Methods for producing nucleic acids

Abstract

Described are methods for producing RNA molecules in an in vitro transcription reaction. Also described are methods for producing an mRNA molecule from a closed end DNA template using an in vitro reaction system.

Description

[0001] METHODS FOR PRODUCING NUCLEIC ACIDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 769,580, filed March 10, 2025, and U.S. Provisional Patent Application Serial No. 63 / 572,156, filed March 29, 2024, the disclosures of which are hereby incorporated by reference in their entirety.

[0004] FIELD

[0005] The present invention relates to an improved process for synthesis of nucleic acid molecules, in particular cell-free enzymatic synthesis of closed end DNA (ceDNA) and synthesis of RNA molecules via in vitro transcription (IVT).

[0006] BACKGROUND

[0007] There is a need in the art for improved and efficient in vitro methods of nucleic acid amplification, specifically closed end DNA (ceDNA) molecules and templates for IVT, without the use of plasmids and bacterial fermentation (e.g., cell-free production of DNA molecules). Such improved methods may, for example, facilitate faster response times to address virus strain changes during a pandemic or seasonal viral changes, since conventional multi-step methods of DNA-template generation from plasmids produced by bacteria fermentation can be time-consuming.

[0008] SUMMARY OF THE INVENTION

[0009] In some embodiments, disclosed herein are circular DNA molecules comprising: (a) an in vitro transcription (IVT) expression cassette; (b) a protelomerase (teIRL) sequence; and (c) a restriction endonuclease (RE) recognition site, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less. In some embodiments, the IVT expression cassette comprises a promoter, a gene of interest, and a poly-A tail. In some embodiments, the circular DNA molecules further comprise an origin of replication. In some embodiments, the circular DNA molecules further comprise an antibiotic resistance gene. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less. In some embodiments, the circular DNA molecules comprises one or more ligated synthetic fragments.

[0010] In some embodiments, disclosed herein are methods of reducing double-stranded RNA (dsRNA) formed in an in vitro transcription (IVT) reaction, comprising: (a) obtaining a circular DNA molecule comprising: a. an IVT expression cassette comprising a promoter, a gene of interest, and a poly- A tail, b. a protelomerase (teIRL) sequence, and c. a restriction endonuclease (RE) recognition site, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less; (b) treating the circular DNA molecule with a protelomerase to obtain a treated composition; (c) contacting the treated composition with an exonuclease to obtain a cleaned composition; (d) purifying the cleaned composition to obtain a first purified composition; (e) contacting the first purified composition with an endonuclease to obtain a digested composition; (f) purifying the digested composition to obtain a second purified composition; and (g) performing an in vitro transcription reaction to obtain an mRNA molecule, thereby reducing dsRNA formed in the IVT reaction. In some embodiments, purifying the cleaned composition comprises a purification selected from the group consisting of proteinase K digestion, ultra-filtration / diafiltration (LIF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation. In some embodiments, purifying the digested composition comprises a purification selected from the group consisting of ultra- filtration / diafiltration (LIF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less. In some embodiments, the endonuclease comprises a type IIS restriction endonuclease. In some embodiments, the endonuclease is selected from the group consisting of Lgul, Eam1104l, and BspQI. In some embodiments, treating the circular DNA molecule with a protelomerase occurs in the absence of a surfactant. In some embodiments, treating the circular DNA molecule with a protelomerase occurs in the absence of Triton X-100. In some embodiments, disclosed herein are methods of reducing double-stranded RNA (dsRNA) formed in an in vitro transcription (IVT) reaction, comprising: (a) obtaining a circular DNA molecule comprising: a. an IVT expression cassette comprising a promoter, a gene of interest, and a poly-A tail, b. a protelomerase (teIRL) sequence, and c. a restriction endonuclease (RE) recognition site, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less; (b) treating the circular DNA molecule with a protelomerase to obtain a treated composition; (c) contacting the treated composition with an exonuclease to obtain a cleaned composition; (d) purifying the cleaned composition to obtain a first purified composition; (e) contacting the first purified composition with an endonuclease to obtain a digested composition; (f) purifying the digested composition to obtain a second purified composition; (g) performing an in vitro transcription reaction to obtain an mRNA molecule, and (h) capturing the mRNA molecule using an oligo dT affinity ligand immobilized toa solid support, thereby reducing dsRNA formed in the IVT reaction. In some embodiments, the solid support is selected from the group consisting of a chromatography resin and a magnetic bead. In some embodiments, treating the circular DNA molecule with a protelomerase occurs in the absence of a surfactant. In some embodiments, treating the circular DNA molecule with a protelomerase occurs in the absence of Triton X-100. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less. In some embodiments, a level of dsRNA formed in the IVT reaction is reduced as compared to a level of dsRNA formed in a standard IVT reaction, wherein the standard IVT reaction is performed with a circular DNA molecule comprising a teIRL sequence downstream of an RE recognition site and separated from the RE recognition site by 301 nucleotides or more. In some embodiments, the level of dsRNA formed is measured by dot blot. In some embodiments, the level of dsRNA formed in the IVT reaction is reduced by at least two-fold. In some embodiments, the circular DNA molecule comprises one or more ligated synthetic fragments.

[0011] In some embodiments, disclosed herein are methods of producing an mRNA molecule comprising: (a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, wherein the circular double-stranded DNA template comprises a teIRL sequence, b. a primer or primase, c. a deoxyribonucleotide triphosphate (dNTP), d. a pyrophosphate, and e. a phi29 DNA polymerase; (b) incubating the composition for a time period between about 20 and 30 hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) treating the incubated composition with a protelomerase to obtain a treated composition; (d) contacting the treated composition with an exonuclease to obtain a cleaned composition; (e) purifying the cleaned composition to obtain a first purified composition; (f) contacting the first purified composition with a restriction endonuclease to obtain a digested composition; (g) purifying the digested composition to obtain a second purified composition; and (h) performing an in vitro transcription reaction, thereby producing the mRNA molecule. In some embodiments, steps (a) to (d) occur in the same reaction vessel. In some embodiments, the methods do not comprise a heat denaturation reaction prior to incubating the reaction. In some embodiments, the protelomerase is immobilized to a substrate. In some embodiments, the substrate is a N- hydroxy succinimide agarose bead. In some embodiments, purifying the cleaned composition comprises a purification selected from the group consisting of proteinase K digestion, ultra- filtration / diafiltration (UF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation. In some embodiments, purifying the digested composition comprises a purification selected from the group consisting of ultra- filtration / diafiltration (UF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation. In some embodiments, treating the incubated composition with a protelomerase occurs in the absence of a surfactant. In some embodiments, the surfactant comprises Triton X-100. In some embodiments, disclosed herein are methods of producing an mRNA molecule comprising: (a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, wherein the circular doublestranded DNA template comprises a teIRL sequence, b. a primer or primase, c. a deoxyribonucleotide triphosphate (dNTP), d. a pyrophosphate, and e. a phi29 DNA polymerase; (b) incubating the composition for a time period between about 20 and 30 hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) treating the incubated composition with a protelomerase to obtain a treated composition; (d) contacting the treated composition with an exonuclease to obtain a cleaned composition; (e) purifying the cleaned composition to obtain a first purified composition; (f) contacting the first purified composition with a restriction endonuclease to obtain a digested composition; (g) purifying the digested composition to obtain a second purified composition; (h) performing an in vitro transcription reaction, and (i) capturing the mRNA molecule using an oligo dT affinity ligand immobilized to a solid support, thereby producing the mRNA molecule. In some embodiments, the solid support is selected from the group consisting of a chromatography resin and a magnetic bead. In some embodiments, the phi29 DNA polymerase comprises a mutation resulting in increased thermostability and processivity. In some embodiments, the methods comprise repeating (a) and (b) one or more times. In some embodiments, the methods comprise repeating steps (a) - (h) using the second purified composition from step (g) as the circular double- stranded DNA template in step (a). In some embodiments, treating the incubated composition with a protelomerase occurs in the absence of a surfactant. In some embodiments, treating the incubated composition with a protelomerase occurs in the absence of Triton X-100. In some embodiments, the circular DNA molecule comprises one or more ligated synthetic fragments.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is an exemplary diagram depicting closed end linear DNA production via multiple stage rolling cycle amplification and TelN protelomerase treatment. The three stages include: 1) a first round of rolling circle amplification (RCA) of a circular DNA containing a TeIRL site to produce concatemeric DNA which is subjected to 2) multiple rounds of RCA to produce extended concatemeric DNA which is subjected to 3) TelN and exonuclease treatment to produce closed end DNA (ceDNA).

[0014] FIG. 2 is an exemplary diagram depicting a “one-pot” reaction to produce closed end linear DNA from a template. As shown in the diagram, Phi29 DNA polymerase, primer / primase, dNTPs, and circular DNA with a teIRL site is combined in a rolling circle amplification (RCA) reaction. TelN protelomerase treatment results in ceDNA formation, followed by T5 exonuclease clean-up, and then EDTA / proteinase K enzyme inactivation and degradation. The ceDNA is then purified, subjected to ultrafiltration / diafiltration, and then polished.

[0015] FIG. 3 is an exemplary agarose gel image analysis of closed end linear DNA synthesis. Lane 1 is undigested plasmid DNA (pDNA); lane 2 is plasmid DNA digested by Lgul; lane 3 is rolling circle amplification (RCA) product using plasmid DNA as the template; lane 4 is RCA product digested by Lgul; lane 5 is closed end linear DNA formation by digesting RCA product with telN protelomerase; lane 6 is clean-up of closed end linear DNA with T5 exonuclease; and lane 7 is purified closed end linear DNA digested with Lgul.

[0016] FIG. 4 is an exemplary agarose gel image depicting pre-treatment of plasmid DNA with exonuclease for a rolling circle amplification (RCA) reaction.

[0017] FIG. 5 depicts an electropherogram of RNA made from ceDNA made with specific primers and a pDNA template with truncations in the poly(A) region, displaying an ideal peak shape. Overlayed with reference standard (lower line).

[0018] FIG. 6 depicts an electropherogram of RNA made from ceDNA made with Tth primase and a pDNA template with truncations in the poly(A) region, displaying a much broader peak shape. Overlayed with reference standard (lower line).

[0019] FIGS. 7A-C show exemplary plasmid configuration designs. FIG. 7A depicts a 5’ TeIRL design with approximately 2000 base pairs between the restriction endonuclease recognition site (RS) and the TeIRL site. FIG. 7B depicts a 3’ TeIRL design with approximately 30 base pairs between the RS and the TeIRL site. FIG. 7C depicts desirable on-target transcription resulting in transcription of the gene of interest (GOI) (top) and undesirable off-target transcription (bottom), which is a possible source of double-stranded RNA initiating from the ceDNA loop portion.

[0020] FIG. 8 is an exemplary diagram depicting how TelN removal in generation 1 results in successful synthesis of ceDNA in generation 2. Generation 1 produces ceDNA from plasmid using rolling circle amplification (RCA) with subsequent addition of DNA relaxing agents including betaine, single strand binding (SSB) protein from E. coli (EtSSB), and single-stranded DNA binding protein from T4 phage (T4G32), followed by removal of TelN with thermolabile protease K digestion (TLProK), magnetic bead removal, or phenol chloroform purification.

[0021] FIG. 9 depicts generation 2 ceDNA yield by varying TelN additives with and without TelN removal (first column TelN, second column T5 for each sample).

[0022] FIG. 10 depicts an exemplary agarose gel image of a single self-ligating fragment design minicircle. From left-to-right, the lanes are: 1kb DNA Standard (lane 1); Synthetic Gene Fragment (lane 2); First Round PCR Amplification (lane 3); Second Round PCR Poly(A) tail addition (lane 4); Bsal Digestion (lane 5); Ligation (lane 6); T5 Exonuclease Treatment (lane 7); and Cleaned & Concentrated Minicircle (lane 8).

[0023] FIG. 11 depicts an exemplary minicircle process using a single self-ligating fragment design. In this process, circular DNA is prepared from a synthetic gene fragment. As shown in the figure, two rounds of PCR are performed to amplify the target sequence and add the sequences containing all desired elements of the DNA fragment (e.g., Poly A site, restriction sites, nucleotides for ligation, etc.).

[0024] DETAILED DESCRIPTION mRNA technology has tremendous potential to treat diseases. To achieve that potential, high- quality mRNA for vaccine or therapeutic purposes is needed. In vitro transcription (IVT) can be carried out to synthesize RNA from a closed end DNA (ceDNA) template.

[0025] Disclosed herein are batch and fed-batch methods for production of RNA molecules, e.g., mRNA, including modified mRNA molecules and / or self-amplifying RNA (saRNA), that are useful for producing clinical grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). The methods use a closed end DNA (ceDNA) template in an in vitro transcription (IVT) reaction to generate the RNA molecule and can be applied to a wide variety of constructs with varying 5’ UTRS, coding sequence lengths, 3’ UTRs, and 3’ ends. The RNA can be mRNA, including modified mRNA molecules, unmodified mRNA molecules, and / or saRNA, that are useful for producing clinical grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP). In some aspects, the RNA molecule is purified via chromatographic methods, e.g., through use of an oligo(dT) substrate. In some aspects, enzymatic capping is used for 5' capping of the RNA molecule.

[0026] Also disclosed herein are methods for various “one pot” reactions for production of closed end linear DNA as the template for in vitro transcription (IVT). The reactions including rolling circle amplification (RCA) of a circular DNA containing a teIRL site followed by TelN protelomerase treatment and exonuclease digestion to synthesize closed end DNA (ceDNA) as shown in FIG. 1. The whole process from RCA to ceDNA (with enzymatic cleanup) can be performed in one reactor by sequentially dosing substrates and enzymes as shown in FIG. 2. The closed end linear DNA can then be purified by ultrafiltration / diafiltration (UF / DF) after proteinase K treatment and can be used as tempalte for in vitro transcription (IVT) to produce mRNA with similar IVT yield and main quality attributes as mRNA made from plasmid DNA.

[0027] Certain Definitions

[0028] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some asoects the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the measurement or quantitation.

[0029] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0030] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0031] The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.

[0032] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. It is contemplated that aspects described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.” Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of” (and any form of consisting of, such as “consist of’ and “consists of’) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0033] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” “a further embodiment,” “some embodiments”, “one aspect,” “an aspect,” “a particular aspect,” “a related aspect,” “a certain aspect,” “an additional aspect,” “a further aspect,” “some aspects” or combinations thereof means that a particular feature, structure or characteristic described in connection with the aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

[0034] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. For example, in some embodiments the methods disclosed herein reduce the amount of double-stranded RNA (dsRNA) formed in an in vitro transcription (IVT) reaction when compared to a standard IVT reaction not incorporating the circular DNA molecules disclosed herein. The methods disclosed herein may reduce the amount of dsRNA formed when compared to a standard IVT reaction, such as a reduction of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 pg / ug. The methods disclosed herein may reduce the fold amount of dsRNA formed when compared to a standard IVT reaction, such as a 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4- fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold reduction in amount of dsRNA formed. The terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.

[0035] As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and / or determined substantially simultaneously and / or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and / or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.

[0036] The term “DNA,” as used herein, means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or thymine (T)). For example, DNA can contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. Without any limitation, DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.

[0037] The term “RNA,” as used herein, means a nucleic acid molecule that includes ribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and / or uracil (II) or N-1-methylpseudouridine). For example, RNA can contain all, or a majority of, ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. In one aspect, RNA can be messenger RNA (mRNA) that relates to an RNA transcript which encodes a peptide or protein. As known to those of skill in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a polypeptide coding region, and a 3' untranslated region (3 -llTR). Without any limitation, RNA can encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, circular RNA, self-amplifying RNA (saRNA), and modified RNA (modRNA).

[0038] As contemplated herein, without any limitations, RNA can be used as a therapeutic modality to treat and / or prevent a number of conditions in mammals, including humans. Methods contemplated comprise administration of the RNA described herein to a mammal, such as a human. For example, in one aspect, such methods of use for RNA include an antigen-coding RNA vaccine to induce robust neutralizing antibodies and accompanying / concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses. The RNA administered is preferably in vitro transcribed RNA.

[0039] An “isolated RNA” is defined as an RNA molecule that can be recombinant or has been isolated from total genomic nucleic acid. A “modified RNA” or “modRNA” refers to an RNA molecule, e.g., an mRNA molecule, having at least one addition, deletion, substitution, and / or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations can refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5' and / or 3' end(s) of RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide. For example, a modified nucleotide can replace one or more uridine and / or cytidine nucleotides. For example, these replacements can occur for every instance of uridine and / or cytidine in the RNA sequence, or can occur for only select uridine and / or cytidine nucleotides. Such alterations to the standard nucleotides in RNA can include nonstandard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide can be replaced with 1-methylpseudouridine in an RNA sequence. Other such altered nucleotides are known to those of skill in the art. Such altered RNAs are considered analogs of naturally-occurring RNA. In some aspects, the RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, the RNA can be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA).

[0040] As used herein, a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues. As used herein, the term “wild-type” or “native” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in many aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0041] The term “isolated” can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (e.g., when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (e.g., when chemically synthesized). Moreover, an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel. Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in the functional state and / or that is altered or removed from the natural state through human intervention. For example, a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered.

[0042] All patents, published patent applications, other publications, and databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

[0043] DNA Template

[0044] In some aspects, the method for producing an RNA molecule, e.g., mRNA, includes providing a sample that includes a linear closed end DNA (ceDNA) template. The ceDNA template includes a sequence coding for a gene of interest that encodes, e.g., a peptide or polypeptide of interest. In some aspects, the ceDNA template includes an RNA polymerase promoter sequence operably linked to the sequence coding for a gene of interest. In some aspects, the DNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. For example, in some preferred aspects, the ceDNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence operably linked to the RdRp gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. In some aspects, a ceDNA template lacks a plasmid backbone.

[0045] In some aspects, the linear ceDNA template used as the template for in vitro transcription is derived from plasmid DNA. In some aspects, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH10B cells, are transformed with the plasmid DNA template. The transformed cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some aspects, the linear ceDNA template is synthesized in a cell-free environment. In some aspects, the linear ceDNA template is synthesized by rolling circle amplification (RCA). In some aspects, the RCA reaction comprises a phi29 DNA polymerase. In some aspects, the RCA reaction comprises a phi29 DNA polymerase comprising a mutation resulting in increased thermostability and processivity, such as one or more of the mutations disclosed in U.S. Patent Application Publication No. US 2014 / 0322759, incorporated by reference herein for purposes of the DNA polymerase mutants described therein. In some aspects, the RCA reaction comprises an EquiPhi29 DNA polymerase. In some aspects, the RCA reaction comprises a phi29 DNA polymerase comprising at least one mutation relative to wild-type phi29 DNA polymerase selected from V51A, M97T, L123S, G197D, K209E, E221 K, E239G, Q497P, K512E, E515A, and F526L. In some aspects, RCA includes an amplification target circle (ATC) that forms a template on which new DNA is made, thereby extending the initial sequence as a continuous sequence of repeated sequences complementary to the circle but generating only about several thousand copies per hour. In some aspects, the linear ceDNA template is provided by exponential RCA, including hyperbranched RCA (also termed ramification amplification).

[0046] In some aspects, the ceDNA template also includes an RNA polymerase promoter sequence, e.g., a T7 promoter, located 5' to and operably linked to the gene of interest. As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like. For example, a gene of interest operably linked to an RNA polymerase promoter allows transcription of the gene of interest. Any RNA polymerase or variants thereof may be used in the methods described here. The RNA polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.

[0047] As used herein, “gene of interest” refers to a polynucleotide which encodes a polypeptide or protein of interest. Depending on the context, the gene of interest refers to a deoxyribonucleic acid, e.g., a gene of interest in a ceDNA template which may be transcribed to an RNA molecule, or a ribonucleic acid, e.g., a gene of interest in an RNA molecule which may be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. As described in more detail below, a polypeptide of interest includes but is not limited to, biologies, antibodies, vaccines, therapeutic proteins or peptides, etc.

[0048] Flanking Regions: Untranslated Regions (UTRs)

[0049] In some aspects, the method for producing an RNA molecule includes (a) providing a sample having a linear ceDNA template, the ceDNA template includes an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a 5' untranslated region (UTR) and / or a 3' UTR. In some aspects, the ceDNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest. For example, in some aspects, the methods for producing an RNA molecule include (a) providing a sample having a linear ceDNA template, the ceDNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5' to and operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest and a 5' untranslated region (UTR) and / or a 3' UTR.

[0050] The ceDNA template and RNA molecule may include UTRs. Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory features of a UTR may be incorporated into the polynucleotides, primary constructs and / or mRNA of the present invention to enhance the stability of the molecule. The specific features may also be incorporated to ensure controlled down- regulation of the transcript in case they are misdirected to undesired organs or sites in the body.

[0051] Poly(A) tail

[0052] In some aspects, the methods for producing an RNA molecule disclosed herein include providing a sample having a ceDNA template, the ceDNA template having an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a poly(A) tail sequence of 20-100 nucleotides. The poly(A) tail can prevent degradation of the RNA molecule in a cell. Accordingly, in some aspects, the plasmid ceDNA template includes a sequence coding for a poly(A) tail located 3' to the gene of interest. As used herein, “poly(A) tail” refers to a chain of adenine nucleotides. In some aspects, the poly(A) tail includes 5-300 adenine nucleotides in length, e.g., at least, at most, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 adenine nucleotides in length, or any range or value derivable therein. In preferred aspects, the ceDNA template includes a poly(A) tail that includes about 40 adenines. In preferred aspects, the ceDNA template includes a poly(A) tail that includes about 80 adenines. In some aspects, the poly(A) tail is encoded in the ceDNA template. In other aspects, the poly(A) tail is added to the RNA molecule by enzymatic treatment with a Poly(A) polymerase. In some aspects, the RNA molecule does not include a poly(A) tail.

[0053] In some aspects, immediately downstream of the poly(A) tail coding sequence on the plasmid ceDNA template is a recognition site for a restriction endonuclease to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional readthrough.

[0054] In some aspects, following linearization, the plasmid ceDNA template is filtered into an appropriate solvent, e.g., a solvent selected from the group consisting of water, HEPES, Tris-CI and EDTA. In a preferred aspect, the solvent includes 10 mM HEPES, 0.1 mM EDTA, and the like. Filtration occurs via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0055] The linear ceDNA template may be purified before use as a template for in vitro transcription. For example, the linear ceDNA template may be purified chromatographically or by ethanol precipitation.

[0056] In vitro Transcription

[0057] In vitro transcription (IVT) refers to a procedure that allows for DNA-directed synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several kilobases. In some aspects, in vitro transcription involves engineering of a ceDNA template to include a bacteriophage promoter sequence (e.g., from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In some aspects, the resulting RNA molecules are subsequently modified (e.g., by capping, splicing, the addition of a poly(A) tail, etc.).

[0058] The methods described herein for producing an RNA molecule, e.g., mRNA, includes contacting a ceDNA template with an in vitro transcription (IVT) reaction system. In some aspects, the IVT reaction system includes an RNA polymerase and ribonucleotides, which may be natural and / or modified ribonucleotides. In some aspects, the IVT reaction system includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and an RNA polymerase. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified, such as, for example, N-1-methylpseudouridine triphosphate) NTPs.

[0059] RNA Polymerase

[0060] In some aspects, the RNA polymerase used to generate the mRNA transcript may also be referred to as a “DNA-dependent RNA polymerase” which transcribes DNA into RNA molecules. Exemplary RNA polymerases include bacteriophage T7, T3, Syn5, and SP6 RNA polymerases, or variants thereof (including thermostable / thermophilic variants), which may be used to transcribe the mRNA or self-amplifying RNA from a ceDNA template. RNA polymerases represent the primary machinery that drives transcription. RNA polymerases have been isolated and purified sufficiently that they are useful for producing RNA in vitro. In some aspects, the RNA polymerase is a T7 RNA polymerase, which refers to a monomeric T7 bacteriophage-encoded DNA directed RNA polymerase that catalyzes the formation of RNA in the 5' to 3' direction. The wild-type T7 RNA polymerase includes 883 amino acids. It is homologous to T3 RNA polymerase and somewhat homologous to SP6 RNA polymerase.

[0061] In some aspects, the RNA polymerase includes an engineered T7 RNA polymerase variant, such as a variant that allows for selective incorporation of the m7G(5')ppp(5')m7G cap analog over GTP at the initiation of in vitro transcription. For example, in some aspects, the RNA polymerase has been modified to preferentially accept a cap (also referred to as an RNA cap, an RNA 7- methylguanosine cap or an RNA m7G cap) or cap analog (e.g., the “Anti Reverse Cap Analog” (3'-O-Me-m7G(5')ppp(5')G; “ARCA”), or a methylated cap analog with one or more nucleotides at the transcription initiation site (e.g., m7G(5')ppp(5')N, wherein N is any nucleotide) to begin transcription during transcription initiation. The 5' cap is an altered nucleotide on the 5' end of some eukaryotic primary transcripts such as precursor messenger RNA. The typical cap structure includes a 7-methylguanosine (m7G) linked to the first nucleotide of the transcript via a 5'-5' triphosphate bridge. Cap analogs may include, for example, one, two or more methyl (or other substitution) groups at specific positions. Cap molecules may be added either upfront in the IVT reaction or after the synthesis of mRNA, by enzymatic capping. Cap molecules added upfront in the IVT reaction can make the mRNA production more straightforward.

[0062] Ribonucleotides

[0063] In the methods described herein, the IVT reaction system includes nucleotides (for example, nonmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates). The nucleotides may be selected from any one of natural nucleotides, e.g., A, G, C, and II ribonucleotides; modified nucleotides (such as, for example, N-1-methylpseudouridine triphosphate); or a combination thereof. In some aspects, the ribonucleotides are Tris buffered, such as a 100 mM aqueous solution of ribonucleotide titrated to pH 7.3-7.5 with Tris base. In some aspects, the ribonucleotides are in sodium salt.

[0064] Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules generated by the IVT reaction system include, for example, m5C (5- methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1-methyladenosine); m2A (2- methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0- ribosyladenosine (phosphate)); I (inosine); mil (1-methylinosine); m'lm (l,2'-0- dimethylinosine); m3C (3-methylcytidine); Cm (2T- O-methylcytidine); s2C (2- thiocytidine); ac4C (N4-acetylcytidine); £5C (5-fonnylcytidine); m5Cm (5,2-0- dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mIG (1- methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-0- methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-0- dimethylguanosine); m22Gm (N2,N2,2'-0-trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7-cyano- 7-deazaguanosine); preQi (7- aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0- dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'- O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5- hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine)); mchm5U (5- (carboxyhydroxymethyl)uridine methyl ester); mcm5U (5-methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5- methoxycarbonylmethyl- 2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2- selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0- methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L- Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5- carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); rn62Am (N6,N6,0-2- trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7- trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-0- methylcytidine); mIGm (l,2'-0-dimethylguanosine); m'Am (1 ,2-0- dimethyl adenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine)); imG- 14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7- substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5- aminouracil, 5-(Ci-C6)-alkyluracil, 5-methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)- alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5- hydroxycytosine, 5-(Ci-C6 )- alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2- C6)-alkynylcytosine, 5- chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2- C6)alkynylguanine, 7-deaza- 8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8- oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8- azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-0-methyl-U. Additional exemplary modified nucleotides include any one of N-1-methylpseudouridine ; pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine.

[0065] In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0066] In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, II, G and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine. In other aspects, the RNA may include a 5' cap comprising a 7'- methylguanosine, and the first 1 , 2 or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.

[0067] Exemplary In Vitro Transcription Reaction Systems

[0068] In some aspects, the in vitro transcription reaction system includes the following: an RNA polymerase, e.g., a T7 RNA polymerase, ceDNA template; nucleotide triphosphates (NTPs); magnesium; and a buffer such as, e.g., HEPES or Tris (or both HEPES and Tris).

[0069] In some aspects, the in vitro transcription reaction system includes the RNA polymerase, e.g., a

[0070] T7 RNA polymerase, at a final concentration of 1000-44000 U / rnL, e.g., at least, at most, or about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 5900, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 20500, 21000, 21500, 22000, 22500, 23000, 23500, 24000, 24500, 25000, 25500, 26000, 26500, 27000, 27500, 28000, 28500, 29000, 29500, 30000, 30500, 31000, 31500, 32000, 32500, 33000, 33500, 34000, 34500, 35000, 35500, 36000, 36500, 37000, 37500, 38000, 38500, 39000, 39500, 40000, 40500, 41000, 41500, 42000, 42500, 43000, 43500, or 44000 ll / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes a T7 RNA polymerase at either 8U / uL, 10U / uL, 13U / uL, 15U / uL, 16U / uL, 17U / uL, 18U / uL, 19U / uL, or 20U / uL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 7000 Ll / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 8000 Ll / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 14000 Ll / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 17000 Ll / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 25000 Ll / mL. In some aspects, the in vitro transcription reaction system includes an RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 40000 Ll / mL.

[0071] In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,

[0072] 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,

[0073] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66,

[0074] 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,

[0075] 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112,

[0076] 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 ,

[0077] 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150,

[0078] 151 , 152, 153, 154, 155, 156, 157, 158, 159, or 160 nM. or any range or value derivable therein.

[0079] In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 40 nM. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 144 nM. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 5-24 nM DNA, e.g., at least, at most, or about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 nM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 36 to 144 nM DNA, e.g., at least, at most, or about 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, or 144 nM, or any range or valuable derivable therein. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of, e.g., at least, at most, or about 0.01 , 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.07, 0.075, 0.08, 0.09, 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.50 mg / mL, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.025 mg / mL. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.05 mg / mL. In some aspects, in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.075 mg / mL. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.1 mg / mL.

[0080] In some aspects, the in vitro transcription reaction system includes each nucleotide triphosphate (NTP) at a final concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription system includes each nucleotide triphosphate (NTP) at a starting concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphates (NTPs) at a final concentration of about 8 mM each. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphate ATP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphate CTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the nucleotide triphosphate GTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the UTP or nucleotide triphosphate modified UTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein. In certain aspects the concentration of NTPs in the reaction is 0.4mM, 0.8mM, 1mM, 1.25mM, 3.mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, or 10mM.

[0081] In some aspects, the IVT reaction system includes magnesium ion, for example, as a magnesium salt, such as any one of magnesium chloride and magnesium acetate. In some aspects, the in vitro transcription reaction system includes the magnesium at a final concentration of, e.g., at least, at most, or about 12, 13, 14, 15, 16, 16.5, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122,

[0082] 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 ,

[0083] 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160,

[0084] 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179,

[0085] 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198,

[0086] 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217,

[0087] 218, 219, or 220 mM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 30 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 40 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 16.5 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 33 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 36 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 50 mM. In some aspects, the in vitro transcription reaction system includes magnesium acetate at 110 mM. In some aspects, the Mg:NTP ratio can be maintained at a ratio of, e.g., at least, at most, or about, 0, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , or 2.2 mM Mg / mM NTP, or any range or value derivable therein. In some aspects, one or more reaction components are added during in vitro transcription by occasional bolus feeds, semi-continuous feeds, or continuous feeds. Bolus feeds can be delivered at intervals of, e.g., at least, at most, or about, 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 minutes, or any range or value derivable therein. These components can include, but are not limited to, one or more NTPs and a cation such as magnesium. These components can be combined into a single feed, or they can be delivered separately in the form of multiple feeds. In some aspects, a continuous feed of at least 1 NTP can be delivered at flow rates of, e.g, at least, at most, or about 0, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, or 4 mL / L / min. In some aspects, a continuous feed of a cation such as magnesium can be delivered at concentrations of, e.g., at least, at most, or about 0, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 .0 mM / min, or any range or value derivable therein

[0088] In some aspects, the in vitro transcription (IVT) reaction system includes a buffer. Exemplary buffers for the IVT reaction system may include Tris and / or HEPES. In some aspects, the in vitro transcription reaction system includes the buffer at a pH of, e.g., at least, at most, or about 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In some aspects, the buffer is Tris-HCI, pH 8.0. In some aspects, the in vitro transcription reaction system includes 40 mM Tris HCI, pH 8.0. Alternative buffers for the IVT reaction system include 40 mM Tris pH 7.5, 80 mM HEPES. In some aspects, the in vitro transcription reaction system does not include HEPES. In some aspects, the in vitro transcription reaction system includes HEPES and Tris. In some aspects, the buffer does not comprise dithiothreitol (DTT).

[0089] In some aspects, an RNase inhibitor is included in the in vitro transcription reaction system. The RNase inhibitor may reduce RNase-induced degradation during the transcription reaction. For example, murine RNase inhibitor may be utilized at a final concentration of 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 U / rnL. In some aspects, the in vitro transcription reaction system comprises RNase inhibitor at a final concentration of 100 U / rnL. In some aspects, the in vitro transcription reaction system comprises RNase inhibitor at a final concentration of 1000 U / rnL.

[0090] In some aspects, a pyrophosphatase is included in the in vitro transcription reaction system. The pyrophosphatase may cleave the inorganic pyrophosphate generated following each nucleotide incorporation into two units of inorganic phosphate, which may reduce the likelihood of magnesium co- precipitating with pyrophosphate to form magnesium pyrophosphate. Pyrophosphatase in certain aspects may be diluted in pyrophosphatase buffer and present in the reaction at concentrations of 0.01mll / uL, 0.02mll / uL, 0.05mll / uL, 0.08mll / uL, 0.1mll / uL, 0.2mll / uL, 0.8mll / uL, or 2mU / uL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 0.25 U / rnL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 0.5 U / rnL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 1 U / rnL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 2 U / rnL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 3 U / rnL. In some aspects, the in vitro transcription reaction system includes an inorganic pyrophosphatase at a final concentration of 6 U / rnL.

[0091] In some aspects, the in vitro transcription reaction system includes a polyamine. Exemplary polyamines include spermine, putrescene, and spermidine. In some aspects 1 mM spermidine is included. In some aspects 2.0 mM spermidine is included. In some aspects 2.15 mM spermidine is included. In some aspects, the in vitro transcription reaction system lacks a polyamine. In some aspects, the in vitro transcription reaction system lacks spermidine.

[0092] In some aspects, the IVT reaction system includes a reducing reagent, such as, for example, DTT (dithiothreitol), e.g., at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 mM, or any range or value derivable therein. In some aspects, the reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythritol (DTE), Tris(2- carboxyethyl)phosphine (TCEP) and beta-mercaptoethanol. In some aspects, the IVT reaction system includes 1 mM DTT. In some aspects, the IVT reaction system includes 5 mM DTT. In some aspects, the IVT reaction system includes 10 mM DTT. In some aspects, the IVT reaction system includes 20 mM DTT. In some aspects, the IVT reaction system lacks a reducing agent. In some aspects, the IVT reaction system does not contain DTT. In some aspects, the IVT reaction system does not contain added DTT beyond the protective amount of DTT present in the T7 RNA polymerase storage solution.

[0093] In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for about 4 hours or about 240 minutes, e.g., at least, at most, or about 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 minutes, or any range or value derivable therein. In some preferred aspects, the in vitro transcription reaction proceeds, for example, at less than 50°C for less than 4 hours, such as for example, at least, at most, or about 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41 °C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31 °C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21 °C, or about 20°C, for at least, at most, or about 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 minutes, or any range or value derivable therein. In some aspects, the in vitro transcription reaction proceeds at about 36°C for about 120 minutes. In some aspects, the in vitro transcription reaction proceeds at about 36°C for about 150 minutes. In some aspects, the in vitro transcription reaction proceeds at about 37°C for about 120 minutes. In some aspects, the in vitro transcription reaction proceeds at about 37°C for about 150 minutes.

[0094] In some aspects, the in vitro transcription reaction proceeds at about 37°C, at greater than 120 minutes and less than 360 minutes, preferably greater than 120 minutes and less than 300 minutes, more preferably greater than 120 minutes and less than 260 minutes. In some preferred aspects, the in vitro transcription reaction proceeds at about 37°C for about 150 minutes.

[0095] As disclosed herein, a reaction system proceeding at about 37°C for less than 4 hours results in high yields of highly pure RNA. In some aspects, yields per in vitro transcription reaction may be at least 0.3 mg of RNA per mL starting volume of IVT reaction to about 20 mg of RNA per mL starting volume of IVT reaction. For example, in some aspects, the total yield of RNA molecule may be at least, at most, or about 0.3 mg RNA / mL, 0.4 mg RNA / mL, 0.5 mg RNA / mL, 0.6 mg RNA / mL, 0.7 mg RNA / mL, 0.8 mg RNA / mL, 0.9 mg RNA / mL, 1.0 mg RNA / mL, 2 mg RNA / mL, 3 mg RNA / mL, 4 mg RNA / mL, preferably at least 5 mg RNA / mL, 6 mg RNA / mL, 7 mg RNA / mL, 8 mg RNA / mL, 9 mg RNA / mL, 10 mg RNA / mL, 11 mg RNA / mL, 12 mg RNA / mL, 13 mg RNA / mL, 14 mg RNA / mL, 15 mg RNA / mL, 16 mg RNA / mL, 17 mg RNA / mL, 18 mg RNA / mL, 19 mg RNA / mL, or 20 mg RNA / mL starting volume of IVT reaction, or any range or value derivable therein. In preferred aspects, the total yield per in vitro transcription reaction of RNA molecule produced having at least 90% of the intended full length transcript may be at least 2 mg RNA / mL, 3 mg RNA / mL, 4 mg RNA / mL, preferably at least 5 mg RNA / mL, 6 mg RNA / mL, 7 mg RNA / mL, 8 mg RNA / mL, 9 mg RNA / mL, 10 mg RNA / mL, 11 mg RNA / mL, 12 mg RNA / mL, 13 mg RNA / mL, 14 mg RNA / mL, 15 mg RNA / mL, 16 mg RNA / mL, 17 mg RNA / mL, 18 mg RNA / mL, 19 mg RNA / mL, or 20 mg RNA / mL starting volume of IVT reaction. In some aspects, the total yield per in vitro transcription reaction of RNA molecule produced having at least 90% of the intended full length transcript is at least 17 mg RNA / mL starting volume of IVT reaction.

[0096] In some aspects, following an IVT reaction using a ceDNA template and an RNA polymerase as described here, a first composition that includes an uncapped RNA molecule is produced. In some aspects, the RNA molecule includes the coding sequence for a gene of interest and a poly(A) tail. As used herein, the RNA molecule includes an mRNA. The RNA molecule may include modifications, such as, modified nucleotides. As used herein, an “RNA molecule” produced by in vitro transcription may be referred to as an “RNA transcript.” An “RNA molecule” and an “RNA transcript” may encompass any one of modified mRNA “modRNA,” unmodified mRNA, and self-amplifying RNA (saRNA). In some aspects, the RNA molecule is a saRNA.

[0097] In some aspects, the methods of producing RNA molecules by contacting the ceDNA sample with the in vitro transcription reaction system described herein produces a first composition having an uncapped RNA molecule. In some aspects, at least 30% of the RNA molecules in the first composition includes uncapped RNA molecules. In some aspects, the first composition includes at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, uncapped RNA molecules.

[0098] RNA Molecule

[0099] The RNA molecule produced by the methods described herein may be a non-coding and / or a coding RNA. A non-coding RNA (ncRNA) molecule includes a functional RNA molecule that is not translated into a peptide or polypeptide. Non-coding RNA molecules may include highly abundant and functionally important RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs such as snoRNAs, microRNAs, siRNAs, snRNAs, guideRNAs, circularRNAs, exRNAs, and piRNAs and long ncRNAs. In a preferred aspect, the RNA molecule is an mRNA molecule that includes a modified nucleotide (herein referred to as a “modified RNA molecule” or “modified mRNA molecule”). In some preferred aspects, the RNA molecule is a selfamplifying RNA molecule.

[0100] Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide. In some aspects, the coding RNA molecule includes at least one open reading frame coding for at least one peptide or polypeptide. The coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs). The coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule or selfamplifying RNA molecule (saRNA, also referred to as a replicon). Preferably, the RNA molecule is an mRNA.

[0101] In some preferred aspects, the RNA molecule is a saRNA. “Self-amplifying RNA,” “self-amplifying RNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.

[0102] In some aspects, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. Preferably epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both. In some aspects, the RNA molecule has a 3' poly(A) tail, that is, a stretch of consecutive adenosine residues, that may be attached to the 3' end of the RNA. The poly(A) tail may increase the half-life of the RNA molecule. The RNA molecule may further include a poly(A) polymerase recognition sequence (e.g., AALIAAA) near its 3' end. In some aspects, the 3' poly(A) tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. Preferably, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some preferred aspects, the RNA molecule includes about 40 consecutive adenosine residues. In some aspects, the RNA molecule includes about 80 consecutive adenosine residues. Poly(A) tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyper-polyadenylation may signal for RNA degradation. Exemplary designs include a poly(A) tails of about 40 As, about 80 As. In some aspects, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly(A) tail sequence.

[0103] In some aspects, the RNA molecule produced by the in vitro transcription reaction described herein is purified, e.g., including filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration / diafiltration.

[0104] Capping of RNA Molecule

[0105] In some aspects, the methods of producing RNA molecules described herein further include capping uncapped RNA molecules by contacting the uncapped RNA molecules with a capping reaction system, which includes any one of guanylyltransferase, s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and 2'-O-methyltransferase, and any combination thereof, to produce a capped RNA molecule. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which may be incorporated during RNA synthesis (co-transcriptional capping) or may be performed enzymatically after RNA transcription (post-transcriptional capping). In some preferred aspects, the 5' end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription. In some aspects, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.

[0106] An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyltransferase and guanine-7- methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures). Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule. The 5' cap of the RNA molecule may be further modified by a 2'-O- Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '-O] N), which may further increase translation efficacy.

[0107] In some aspects, the RNA molecule may be enzymatically capped at the 5' end using Vaccinia guanylyltransferase, guanosine triphosphate and S-adenosyl-L-methionine to yield cap 0 structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, use of a 2'-O-methyltransferase with Vaccinia guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2'-OH group is methylated on the first transcribed nucleotide. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. In some preferred aspects, RNase inhibitor is not included in the enzymatic capping reaction. In a preferred aspect, the enzymatic capping reaction step is performed under constant mixing. In another aspect, the RNA molecule is not co-transcriptionally capped.

[0108] In one aspect, the capping reaction system includes enzymatic 5' capping that is performed as follows. The final 1X buffer conditions includes the following: at least, at most, or about 50 mM Tris HCI, pH 8, 5 mM KCI, 1 mM MgCI2, 0.5 mM GTP, 0.2 mM S-adenosyl-methionine and 1 mM dithiothreitol. In some aspects, the final 1X buffer does not include dithiothreitol.

[0109] In some aspects, the capping reaction occurs in the vessel in which the IVT reaction was performed. In such aspects, the IVT reaction is diluted between 3-fold and 10-fold, e.g., at least, at most, or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, before the capping reaction. In some aspects, the IVT reaction is diluted with Tris pH 7.0 buffer.

[0110] To degrade residual ceDNA template from the IVT reaction, DNase I can be added. In some aspects, DNase I is added at a concentration between at least, at most, or about 1 ll / pg of DNA to 10 ll / pg of DNA, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 ll / pg of DNA, or any range or value derivable therein. In addition to DNase I, CaCh can be added as a co-factor for DNase I at a concentration between at least, at most, or about 0.1 mM to 4 mM, e.g., 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mM, or any range or value derivable therein.

[0111] In some aspects, pyrophosphatase is added into the capping reaction. Pyrophosphatase assists with degrading pyrophosphate, which is the inhibitory by-product that is generated by the IVT reaction or by the capping reaction.

[0112] In some aspects, the capping reaction is conducted under 37 °C for 30 minutes to 2 hours, e.g., at least, at most, or about 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,

[0113] 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72,

[0114] 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,

[0115] 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117,

[0116] 118, 119, or 120 minutes, or any range or value derivable therein. In some aspects, the capping reaction is conducted at a temperature greater than 20°C and less than 50°C, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50°C.

[0117] In some aspects, the step of capping the uncapped RNA molecules results in at least, at most, or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% capped RNA molecules of the total of RNA molecules (capped and uncapped), or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Fragment analyzer or Bioanalyzer chip-based electrophoresis and measured by, e.g., peak area of full-length RNA molecule relative to total peak.

[0118] Purification

[0119] In some aspects, the RNA molecule produced by the methods described herein may be contacted with DNase I and CaCl2 to enzymatically digest ceDNA template following the in vitro transcription reaction. In some aspects, such as during a large scale (reaction volumes above 10 mL) operation, the IVT reaction may include DNase I and CaCl2 additions as well as an additional treatment with EDTA and proteinase K. The EDTA may quench any cationic metal species, including magnesium, and the proteinase K may digest proteins present in the IVT reaction, reducing their size.

[0120] In some aspects, the methods described herein do not include contacting the RNA molecule produced by the methods described herein with DNase I to enzymatically digest ceDNA template following the in vitro transcription reaction.

[0121] In some aspects, the linear ceDNA template is removed from the in vitro transcription reaction system, for example, the ceDNA template is separated from the RNA molecule via chromatography. In some aspects, the RNA molecule binds to an affinity substrate while the ceDNA template flow through and is removed. In some aspects, the poly(A) capture-based affinity purification is oligo(dT) purification. For example, a polythymidine ligand may be immobilized to a derivatized chromatography resin. The mechanism of purification may involve hybridization of the poly(A) tail of the RNA molecule to the oligonucleotide ligand, wherein the ceDNA template will not bind. In preferred aspects, the RNA molecules that do not include Poly(A) stretches (abortive transcripts and other truncates formed during in vitro transcription) will not bind to the resin and will not form a duplex with the affinity ligand. Poly-adenylated RNA may then be eluted from the resin utilizing a low ionic strength buffer or a competitive binding oligonucleotide solution.

[0122] Preferably, purified material is substantially free of one or more impurities or contaminants including the linear ceDNA template and / or reverse complement transcription products described herein and for instance is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% pure; more preferably, at least 98% pure, and more preferably still at least 99% pure.

[0123] The method for production of an RNA molecule may include additional purification steps after the in vitro transcription, e.g., an ion exchange chromatography step, a hydrophobic interaction chromatography (HIC) step, a ceramic hydroxyapatite (CHA) chromatography step, and / or an ultrafiltration / diafiltration (LIF / DF) step. In a preferred aspect, said synthesizing of an RNA molecule of a given sequence is performed as a large-scale synthesis.

[0124] Characterization and Analysis of the RNA Molecule

[0125] The RNA molecule produced by the methods described herein may be analyzed and characterized using various methods. Analysis may be performed before or after capping. Alternatively, analysis may be performed before or after poly(A) capture-based affinity purification. In another aspect, analysis may be performed before or after additional purification steps, e.g., anion exchange chromatography and the like. For example, RNA transcript integrity may be determined using electrophoresis (using the fragment analyzer capillary or Bioanalyzer chip systems) or through a reverse phase HPLC method. The fragment analyzer can in some instances automate capillary electrophoresis and HPLC. In other aspects, RNA template purity is analyzed using analytical reverse phase HPLC respectively. Capping efficiency may be analyzed using, e.g., total nuclease digestion followed by LC-UV or LC-MS quantitation of the dinucleotide cap species vs. uncapped GTP species. In vitro efficacy may be analyzed by, e.g., transfecting RNA molecule into a human cell line. Protein expression of the polypeptide of interest may be quantified using methods such as ELISA or flow cytometry. Immunogenicity may be analyzed by, e.g., transfecting RNA molecules into cell lines that indicate innate immune stimulation, e.g., PBMCs. Cytokine induction may be analyzed using, e.g., methods such as ELISA to quantify a cytokine, e.g., Interferon-a.

[0126] The method of producing RNA molecules described herein may produce an RNA molecule that is at least 30% full-length transcript, or at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% full-length transcript, or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measure by, e.g., peak area of full-length RNA molecule relative to total peak.

[0127] Genes of Interest

[0128] The DNA template and resulting RNA molecule of the present invention include a gene of interest. The gene of interest encodes a polypeptide of interest selected from, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. The sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GenBank.

[0129] In some aspects, the RNA molecule includes a coding region for an antigen preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Area no bacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean- Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DENV-1 , DENV-2, DENV-3 and DENV-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (including Influenza such as avian influenza and human influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, Coronavirus (e.g., SARS-CoV-2), Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis.

[0130] In some aspects, the RNA molecules of the present disclosure encode a viral polypeptide or fragment thereof, including naturally occurring or engineered variants thereof, for prophylaxis against a virus in humans. In some aspects, the viral polypeptide does not comprise a coronavirus polypeptide. In some aspects, the viral polypeptide does not comprise a severe acute respiratory syndrome (SARS) virus polypeptide. In some aspects, the viral polypeptide does not comprise a SARS-CoV-2 polypeptide.

[0131] Thus, in some aspects, the RNA molecules of the present disclosure do not encode a coronavirus polypeptide or fragment thereof, including naturally occurring or engineered variants thereof. In some aspects, the RNA molecules of the present disclosure do not encode a SARS virus polypeptide or fragment thereof, including naturally occurring or engineered variants thereof. In some aspects, the RNA molecules of the present disclosure do not encode a SARS-CoV-2 virus polypeptide or fragment thereof, including naturally occurring or engineered variants thereof.

[0132] In further aspects, the RNA molecules of the present disclosure are not used for prophylaxis against a coronavirus in humans. In some aspects, the RNA molecules of the present disclosure are not used for prophylaxis against a SARS virus in humans. In some aspects, the RNA molecules of the present disclosure are not used for prophylaxis against SARS-CoV-2 in humans.

[0133] EXAMPLES

[0134] Below are examples of specific aspects for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0135] EXAMPLE 1

[0136] CREATION OF A CIRCULAR DNA CONTAINING A TELRL SITE BY DNA ASSEMBLY

[0137] As an alternative to using a difficult to expand plasmid, insertion of a teIRL recognition sequence (56 bp; Deneke et al., The protelomerase of temperate Escherichia coli phage N15 has cleavingjoining activity, PNAS 97(14):7721-7726 (2000)) into a plasmid can enable the plasmid to perform rolling-circle-amplification (RCA) and lead to a closed-end DNA (ceDNA) construct. The teIRL sequence was inserted upstream of 5’ to the Sbfl site in a self-amplified RNA (saRNA) construct or upstream of the T7 promoter in a Xcml site (20 bp) of a modRNA construct. PCR was performed on the VSV-G (Vesicular Stomatitis Virus Glycoprotein) gene with flanking sequences to match the regions surrounding the Pmel insertion site using InFusion. In addition, teIRL sequences were also inserted at 402bp downstream of the poly(A) region of a modRNA plasmid. The VSV-G sequence was subcloned into the Pmel insertion site. These teIRL containing plasmids were used in the following examples.

[0138] EXAMPLE 2

[0139] RCA REACTION WITH CIRCULAR DNA OR RCA AS THE TEMPLATE

[0140] A plasmid or circular DNA containing self-amplifying RNA with an 80 poly(A) tail and at least one teIRL site were used to run the reaction shown in Table 1 :

[0141] Table 1 : RCA Reaction with circular DNA as the template (Reaction 1)

[0142] As an alternative to the random hexamers or specific primers, Thermus thermophilus (Tth) primase was used at a final concentration of 10 ug / mL. Tth primase was prepared using a 21 amino acid C-terminal truncated primase sequence from Thermus thermophilus cloned into a vector and expressed as a N-terminal His tag fusion protein in E. coli DE3 host and purified using nickel resin. The above mixture was gently mixed for 15 minutes on a thermo block shaker and then incubated at 30C for 20-30 hours (alternatively, the mixture can be agitated at 64-170 rpm with a pitched blade impellor for 10-30 minutes in a stirred-tank reactor). This RCA reaction could achieve about 1-2 mg / ml amplified DNA based on analysis via the Qubit DNA assay kit. A comparison of the three different priming strategies with flu constructs encoding four different strains is shown in Table 2:

[0143] EXAMPLE 3 CREATION OF CLOSED END DNA WITH “ONE-POT” REACTION

[0144] The RCA reaction described in Example 2 was treated with TelN protelomerase to generate closed end linear DNA. 1 ml of the RCA reaction was combined with 0.2ml of TelN protelomerase (NEB, 5000U / ml) and 1.3ml protelomerase reaction buffer (containing 20 mM Tris-HCI, 10 mM ((NH4)2SO4, 10 mM KCI, 2 mM MgSO4, pH8.8). The mixture was gently mixed while incubated at 30C for 16hours (or 37C for 4-6 hours).

[0145] After protelomerase treatment, T5 exonuclease was added to reach a value of 200 U per mL of reaction and the solution was incubated at 37C for 4-6 hrs to remove non-closed end DNA or nicked DNA so that the intact closed end linear DNA could be obtained. Based on the initial template size, this “one-pot” reaction can achieve >0.5mg ceDNA / ml RCA reaction. An example of agarose gel analysis from samples obtained from Example 2 is shown in FIG. 3.

[0146] EXAMPLE 4

[0147] PURIFICATION OF CLOSED END DNA

[0148] Resulting closed end linear DNA can be further purified by phenol-chloroform extraction followed by ammonium acetate and alcohol precipitation at benchtop scale to remove enzymes and nucleotides so that it is ready for other application, such as sequencing or IVT. It can also be purified by commercial DNA purification kits (including Meg-Bind TotalPure NGS, OMEGA BIO- TEK) or ammonium acetate and alcohol precipitation. In addition, for scale-up purposes, the resulting closed end linear DNA can be purified by ultrafiltration / diafiltration (UF / DF) after treatment with 40mM EDTA and 1-2 U / ml of proteinase K at 37C for 18-24 hours. These purification steps and the RCA reaction can be performed in a “one-pot” reaction without changing the reaction vessel, as depicted in FIG. 2.

[0149] The reaction from Example 3 was treated with proteinase K to reach a value of 1.2 U per mL of reaction and then concentrated ammonium sulfate solution was added to a final concentration of 250mM ammonium sulfate. The solution was loaded into a tangential filtration system with 300KDa Hydrosart membrane equilibrated with DF1 buffer as 250 mM Ammonium Sulfate, 10 mM Tris, 1mM EDTA, pH 7.0. The solution was concentrated ~4X to achieve DNA concentration ~1.2 - 1.5 g / L. Then concentrated solution was diafiltrated for 10 diafiltration volumes (DV) of DF1 buffer followed by 10 DV of DF2 buffer (10DV of 10mM Tris, pH7). Target final DNA concentration was -1.2 g / L. The purified pool was filtrated via 0.2 um PES member for sterile filtration. This LIF / DF protocol can achieve recovery yields as high as >80%. The final closed end DNA was ready for DNA linearization and use in an in vitro transcription reaction.

[0150] EXAMPLE 5

[0151] USE OF CLOSED END LINEAR DNA AS A TEMPLATE FOR IVT

[0152] The purified, closed end linear DNA from Example 4 was used as a template for in vitro transcription (IVT) to generate mRNA. Plasmid or purified, closed end linear DNA was digested by Lgul endonuclease per manufacturer instruction at 37C overnight. The digested plasmid or closed end linear DNA can be purified by ethanol purification (or methods used for large-scale DNA purification) or can be used directly in IVT reactions, as shown in Table 3:

[0153] Table 3: IVT using closed end linear DNA as template

[0154] 200ul of the mixture was incubated at 37 °C (for mRNA or 25 °C for saRNA) for 120 min. 50 U / ul of DNase I and 1 M CaCh were added to final concentration as 8U of DNase l / ug DNA and 2 mM of CaCh. After incubating for an additional 30min at 37 °C, 50% LiCI was added in 1 :1 ratio. The reaction solution was frozen at -80C for at least 12 hours, and then centrifuged for 1 hour. 1 ml of cold 200 proof ethanol was added, and then the mixture centrifuged for 10 minutes. The supernatant was removed and the process repeated. The pellet was dried and resuspended in nuclease free water. The IVT yield was measured with a Nanodrop Spectrophotometer (THERMO) and mRNA integrity was measured by Fragment Analyzer (AGILENT) and showed no significant difference using plasmid or closed end linear DNA as template as shown in Table 4:

[0155] EXAMPLE 6 PRE-TREATMENT OF PLASMID DNA WITH EXONUCLEASE PRIOR TO RCA REACTION

[0156] The plasmid DNA (pDNA) template used for the rolling circle amplification (RCA) reaction can be treated with exonuclease to remove nicked or linear DNA species and improve ceDNA yield, as shown in Table 5:

[0157] Table 5: Pre-treatment of plasmid DNA with exonuclease

[0158] The reaction was incubated at 37C for 2-16 hours and then dsDNA concentration was measured using the Qubit dsDNA assay kit (THERMO FISHER). The agarose gel in FIG. 4 shows that the plasmid DNA treated with exonuclease has reduced nicked and linear plasmid content. Exonuclease-treated pDNA that is unpurified or purified can then be used as starting material for the reactions described above. The data in Table 6 indicates that plasmid template treated with exonuclease shows improvement in closed end linear DNA yield.

[0159] Table 6: Closed end linear DNA yield after treating plasmid template with exonuclease

[0160] EXAMPLE 7 APPLICATION OF SPECIFIC-PRIMER PRIMED RCA TO A VARITY OF DNA CONSTRUCTS FOR THE REDUCTION OF TRUNCATIONS IN CLOSED END DNA POLY(A) REGION pDNA constructs containing different Influenza antigens were pretreated with Exonuclease III as described in Example 6, amplified as described in Example 1 , converted to closed end DNA as described in Example 3, and purified as described in Example 4. Table 7 shows improved integrity of the polyA tail region of ceDNA when RCA reactions used specific primers compared to reactions using Tth primase.

[0161] Table 7: Improved reliability of polyA integrity in ceDNA made using specific primers compared to ceDNA made using primase in various constructs Conditions 1, 2, 3, 4 and 8 as described in Table 7 were used to create RNA using the process described in Example 5, with the results displayed in Table 8.

[0162] Table 8: RNA made with ceDNA from a variety of modFlu constructs

[0163] A pDNA construct containing an Influenza antigen (p130) was pretreated with Exonuclease III as described in Example 6. The pDNA lot used had known truncations in its polyA region. The pretreated pDNA was amplified as described in Example 1 using either Tth primase or specific primers. The resulting RCA DNA was converted to closed end DNA as described in Example 3 and purified as described in Example 4. Results along with starting pDNA quality are displayed in Table 9. This data displays the ability of specific primers to mitigate poor polyA integrity from starting pDNA template compared with Tth primase which exacerbates the issue.

[0164] Table 9: Improved reliability of polyA integrity with ceDNA made using specific primers compared to ceDNA made using primase from template with poor polyA integrity

[0165] Conditions 9 and 10 in T able 9 were used to create RNA (using the process described in Example 5) and the results are displayed in Table 10. Electropherograms for RNA from conditions 9 and 10 can be found in FIG. 5 and FIG. 6, respectively, demonstrating a broader peak from the RNA made using condition 10 compared to a thinner peak for condition 9. This shows that the improved DNA polyA integrity from ceDNA made with specific primers led to improved RNA size homogeneity.

[0166] Table 10: RNA made with ceDNA using a pDNA template with poor polyA i nteg rity . 2 | p130 | Tth primase | 6.4 | 94% | 90% |

[0167] EXAMPLE 8

[0168] RANGE STUDIES FOR DETERMINATION OF PROCESS RANGES FOR TELN DIGESTION OF RCA DNA FOR THE CREATION OF CLOSED END DNA

[0169] A pDNA construct containing self-amplifying RNA sequences and a VSV-G antigen (R048) was amplified as described in Example 1 using random primers. Temperature and reaction duration were held consistent at 30C and 18 hours, respectively. Dilution (ratio of TelN reaction volume to RCA reaction volume) and TelN protelomerase dose (units per ug of DNA input) were varied from 2.5X-8X and 1-2 hours, respectively. The ceDNA was purified as described in Example 4. Results can be found in Table 11 demonstrating achieving an effective ceDNA yield with the variety of conditions.

[0170] Table 11 : Using a variety of conditions to digest RCA DNA into ceDNA with similar ceDNA yields.

[0171] In a follow up experiment, a pDNA construct containing an Influenza antigen (p232) was pretreated with Exonuclease III as described in Example 6 and amplified as described in Example 1 using specific primers. Temperature, duration, dilution, and TelN protelomerase dose (units per uL of TelN reaction volume) were all varied around the center point (37 C, 6 hours, 2.5X dilution, 0.4 U / uL TelN). The ceDNA was purified as described in Example 4. Results can be found in Table 12 demonstrating effective ceDNA yield and identical polyA quality across the variety of conditions.

[0172] Table 12: Using a variety of conditions to digest RCA DNA in to ceDNA with similar ceDNA yield and quality.

[0173] EXAMPLE 9

[0174] CREATION OF CLOSED END DNA FROM RCA DNA USING BUFFERS WITH AND WITHOUT TRITON

[0175] Commercially available reaction buffers for TelN digestion use Triton X-100 as a surfactant. Reaction buffers for TelN digest were formulated with and without Triton X-100 for comparison. A pDNA construct containing self-amplifying RNA sequences and VSV-G antigen (R048) was amplified as described in Example 1 using random primers, converted to close-end DNA as described in Example 3, and purified as described in Example 4. Results can be found in Table 13, demonstrating effective ceDNA yield using buffers with and without Triton X-100.

[0176] Table 13: ceDNA made using buffers with and without Triton X-100 have similar ceDNA yield

[0177] EXAMPLE 10

[0178] CLOSED END DNA PLASMID DESIGN

[0179] High purity is essential in mRNA vaccine manufacturing. During the in vitro-transcription (IVT) process, double-stranded RNA (dsRNA) is a major contaminant which induces severe anti-viral immune responses. dsRNA can be introduced by various undesired off-target transcription by T7 polymerase, including cis self-priming, random priming of abortive transcripts, and other nonspecific promoter-independent transcription initiation. This example describes a novel DNA plasmid configuration that significantly reduces dsRNA impurity in mRNA production.

[0180] DNA plasmid templates for use in IVT typically contain a replication origin, antibiotic resistance gene (AbxR), TeIRL site, T7 promoter, gene-of-interest (GOI), poly(A) region, and a restriction site (RS) for creating an open-end linear dsDNA prior to IVT (FIG. 7). Closed-end DNA (ceDNA) synthesized from those plasmid templates are also depicted, where the teIRL site forms singlestranded loops at the end of ceDNA.

[0181] Among different ceDNA configurations, the location of teIRL sites on the plasmid plays an important role. When the teIRL site was placed at the 5’ upstream of T7 promoter (FIG. 7 A), two DNA fragments are generated after digestion at the restriction site (RS). One bearing the IVT cassette, the other consisting of the antibiotic resistance gene (AbxR) and replication origin. Although this large backbone (over 2000bp) doesn’t contain a T7 promoter, it's speculated that long dsRNA can be generated from the teIRL loop by off-target turn-around transcription (FIG.

[0182] 7C). On the other hand, when the teIRL site is located at the 3’ end of the IVT cassette (FIG. 7B), the fragment without the GOI is much smaller after restriction digestion. As shown in FIG. 7C, the final design places the teIRL sequence preferably 30bp downstream of the restriction site. Hence after restriction digestion, the shorter non-IVT fragment is unlikely to generate long dsRNA as an impurity during mRNA production.

[0183] This was tested with four influenza strain vaccine GOIs, building each construct with the 5’ teIRL design and the 3’ teIRL design (Table 14). Using ceDNA and precursor plasmid as control, IVT was performed and dsRNA level was measured with dot blot. The 3’ teIRL design significantly reduced dsRNA level in IVT for all 4 constructs, from 500-1000 to <250 (pg / ug). This suggested that the 3’ teIRL design renders cleaner mRNA with more consistent quality control.

[0184] Table 14: dsRNA levels in mRNA produced from ceDNA constructs and p lasmid controls EXAMPLE 11 TELN IMMOBILIZATION

[0185] It is desirable for plasmids carrying an IVT cassette to be propagated in vivo for many generations to support large-scale DNA production. Accordingly, the possibility of continuing to produce ceDNA from a ceDNA template in vitro was explored. However, the current ceDNA synthesis method using plasmid as the starting material results in less production. For example, using p095 as a proof of concept, comparing to the 1 mg / ml yield using plasmid as template, only about 5ug / ml ceDNA was able to be generated from its seed ceDNA.

[0186] Since TelN protein has extremely slow or no turnover due to its strong DNA binding affinity, it was speculated that the enzyme almost becomes inactive after each round of reaction. It was hypothesized that this inert form of TelN-DNA hybridization in the 1st generation (Genl) ceDNA production can inhibit the access of other enzymes to their DNA substrate in the following reactions, thus reducing the efficiency of 2nd generation (Gen2) ceDNA propagation.

[0187] To test this hypothesis, two scenarios to loosen the binding of TelN-DNA complex after TelN reaction in the 1st generation of ceDNA production were investigated: (i) adding DNA relaxing reagents such as betaine (which loosens up the DNA structure and reduces binding of TelN), and single strand binding (SSB) proteins from E. coli (EtSSB) or from T4 phage (T4G32) (both of which compete with TelN for ceDNA binding); and (ii) removing TelN protein with thermolabile protease K digestion (TLProK) which digests the TelN protein, followed by DNA purification (FIG. 8). As shown in Table 15, additives were added during Genl TelN reaction after RCA and samplel is a negative control, where no additional modifications were made to the existing process. Samples 3, 5, 7, and 9 were tested with various additives, and samples 2, 4, 6, 8, and 10 all underwent an extra TelN removal step. During TelN removal, thermolabile protease K (TLProK) was added to the reaction after TelN treatment to completely break down the TelN enzyme. 1 ul TLProK was added to the 50ul reaction at 37C for 3 hours, then TLProK was heat inactivated at 55C for 20mins in a thermomixer, followed by magnetic bead clean up to purify DNA and eliminate all residual proteins. Then the purified Genl ceDNA was used as seed template in Gen2 ceDNA synthesis. Interestingly, both approaches led to significant improvement in 2nd generation ceDNA production. All samples (other than the negative control) showed improved Gen2 ceDNA yield, which indicates that reducing the TelN bidning affinity of Genl ceDNA is key to Gen2 ceDNA production. Without additives, the TelN removal method by TLProK produced 0.388 mg / ml ceDNA, resulting in over 70-fold increase in yield with respect to control. Alternatively, with additives and no TelN removal, 0.173-0.24 mg / ml ceDNA production was observed, while combining the two approaches generated 0.198-0.34 mg / ml ceDNA (FIG. 9 and Table 15).

[0188] Table 15: ceDNA yield from 2ndgeneration with varying TelN additives

[0189] To examine the mRNA quality resulting from the modified TelN processes, construct p292 was selected with the 3’ teIRL design for 2nd generation ceDNA production, considering this new configuration rendered low dsRNA impurity during IVT procedure (Table 16).

[0190] Table 16: mRNA quality attributes from 2ndgeneration ceDNA as IVT template

[0191] In this study, 4 conditions were tested: for conditions without any additives: (1) TLProK digestion or (2) TLProK combined with phenol chloroform (PC) extraction was used for TelN removal. For conditions with additives: (3) 1.5M betaine and (4) 10ng / ul EtSSB plus TLProK digestion were chosen based on previous observations. Consequently, all 4 conditions produced 0.2-0.24 mg / ml ceDNA which further generated high quality mRNA with low dsRNA, full poly(A) and >88% FA. Hence, it was concluded that TelN removal, with or without DNA relaxing additives, is a key process contributing to high 2nd generation ceDNA yield and high-quality mRNA. Among the 3 TelN removal methods tested, TLProK digestion plus phenol chloroform extraction showed the most consistent results (data not shown). However, this process requires additional time for digestion, meanwhile phenol chloroform extraction subjects to about 50% loss in ceDNA production. To tackle this challenge, immobilizing TelN enzyme using NHS resin was explored.

[0192] Table 17 shows ceDNA produced with constructs p292, containing 3’ teIRL and poly 30L70 and p095 containing 3’ teIRL and a poly 80A where TelN enzyme is immobilized using NHS (N- hydroxy succinimide) agarose.

[0193] Table 17: Immobilized TelN in 1stgeneration and 2ndgeneration ceDNA and mRNA quality The experiment for p292 was a comparison between using 10 mg TelN protein vs 5 mg TelN protein with NHS immobilization of the TelN during incubation with the RCA concatemer during production of first generation ceDNA. The experiment for p095 was a comparison between using specific primers vs primase in the first-generation RCA step before the (immobilized) TelN step.

[0194] In the first generation, the 1 :2.5 diluted RCA concatemer was incubated at 37°C for 6 hours with TelN immobilized using NHS beads on a rotating platform. The DNA material was then eluted from the column, leaving the TelN on the NHS beads. T5 exonuclease was added to the material post TelN and incubated at 37°C for 4 hours to remove open ended DNA. The resulting ceDNA was extracted from a phenol chloroform solution to remove protein and precipitated with NH4OAC and pure ethanol then resuspended in EB buffer. The 1stgeneration ceDNA yield was > 0.5 mg ceDNA / ml RCA.

[0195] The first generation ceDNA was denatured and used as a template for 2nd generation RCA, TelN and T5 exonuclease. During the production of 2nd generation ceDNA, TelN was not removed prior to the T5 exonuclease step. After T5 exonuclease, purified ceDNA was extracted with phenol chloroform and precipitated with NH4OAc and pure ethanol and resuspended in EB buffer. The resulting 2nd generation ceDNA was then linearized and transcribed to produce mRNA.

[0196] EXAMPLE 12

[0197] CREATION OF A CIRCULAR DNA FROM SYNTHETIC FRAGMENTS

[0198] This example was performed with a single self-ligating fragment design. A synthetic gene fragment served as the initial template (sourced from a commercial vendor). This gene fragment was flanked on both the 5’ and 3’ ends by approximately 250 base pair sequences. Within the 5’ and 3’ flanking regions, there was a TeIRL sequence (56 bp; Deneke et al., The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity, PNAS 97(14):7721-7726 (2000)), a T7 promoter sequence, a 5’ UTR, the gene of interest (GOI), and a 3’ UTR. A polymerase chain reaction using ~0.5 pM of specifically-designed forward & reverse primers, 0.02 ng / pl DNA template, 1X Q5 High-Fidelity 2X Master Mix (NEW ENGLAND BIOLABS), and water (qs) was performed (2min denature at 98°C, 20 cycles of [10sec at 98°C, 30sec at 69°C, 4min 30sec at 72°C], 5min at 72°C, then hold at 4°C) to amplify the gene fragment and remove the flanking regions.

[0199] The product of the first PCR was used in a second round of PCR using explicitly-designed primers to add a poly A tail, a BspQI restriction site for IVT linearization, and Bsal restriction sites, along with four GC-rich nucleotide ligation site sequence on both the 5’ and 3’ ends. This round of PCR required longer oligonucleotides, which have only partial binding to the amplicon, while the nonbinding sequences promote addition of the complement to the 5’ and 3’ ends of the amplicon. The forward primer included sequences for the addition of a 5’ Bsal restriction site (GGTCTCN) followed by a four nucleotide GC-rich sequence compatible with T7 ligase to promote hybridization of GC-rich sticky ends. The Ultramer reverse primer (>1OOnt; INTEGRATED DNA TECHNOLOGIES) partially binds to the 3’IITR and included non-binding complementary sequences for the addition of a poly A tail, BspQI restriction site (GCTCTTC), a four nucleotide GC-rich sequence, followed by a sequence complementary to a 3’ Bsal restriction site.

[0200] Fragment design was such that digestion with Bsal restriction endonuclease cleaved the double strand on both ends, leaving GC-rich complementary overhangs (sticky ends) on the 5’ ends of the sense and anti-sense strands. Addition of T7 ligase hybridizes the complementary GC-rich sticky ends together resulting in a self-ligated minicircle containing a TeIRL sequence, 5’ UTR, GOI, 3’ UTR, poly A tail and a BspQI restriction site. An exemplary minicircle using this single self-ligating fragment design was used in an IVT reaction and compared to IVT reactions using plasmid templates, as shown in Table 18.

[0201] Table 18: Data for comparison of ceDNA mRNA prepared from minicircle template in RCA versus other DNA template for IVT :

[0202] The data in Table 18 demonstrates that the ceDNA yield prepared from a minicircle of synthetic fragments is comparable to the ceDNA yield from plasmid. In addition, comparison of the mRNA quality attributes of yield, FA%, cap% and poly A confirmation from in vitro transcription of plasmid DNA, plasmid ceDNA and minicircle ceDNA are comparable and are within acceptable specifications. % circularization is estimated using densitometry of the gel image, where intensity of product band + intensity of total bands in lane * 100 = % circularization.

[0203] This method can also be performed with a multiple fragment design for ligation. In this example, two or more synthetic fragments (synthesized in-house or commercially sourced) are designed so that the double stranded fragments hybridize GC-rich overhangs at the 5’ ends of each strand. Regardless of the number of fragments, in order for ligation to occur and form circular DNA, all fragments must contain on both 5’ ends of the sense and anti-sense strands: a) GC-rich four nucleotide sequences specifically complementary to the opposite strand of another fragment and b) Bsal restriction sites outside of the GC-rich nucleotide sequences (with 1 spacer nucleotide). Fragments may then contain: a) >25 bp flanking sequences on both ends (outside all other sequences), b) TeIRL sequence, c) T7 promoter sequence, d) 5’UTR, d) GOI, f) the 3’UTR, and e) any other sequences needed. One of the fragments will be a duplex Ultramer (INTEGRATED DNA TECHNOLOGIES) (complementary strands of olioonucleotides annealed together) that contains a poly A tail, GC-rich four nucleotide sequences and Bsal restriction site sequences on both ends. The fragments may be amplified via PGR.

[0204] Digestion with Bsal cleaves both ends of each fragment, leaving GC-rich four nucleotide overhangs at the 5’ ends of each sense and anti-sense strand (sticky ends). Bsal digestion can include 0.1 pg / pl DNA template, 1X Cutsmart Buffer (NEW ENGLAND BIOLABS), 5 U / pg Bsal- HF v2 (NEW ENGLAND BIOLABS) and water (qs) and can be run at 37°C for > 1 hour, static. The GC-rich nucleotide sequences are designed to promote hybridization of the 5’ overhang of the sense strand of one fragment to only the 5’ overhang of the anti-sense strand of a second fragment that is complementary. Each 5’ overhang sense strand must be explicitly complementary to only one 5’ overhang anti-sense strand.

[0205] During the ligation step, the 5’ sticky end of the sense strand hybridizes with the complementary 5’ sticky end of the anti-sense strand, and then T7 DNA ligase forms phosphodiester bonds linking the hybridized nucleotides in the strands together to form a single fragment self-ligated minicircle or circular DNA from multiple ligated fragments. This minicircle can contain: a) the TeIRL site, b) T7 promoter, c) 5’ UTR, d) GOI, e) 3’ UTR, f) restriction site for IVT linearization, g) the poly A tail, h) ligation site(s) - 5’ end sense strand GC-rich four nucleotide sticky ends joined with their complementary sequences from the 5’end anti-sense strand, and j) any other sequences needed. Ligation can occur at 4°C-37°C depending on fragment size(s) (bp) for > 2 hours, static. Ligation components can include variable amounts of DNA (-2-12 ng / pl or <1 pM), fragments in a 1 :1 molar ratio, 0.2 U / pl Bsal-HF v2 (NEW ENGLAND BIOLABS), 25 U / pl T7 DNA ligase, 0.2X-1X StickTogether DNA ligase buffer (NEW ENGLAND BIOLABS), variable amounts (2-15%) of 1 ,2- propanediol, and water (qs).

[0206] For purification, T5 exonuclease is added after ligation to remove non-circular (linear or nicked) double stranded DNA leaving only circular DNA components. T5 exonuclease can be added at a final concentration of 0.1 U / pl. Digest at 37°C for 1-2 hours. Purification of DNA (for example, using a commercial cleanup kit) can occur after PCR steps, after digestion, and / or after T5 exonuclease treatment to remove non-nucleic acid components (such as protein) (FIG. 10). An exemplary schematic for the minicircle process using a single self-ligating fragment design is depicted in FIG. 11.

[0207] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to person skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMS1. A circular DNA molecule comprising:(a) an in vitro transcription (IVT) expression cassette;(b) a protelomerase (teIRL) sequence; and(c) a restriction endonuclease (RE) recognition site, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less.

2. The circular DNA molecule of claim 1 , wherein the IVT expression cassette comprises a promoter, a gene of interest, and a poly-A tail.

3. The circular DNA molecule of any one of claims 1 or 2, further comprising an origin of replication.

4. The circular DNA molecule of any one of claims 1 to 3, further comprising an antibiotic resistance gene.

5. The circular DNA molecule of any one of claims 1 to 4, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less.

6. The circular DNA molecule of any one of claims 1 to 5, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less.

7. The circular DNA molecule of any one of claims 1 to 6, wherein the circular DNA molecule comprises one or more ligated synthetic fragments.

8. A method of reducing double-stranded RNA (dsRNA) formed in an in vitro transcription (IVT) reaction, comprising:(a) obtaining a circular DNA molecule comprising: a. an IVT expression cassette comprising a promoter, a gene of interest, and a poly-A tail, b. a protelomerase (teIRL) sequence, and c. a restriction endonuclease (RE) recognition site, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less;(b) treating the circular DNA molecule with a protelomerase to obtain a treated composition;(c) contacting the treated composition with an exonuclease to obtain a cleaned composition;(d) purifying the cleaned composition to obtain a first purified composition;(e) contacting the first purified composition with an endonuclease to obtain a digested composition;(f) purifying the digested composition to obtain a second purified composition; and(g) performing an in vitro transcription reaction to obtain an mRNA molecule, thereby reducing dsRNA formed in the IVT reaction.

9. The method of claim 8, wherein purifying the cleaned composition comprises a purification selected from the group consisting of proteinase K digestion, ultra- filtration / diafiltration (LIF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.

10. The method of any one of claims 8 or 9, wherein purifying the digested composition comprises a purification selected from the group consisting of ultra-filtration / diafiltration (LIF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.

11. The method of any one of claims 8 to 10, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less.

12. The method of any one of claims 8 to 11, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less.

13. The method of any one of claims 8 to 12, wherein the endonuclease comprises a type IIS restriction endonuclease.

14. The method of any one of claims 8 to 13, wherein the endonuclease is selected from the group consisting of Lgul, Eam1104l, and BspQI.

15. The method of any one of claims 8 to 14, further comprising:(h) capturing the mRNA molecule using an oligo dT affinity ligand immobilized to a solid support.

16. The method of claim 15, wherein the solid support is selected from the group consisting of a chromatography resin and a magnetic bead.

17. The method of any one of claims 8 to 16, wherein treating the circular DNA molecule with a protelomerase occurs in the absence of a surfactant.

18. The method of any one of claims 8 to 17, wherein treating the circular DNA molecule with a protelomerase occurs in the absence of Triton X-100.

19. The method of any one of claims 8 to 18, wherein a level of dsRNA formed in the IVT reaction is reduced as compared to a level of dsRNA formed in a standard IVT reaction, wherein the standard IVT reaction is performed with a circular DNA molecule comprising a teIRL sequence downstream of an RE recognition site and separated from the RE recognition site by 301 nucleotides or more.

20. The method of claim 19, wherein the level of dsRNA formed is measured by dot blot.

21. The method of any one of claims 19 or 20, wherein the level of dsRNA formed in the IVT reaction is reduced by at least two-fold.

22. The method of any one of claims 8 to 21 , wherein the circular DNA molecule comprises one or more ligated synthetic fragments.

23. A method of producing an mRNA molecule comprising:(a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, wherein the circular double-stranded DNA template comprises a teIRL sequence, b. a primer or primase, c. a deoxyribonucleotide triphosphate (dNTP), d. a pyrophosphate, and e. a phi29 DNA polymerase;(b) incubating the composition for a time period between about 20 and 30 hours at a temperature between about 30 and 45 °C to obtain an incubated composition;(c) treating the incubated composition with a protelomerase to obtain a treated composition;(d) contacting the treated composition with an exonuclease to obtain a cleaned composition;(e) purifying the cleaned composition to obtain a first purified composition;(f) contacting the first purified composition with a restriction endonuclease to obtain a digested composition;(g) purifying the digested composition to obtain a second purified composition; and(h) performing an in vitro transcription reaction, thereby producing the mRNA molecule.

24. The method of claim 23, wherein steps (a) to (d) occur in the same reaction vessel.

25. The method of any one of claims 23 or 24, wherein the method does not comprise a heat denaturation reaction prior to incubating the reaction.

26. The method of any one of claims 23 to 25, wherein the protelomerase is immobilized to a substrate.

27. The method of claim 26, wherein the substrate is a N-hydroxy succinimide agarose bead.

28. The method of any one of claims 23 to 27, wherein purifying the cleaned composition comprises a purification selected from the group consisting of proteinase K digestion, ultra-filtration / diafiltration (LIF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.

29. The method of any one of claims 23 to 28, wherein purifying the digested composition comprises a purification selected from the group consisting of ultra-filtration / diafiltration (LIF / DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.

30. The method of any one of claims 23 to 29, further comprising:(i) capturing the mRNA molecule using an oligo dT affinity ligand immobilized to a solid support.

31. The method of claim 30, wherein the solid support is selected from the group consisting of a chromatography resin and a magnetic bead.

32. The method of any one of claims 23 to 31 , wherein the phi29 DNA polymerase comprises a mutation resulting in increased thermostability and processivity.

33. The method of any one of claims 23 to 32, further comprising repeating (a) and (b) one or more times.

34. The method of any one of claims 23 to 33, wherein steps (a) - (h) are repeated using the second purified composition from step (g) as the circular double-stranded DNA template in step (a).

35. The method of any one of claims 23 to 34, wherein treating the incubated composition with a protelomerase occurs in the absence of a surfactant.

36. The method of any one of claims 23 to 35, wherein treating the circular DNA molecule with a protelomerase occurs in the absence of a surfactant Triton X-100.

37. The method of any one of claims 23 to 36, wherein the circular DNA molecule comprises one or more ligated synthetic fragments.

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