.e. 10pL into 10OpL).• Add 2.5x the total sample volume of pure EtOH (i.e. 275pL to 110pL) and incubate at -20°C overnight• Spin at >12000 x g for 30 min at 4°C. Can be done in microcentrifuge or large fixed angle centrifuge• Resuspend pellet in H20 by pipetting up and down. Use a volume that will be convenient for future steps. No need to further purify before Poly-A step.• Quantify yield by A260.Poly-A tailing of linear contaminants

[0127] Prepare poly-A reaction as below0.25 U per pg RNA (enzyme concentration is 5u / pL)• Incubate poly-A reaction at 37°C for 30min• Purify Dynabead carboxylic acid beads or comparable method. Assume the [RNA] will have increased slightly above the input (540 pg instead of 500) when deciding the bead capacityRNaseR and rSAP treatment-500 pg per reactionRNaseR = 0.125 units per 1 pg RNA (RNaseR concentration is 20U / pL)rSAP = 0.067 units per 1 pg RNA (rSAP concentration is 1 U / pL)• Incubate RNase R reaction at 37°C for 30min• Purify using NEB monarch kit or comparable method.• Store RNAs at -80 °CEX gel analysis of circRNA

[0128] Prepare gel samples for each IVT reaction. Mix 13 µL formamide, 200 ng RNA, 2 µL of 2X RNA loading dye and adjust to 20 pL with water, incubate 70 °C for 3 min and cool down on ice for 2 min. Load the gel using the full 20pL in each sample. Run the EX 1%-2% program on the gel chamber.

[0129] FIG. 2A is an agarose gel that shows the relative purity of circular RNA after each purification step. The top band corresponds to circular RNA, middle bands correspond to linear RNAs (precursor, nicked RNA) and lower bands are the introns. The final step (IVT+circ+PolyA+RNAseR) shows a single band of about 80% or greater purity.

[0130] FIG. 2B is a table that shows the relative percentage of circular, linear and intron RNA. Densitometry is shown after each step. The final step (Circ_T4+PolyA+RNAseR) shows 78.26% purity.

[0131] FIG. 3A is a dot blot that shows the removal of double stranded RNA. 2.5 µg of RNA was used (top) with decreasing amount to 150 ng (bottom). FIG. 3B is a graphical depiction of the total amount of double stranded RNA from the dot blot. Percent of total prME positive cells is compared in cellulose and no cellulose.

[0132] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0133] Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0134] Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term "about." As used herein, the term "about" means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.

[0135] The terms "a," "an," "the" and similar referents used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0136] All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely fortheir disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

[0137] In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and / or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

What is claimed is:

1. A method of producing and purifying circular RNA (circRNA) in a solution, the method comprising steps of:a) obtaining a solution containing a plasmid of interest,b) linearizing the plasmid with one or more restriction enzymes,c) conducting in vitro-transcription to generate RNA from the plasmid DNA, d) treating with DNAse I to remove plasmid DNA,e) incubating the solution containing the RNA at about 55°C,f) exposing the solution to cellulose chromatography to remove double stranded RNA,g) performing ethanol precipitation to concentrate the RNA,h) desalting and purifying the solution with magnetic carboxylic acid beads to remove excess salts and / or adjust pH,i) poly-A polymerase treatment to make contaminant RNA more accessible for RNase treatment,j) desalting and the solution with magnetic carboxylic acid beads to remove poly A reaction components,k) performing RNase R treatment to remove linear RNA, andl) desalting and purifying the solution to yield purified circular RNA.

2. The method of claim 1, wherein the contaminants comprise plasmid DNA, linear RNA and doubled stranded RNA.

3. The method of claim 1, wherein the method results in circular RNA that is >80% circular.

4. The method of claim 1, wherein the method results in less than 0.1% double-stranded (dsRNA).

5. The method of claim 1, further comprising a step of vaccine production, wherein the circular RNA comprises one or more antigenic sequences.

6. The method of claim 5, wherein the vaccine is an mRNA vaccine.

7. The method of claim 6, wherein the mRNA vaccine is a circular mRNA vaccine.

8. The method of claim 5, wherein the vaccine is a vaccine against a bacteria, a virus, a parasite or a cancer.

9. A method of producing a plasmid, the method comprising:a) obtaining a plasmid of interest, andb) linearizing the plasmid with one or more restriction enzymes.

10. The method of claim 9, wherein the one or more restriction enzymes comprises two restriction enzymes.

11. The method of claim 9, wherein the step of linearizing the plasmid with one or more restriction enzymes comprises incubation for six to twelve hours at about 37°C.

12. The method of claim 9, further comprising a step of:c) performing in vitro-transcription to generate RNA and subsequently treating with DNAse I to remove plasmid DNA.

13. The method of claim 12, wherein the transcribed RNA is circular RNA (circRNA).

14. The method of claim 12, further comprising purifying circular RNA, the method comprising steps of:d) incubating the solution containing the circular RNA at about 55°C,e) exposing the solution to cellulose chromatography to remove double stranded RNA,f) ethanol precipitation to concentrated RNA,g) desalting and purifying the solution with magnetic carboxylic acid beads to remove excess salts and / or adjust pH,h) poly-A polymerase treatment to make contaminant RNA more accessible for RNase treatment,i) desalting and the solution with magnetic carboxylic acid beads to remove poly A reaction components,j) RNase R treatment to remove linear RNA, andk) desalting and purifying the solution to yield purified circular RNA.

15. The method of claim 14, wherein the contaminants comprise plasmid DNA, linear RNA and doubled stranded RNA.

16. The method of claim 14, wherein the method yields more than 80% circular RNA.

17. The method of claim 14, wherein the method yields less than 0.1% doublestranded RNA.

18. The method of claim 14, wherein the 2mM GTP is added to the solution before the step of incubation at about 55°C.

19. The method of claim 14, further comprising a step of vaccine production, wherein the circRNA comprises one or more antigenic sequences.

20. The method of claim 19, wherein the vaccine is an mRNA vaccine.

21. The method of claim 20, wherein the mRNA vaccine is a circular mRNA vaccine.

22. The method of claim 20, wherein the vaccine is a vaccine against a bacteria, a virus, a parasite or a cancer.

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