DNA plasmids

Abstract

Described herein are plasmid backbones and constructs wherein non-essential nucleotide sequences have been substantially removed while still supporting robust manufacturing processes. These plasmids and constructs are particularly useful in synthesis of RNA molecules via in vitro transcription (IVT).

Description

[0001] PC073234A

[0002] DNA PLASMIDS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 753,756, filed February 4, 2025, the disclosure of which is hereby incorporated by reference in its entirety.

[0005] REFERENCE TO SEQUENCE LISTING

[0006] The instant application contains a sequence listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml file, named “PC073234A_SEQListing_ST26.xml”, was created on January 27, 2026, and is 18 KB in size

[0007] FIELD

[0008] The present compositions and methods relate to improved DNA plasmid backbones useful as vectors supporting propagation in bacteria for the purpose of producing template for synthesis of RNA molecules via in vitro transcription (IVT).

[0009] BACKGROUND

[0010] Plasmids are an essential element in biotechnology. Plasmids are DNA molecules that can be introduced or transfected into bacterial cells and will replicate autonomously in the cell. Plasmids allow for the amplification of cloned DNA present in the plasmid. High quality plasmids are especially useful as the template for in vitro transcription (IVT). IVT can be used to manufacture mRNA for vaccines and therapeutic applications. T7 RNA polymerase (T7 RNAP), a DNA-dependent RNA polymerase from the T7 bacteriophage, is often used in IVT to manufacture target mRNA. In addition to T7 RNAP, an IVT reaction often includes each of the four NTPs, a linear DNA template (often a linear plasmid DNA template), a pyrophosphatase enzyme and a buffer system.

[0011] SUMMARY OF THE INVENTION

[0012] In some embodiments, disclosed herein are DNA molecules comprising a nucleic acid sequence having at least 85% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the nucleic acid sequence has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs:1-5. In some embodiments, the nucleic acid sequence has at least 90% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the nucleic acid sequence has at least 95% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the nucleic acid sequence has at least 98% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs: 1-5. In some embodiments, disclosed herein are host cells comprising a DNA molecule comprising a nucleic acid sequence having at least 85, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs:1-5. In some embodiments, the DNA molecules disclosed herein further comprise an expression cassette. In some embodiments, the expression cassette comprises a transcription start site, a 5’ UTR, a gene of interest, a 3’ UTR, and a poly(A) tail. In some embodiments, the gene of interest comprises a coding region for an antigen derived from a pathogen associated with an infectious disease. In some embodiments, disclosed herein are methods of transfecting a cell comprising contacting a cell with a DNA molecule comprising a nucleic acid sequence having at least 85, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1-5 in vitro, such that the DNA molecule enters the cell.

[0013] DETAILED DESCRIPTION RNA therapeutics and vaccines have tremendous potential to treat and prevent diseases. To achieve that potential, high-quality RNA for vaccine or therapeutic purposes is needed. In vitro transcription (IVT) can be carried out to synthesize RNA from a DNA template using a variety of natural or engineered RNA polymerases including SP6, T7 or T3 RNA polymerases.

[0014] Disclosed herein are DNA molecules comprising a nucleic acid sequence having at least 70% identity to any one of SEQ ID NOs:1-5. In some embodiments, the nucleic acid sequence has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 1-5. In some embodiments, the nucleic acid sequence comprises any one of SEQ ID NOs:1-5. In some embodiments, disclosed herein are host cells comprising a DNA molecule comprising a nucleic acid sequence having at least 85% identity to any one of SEQ ID NOs:1-5. In some embodiments, disclosed herein are DNA molecules comprising a nucleic acid sequence having at least 70% identity to any one of SEQ ID NOs:1-5, wherein the DNA molecule further comprises a gene of interest. In some embodiments, the gene of interest comprises a coding region for an antigen derived from a pathogen associated with infection disease. In some embodiments, disclosed herein are methods of transfecting a cell comprising contacting a cell with a DNA molecule comprising a nucleic acid sequence having at least 70% identity to any one of SEQ ID NOs: 1-5 in vitro, such that the DNA molecule enters the cell.

[0015] Certain Definitions

[0016] 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 orstandard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some aspects, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the measurement or quantitation.

[0017] 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.”

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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, theappearances 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.

[0022] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result. The terms “improve,” “promote,” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.

[0023] 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.

[0024] 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.

[0025] 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-LITR), 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), guide RNA (gRNA), and modified RNA (modRNA).

[0026] The term “RNA drug substance,” as used herein, means a purified RNA that is solubilized in any form of aqueous solution appropriate to permit subsequent encapsulation of the RNA withinencapsulating agents as described below.

[0027] The term “drug substance RNA,” as used herein, means the RNA component of the RNA drug substance.

[0028] The term “RNA drug product,” as used herein, means a purified RNA that has been encapsulated in any form of encapsulating agents (e.g., lipid nanoparticles) described herein and forms a colloidal dispersion (e.g., RNA-loaded LNP dispersion) and where the resulting colloidal dispersion has been adjusted and purified to stabilize the encapsulated RNA.

[0029] 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. In some aspects, the RNA is administered to edit, repair, restore the function of, or reduce the level of a gene or protein in a mammal, such as a human.

[0030] 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 N1-methylpseudouridine in an RNA sequence. Other 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). In some aspects, the RNA can be a guide RNA (gRNA) or circular RNA.

[0031] 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 endogenousversion 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.

[0032] 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.

[0033] 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.

[0034] DNA Template

[0035] In some aspects, the DNA templates described herein include a sequence coding for a gene of interest that encodes, e.g., a peptide or polypeptide of interest. In some aspects, the DNAtemplate 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 DNA 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 DNA template has a minimal plasmid backbone. In some aspects, a DNA template lacks a plasmid backbone.

[0036] In some aspects, the DNA template is a linearized plasmid DNA used as the template for in vitro transcription. In some aspects, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH10B cells, are transfected with the plasmid DNA template. The transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some aspects, the linear DNA template is synthesized in a cell-free environment. In some aspects, the linear DNA template is synthesized by rolling circle amplification (RCA). 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 DNA template is provided by exponential RCA, including hyperbranched RCA (also termed ramification amplification).

[0037] In other aspects, the DNA template is provided by a cell-free process for synthesizing DNA that includes contacting a DNA template with at least one polymerase in the presence of nucleotides to form a reaction mixture, wherein the DNA template is amplified by strand displacement replication, and wherein further nucleotides are supplied to the reaction mixture continuously or at intervals during the process. In some aspects the nucleotides can include an aminoallyl-dNTP (e.g. aminoallyl-dllTP) so that nucleophilic primary amines are inserted into the amplified DNA to enable subsequent covalent immobilization of the resulting linear DNA template. In some aspects, the nucleotides can include other functional groups (including, but not limited to amines, carboxyls, sulfhydryls, aldehydes, hydroxyls, azides, epoxides, or biotin) within their structure to facilitate covalent immobilization, adsorption, entrapment, or co-polymerization of the resulting DNA template on or within a substrate.

[0038] In some aspects, the DNA template also includes an RNA polymerase promoter sequence, e.g., a T7 promoter, located 5' to and operably linked to the 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. In some preferred aspects, the DNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5' toand operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a 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.

[0039] 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 DNA 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.

[0040] In some aspects, the vectors described herein may incorporate a nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors. In some aspects, the vector is a DNA molecule. In some aspects, the vector is a plasmid vector. In some aspects, the vector is a viral vector. Typically, an expression vector will contain a desired coding sequence and appropriate other sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired fragment (typically a DNA fragment) and may lack functional sequences needed for expression of the desired fragment(s). The DNA molecules described herein may be useful in molecular cloning, including molecular cloning techniques such as restriction enzyme cloning, polymerase chain reaction cloning, sticky end ligation, cell-free cloning, gBIocks fragment assembly (IDT), BioBrick assembly (NEW ENGLAND BIOLABS), site-directed mutagenesis, sequence and ligase independent cloning (SLIC), circular polymerase extension cloning (CPEC), seamless ligation cloning extract (SLICE), topoisomerase mediated ligation, homologous recombination, Gateway cloning, In-Fusion cloning (TAKARA), Golden Gate Assembly, Gibson Assembly (NEW ENGLAND BIOLABS), NEBuilder HiFi DNA Assembly (NEW ENGLAND BIOLABS), GeneArt Seamless Cloning and Assembly (THERMO FISHER), and combinations thereof.

[0041] Flanking Regions: Untranslated Regions (UTRs)

[0042] In some aspects, the DNA templates described herein include an RNA polymerase promotersequence 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 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 aspects, the RNA molecule is synthesized from a sample having a linear DNA template, the DNA 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.

[0043] The DNA 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 sites. In some aspects, the DNA templates described herein comprise a 5’ UTR and / or a 3’ UTR as described in International Patent Application Publication No. WO 2024 / 154061, which is herein incorporated by reference for purposes of the untranslated regions disclosed therein.

[0044] Natural 5' UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5' UTR also have been known to form secondary structures which are involved in elongation factor binding. By engineering the features typically found in abundantly expressed genes of specific target organs, one may enhance the stability and protein production of the polynucleotides, primary constructs. For example, use of 5' UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), etc.

[0045] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTRs. For example, introns or portions of introns sequences may be incorporated into the flanking regions of the polynucleotides, primary constructs or mRNA described here. Incorporation of intronic sequences may increase protein production as well as mRNA levels. Cap-dependent translation involves recruitment of the pre-initiation complex (PIC) to the 5' end of an mRNA followed by scanning to find an AUG initiation codon in an optimum sequence context. AUG recognition promotes scanning cessation, release of most initiation factors, and recruitment of the large ribosomalsubunit to initiate elongation. Efficient recognition of an initiation codon depends on its surrounding sequence. In some aspects, the sequence CRCCaugG (R = purine, A or G) may provide optimal context for AUG recognition in eukaryotes.

[0046] 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) may be separated into three classes: Class I AREs include several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD include class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules including this type of AREs include GM-CSF and TNF-alpha. Class III ARES are less well defined. These U rich regions do not include an AUUUA motif c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the EI_AV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3' UTR AREs may be used to modulate the stability of polynucleotides, and primary constructs.

[0047] When engineering specific polynucleotides, and / or primary constructs, one or more copies of an ARE may be introduced to make polynucleotides, and / or primary constructs less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs may be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments may be conducted in relevant cell lines, using polynucleotides, and / or primary constructs and protein production may be assayed at various time points post-transfection. For example, cells may be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

[0048] Poly(A) tail

[0049] In some aspects, the DNA templates disclosed herein comprise 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 DNA 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 some aspects, the poly(A) tail is interrupted by a sequence of the four nucleotides (dA, dC, dG, and dT), and the sequence may be 1 to 2, 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. In some aspects, the poly(A) tail is interrupted by one or more sequences of the four nucleotides, such as a poly(A) tail comprising five or more stretches of 40 adenine nucleotides interrupted by one or two guanine nucleotides. Poly(A) tails containing such interruptions are described in WO 2016 / 005324, hereby incorporated by reference. In preferred aspects, the DNA template includes a poly(A) tail that includes about 40 adenines. In preferred aspects, the DNA template includes a poly(A) tail that includes about 80 adenines. In some aspects, the poly(A) tail is encoded in the DNA 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.

[0050] In some aspects, immediately downstream of the poly(A) tail coding sequence on the plasmid DNA template is a recognition site for a restriction endonuclease to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional readthrough.

[0051] In some aspects, following linearization, the plasmid DNA template is filtered into an appropriate solvent, e.g., water, HEPES, 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.

[0052] The linear DNA template may be purified before use as a template for in vitro transcription. For example, the linear DNA template may be purified chromatographically or by ethanol or isopropanol precipitation.

[0053] In vitro Transcription

[0054] In vitro transcription (IVT) (whether fed-batch, continuous-flow, or otherwise) 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 DNA 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.).

[0055] The methods described herein for synthesis of an RNA molecule, e.g., mRNA, include contacting a DNA 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, nucleoside 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 andunnatural (modified, such as, for example, N1-methylpseudouridine-5’-triphosphate) NTPs. RNA Polymerase

[0056] 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, self-amplifying RNA, or guide RNA from a DNA 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.

[0057] In some aspects, the RNA polymerase includes an engineered T7 RNA polymerase variant, such as a variant that allows for selective incorporation of a 5’ cap analog (e.g. m7G(5')ppp(5')m7G, m7G(5’)ppp(5’)(2’OMeA)pG, or m7G(5')ppp(5')(2'OMeA)pll cap analogs) 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. Exemplary 5’ cap analogs include, but are not limited to, m7G(5')ppp(5')m7G, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')G, G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')A, m7G(5’)ppp(5’)(2’OMeA)pG, m7(3’OMeG)(5’)ppp(5’)(2’OMeA)pG, m7G(5')ppp(5')(2'OMeA)pll, or m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG. Cap molecules or 5’ cap analogs 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.

[0058] Ribonucleotides

[0059] In the methods described herein, the IVT reaction system includes nucleotides (for example, nonmodified ribonucleoside triphosphates or modified ribonucleoside 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, N1-methylpseudouridine-5’-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.

[0060] 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); m3llm (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)-alkynyl uracil, 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 N1-methylpseudouridine; pseudouridine, N6-methyladenosine, 5-methylcytidine, and 5-methyluridine.

[0061] In some aspects, the RNA molecule may include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0062] 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 35' ribonucleotides may be methylated at the 2' position of the ribose.

[0063] Exemplary In Vitro Transcription Reaction Systems

[0064] In some aspects, the DNA molecules described herein are for use in an in vitro transcription (IVT) reaction system, wherein the IVT reaction system includes the following: an RNA polymerase, e.g., a T7 RNA polymerase, DNA template; nucleoside triphosphates (NTPs); magnesium; and a buffer such as, e.g., HEPES or Tris (or both HEPES and Tris). In some aspects, the in vitro transcription reaction system does not include RNA polymerase. In some aspects, the in vitro transcription reaction system does not include DNA template. In some aspects, the in vitro transcription reaction system includes neither RNA polymerase nor DNA template. In some aspects, the RNA polymerase is stationary. In some aspects, the DNA template is stationary. In some aspects, both RNA polymerase and DNA template are stationary. In some aspects, the in vitro transcription reaction system is flowing across a substrate containing stationary RNA polymerase and / or DNA template with a residence time within the substrate of between 1 secondand 3 hours at a temperature between about 20 °C and 50 °C. In some aspects, the total time that the in vitro transcription reaction system is flowing across a substrate, i.e., the total amount of time the in vitro transcription reaction proceeds, is between 1 second and 365 days. In some aspects, the total amount of time the in vitro transcription reaction proceeds is more than 365 days.

[0065] In some aspects, the in vitro transcription reaction system includes the RNA polymerase, e.g., a 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 U / rnL, 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, or 15U / 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 U / rnL. 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 U / rnL. 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 U / rnL. 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 U / rnL. 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 U / rnL. In some aspects, the in vitro transcription reaction system includesan RNA polymerase, e.g., a T7 RNA polymerase, at a final concentration of 40000 U / rnL.

[0066] In some aspects, the in vitro transcription reaction system includes the DNA 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, 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, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 40 nM. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 144 nM. In some aspects, the in vitro transcription reaction system includes the DNA 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, or24 nM, or any range or value derivable therein. In some aspects, the in vitro transcription reaction system includes the DNA 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 DNA 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 DNA template at a final concentration of 0.025 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.05 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.075 mg / mL. In some aspects, the in vitro transcription reaction system includes the DNA template at a final concentration of 0.1 mg / mL. In some aspects, the in vitro transcription reaction system includes each nucleoside 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 eachnucleoside 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 nucleoside triphosphates (NTPs) at a final concentration of about 8 mM each. In some aspects, the in vitro transcription reaction system includes the nucleoside 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 nucleoside 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 nucleoside 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 nucleoside 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.

[0067] In some aspects, the in vitro transcription reaction system includes a 5’ cap analog. Exemplary 5’ cap analogs include, but are not limited to, m7G(5')ppp(5')m7G, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')G, G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')A, m7G(5’)ppp(5’)(2’OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, or m7(3’OMeG)(5’)ppp(5’)m6(2’OMeA)pG. In some aspects, the in vitro transcription reaction system includes a 5’ cap analog 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 a 5’ cap analog 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 a 5’ cap analog at a final concentration of about 4 mM. In certain aspects the concentration of 5’ cap analog 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.

[0068] In some aspects, the IVT reaction system includes magnesium ion, for example, as a magnesium salt, such as any one of magnesium sulfate, 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, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 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, or2.2 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

[0069] In some aspects, the in vitro transcription (IVT) reaction system includes a buffer. Exemplary buffers for the in vitro transcription 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, or8.5, 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. In some aspects, the in vitro transcription reaction system includes 40 mM Tris HCI, pH 7.0. In some aspects, the in vitrotranscription reaction system includes PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) pH 7.0, HEPPSO (2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid), orMES (2-(N-morpholino)ethanesulfonic acid). 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 PIPES. In some aspects, the in vitro transcription reaction system includes PIPES and Tris.

[0070] 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. 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 2mll / 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. In some aspects, the in vitro transcription reaction system includes a polyamine. Exemplary polyamines include spermine, putrescene, and spermidine. In some aspects, 0.2 mM spermidine is included. 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 0.53 mM spermidine is included. In some aspects, 10 mM spermidine is included.

[0071] 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 selectedfrom 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 includes 25 mM DTT. In some aspects, the IVT reaction system includes a monovalent salt. In some aspects, the monovalent salt is selected from the group consisting of KCI, NaCI, RbCI, CsCI, NaCICU, NaF, NaBr, and NH4CI. In some aspects, the monovalent salt is present in the IVT reaction system in an amount between about 10 mM and about 150 mM. In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM potassium chloride (KCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM sodium chloride (NaCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM cesium chloride (CsCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM rubidium chloride (RbCI). In some aspects, the IVT reaction system includes about 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mM ammonium chloride (NH4CI).

[0072] In some aspects, the IVT reaction system includes dimethylsulfoxide (DMSO). In some aspects, the DMSO is present in the IVT reaction system in an amount between about 4% and about 15%. In some aspects, the IVT reaction system includes about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% DMSO. In some aspects, the IVT reaction system includes 5% DMSO. In some aspects, the IVT reaction system includes 10% DMSO.

[0073] In some aspects, the IVT reaction system includes dihydrolevoglucosenone (CYRENE™). In some aspects, the dihydrolevoglucosenone is present in the IVT reaction system in an amount between about 4% and about 15%. In some aspects, the IVT reaction system includes about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% dihydrolevoglucosenone. In some aspects, the IVT reaction system includes 5% dihydrolevoglucosenone. In some aspects, the IVT reaction system includes 10% dihydrolevoglucosenone.

[0074] 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.

[0075] 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.

[0076] In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for about 12 days, e.g., at least, at most, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days, or any range or value derivable therein. In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for about 365 days, e.g., at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 365 days or any range or value derivable therein. In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for any number of days less than 0.1 days. In some aspects, the in vitro transcription reaction proceeds, for example, at about 37°C for any number of days greater than 365 days. In some aspects, the in vitro transcription reaction proceeds, for example, at less than 50°C for less than 365 days, 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, or 365 days or any range or value derivable therein.

[0077] In some aspects, the IVT reactions described herein result 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, thetotal 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.

[0078] In some aspects, the IVT reactions described herein result in high productivity of RNA. Productivity in the case of in vitro transcription refers to the amount of RNA produced per volume of reactor per unit time. In some aspects, productivity of the IVT reaction may be at least 0.1 mg / mL / h to about 1000 mg / mL / h, where volume refers to the reaction volume. For example, in some aspects, the productivity of the IVT reaction may be at least, at most, or about 0.1 mg / mL / h, 0.2 mg / mL / h, 0.3 mg / mL / h, 0.4 mg / mL / h, 0.5 mg / mL / h, 0.6 mg / mL / h, 0.7 mg / mL / h, 0.8 mg / mL / h, 0.9 mg / mL / h, 1.0 mg / mL / h, 2 mg / mL / h, 3 mg / mL / h, 4 mg / mL / h, 5 mg / mL / h, 6 mg / mL / h, 7 mg / mL / h, 8 mg / mL / h, 9 mg / mL / h, 10 mg / mL / h, 11 mg / mL / h, 12 mg / mL / h, 13 mg / mL / h, 14 mg / mL / h, 15 mg / mL / h, 16 mg / mL / h, 17 mg / mL / h, 18 mg / mL / h, 19 mg / mL / h, 20 mg / mL / h, 25 mg / mL / h, 30 mg / mL / h, 35 mg / mL / h, 40 mg / mL / h, 45 mg / mL / h, 50 mg / mL / h, 55 mg / mL / h, 60 mg / mL / h, 65 mg / mL / h, 70 mg / mL / h, 75 mg / mL / h, 80 mg / mL / h, 85 mg / mL / h, 90 mg / mL / h, 95 mg / mL / h, 100 mg / mL / h, 150 mg / mL / h, 200 mg / mL / h, 250 mg / mL / h, 300 mg / mL / h, 350 mg / mL / h, 400 mg / mL / h, 450 mg / mL / h, 500 mg / mL / h, 600 mg / mL / h, 700 mg / mL / h, 800 mg / mL / h, 900 mg / mL / h, or 1000 mg / mL / h, or any range or value derivable therein, where volume refers to the reaction volume. In some aspects, the productivity of the IVT reaction may be any value less than 0.1 mg / mL / h, where volume refers to the reaction volume. In some aspects, the productivity of the IVT reaction may be any value greater than 1000mg / mL / h, where volume refers to the reaction volume. In some preferred aspects, the productivity of the IVT reaction producing RNA having at least 90% of the intended full length transcript is at least 30 mg / mL / h, where volume refers to the reaction volume.

[0079] In some aspects, the IVT reactions described herein are carried out in a reaction volume under specified conditions. In some aspects, the reaction volume ranges from at least 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, or 200 liters or more, or any volume in between. In some aspects, the volume of the IVT reaction is at least 10L, 30L or 50L. In some aspects, the volume of the IVT reaction is at least 0.01 L. In some aspects, the volume of the IVT reaction is at least 0.08L. In some aspects, the volume of the IVT reaction is at least 0.1L. In some aspects, the volume of the IVT reaction is at least 100L.

[0080] In some aspects, following an IVT reaction using a DNA template and an RNA polymerase as described here, a first composition that includes an uncapped RNA molecule is produced. Insome 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.

[0081] As used herein, an “RNA molecule” produced by in vitro transcription may be referred to as an “RNA transcript” or an “in vitro transcribed RNA.” An “RNA molecule,” an “RNA transcript,” or “in vitro transcribed RNA” may encompass any one of modified mRNA “modRNA,” unmodified mRNA, self-amplifying RNA (saRNA), and guide RNA (gRNA).

[0082] Capping of RNA Molecule

[0083] In some aspects, the methods 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 (e.g., vaccinia capping enzyme or faustovirus capping enzyme), s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and 2-0-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 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.

[0084] 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'-0-Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '-O] N), which may further increase translation efficacy.

[0085] In some aspects, the RNA molecule may be enzymatically capped at the 5' end using Vaccinia or Faustovirus guanylyltransferase, guanosine triphosphate and S-adenosyl-L-methionine to yield cap 0 structure. An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge. Alternatively, use of a 2'-0-methyltransferase with Vaccinia or Faustovirus guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2'-0H group is methylated on the first transcribed nucleotide. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. In some aspects, RNase inhibitor is not included in the enzymatic capping reaction. In another aspect, the enzymatic capping reaction step is performed under constant mixing. In another aspect, the RNA molecule is not co-transcriptionallycapped.

[0086] Non-limiting examples of 5' cap structures are those which, among other things, have enhanced binding of cap binding polypeptides, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5' de-capping, as compared to synthetic 5' cap structures known in the art (or to a wild-type, natural or physiological 5' cap structure). For example, recombinant Vaccinia Virus or Faustovirus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme may create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine includes an N7 methylation and the 5'-terminal nucleotide of the mRNA includes a 2'-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5' cap analog structures known in the art. Cap structures include, but are not limited to, m7G(5')ppp(5')N (cap 0) and m7G(5')ppp(5')Nm (cap 1). Cap 0 is a N7-methyl guanosine connected to the 5' nucleotide through a 5' to 5' triphosphate linkage, typically referred to as m7G cap or m7Gppp or m7G(5')ppp(5')N. In the cell, the cap 0 structure is essential for efficient translation of the mRNA that carries the cap. An additional methylation on the 2-0 position of the initiating nucleotide generates Cap 1, sometimes referred to as m7GpppNm- or m7G(5')ppp(5')Nm, wherein Nm denotes any nucleotide with a 2'-0 methylation. In some aspects, the 5' terminal cap includes a cap analog. Exemplary 5’ cap analogs include, but are not limited to, m7G(5')ppp(5')m7G, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')G, G(5')ppp(5')G, m7G(5')ppp(5')A, G(5')ppp(5')A, m7G(5’)ppp(5’)(2’OmeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7G(5’)ppp(5’)(2’OmeA)pll, or m7(3’OMeG)(5’)ppp(5’)m6(2’OmeA)pG. In some aspects, a 5' terminal cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0087] Genes of Interest

[0088] The DNA template and resulting RNA molecules described herein may comprise 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 a 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.

[0089] 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 antigensderived 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), 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.

[0090] In some aspects, the DNA molecules described herein encode RNA molecules that encode a viral polypeptide or fragment thereof, including naturally occurring or engineered variants thereof, for prophylaxis against a virus in humans.

[0091] Entrapment of RNA in Nanoparticles and Processing to Drug Product

[0092] In some aspects, the DNA molecules described herein are in vitro transcribed to create RNA molecules that may be encapsulated to form colloidal dispersions (e.g. RNA-loaded LNP dispersion) comprising at least one encapsulating agent. In one aspect, the encapsulating agent comprises one or more lipids, a lipid nanoparticle (LNP), lipoplexes, one or more polymers, polymeric particles, polyplexes, monolithic delivery systems, or a combination thereof. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing elements may be excluded as an encapsulating agent.

[0093] In one aspect, the encapsulating agent is a lipid, and produced is an RNA-loaded LNP dispersion. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and / or the cationic polymer combine together with the nucleic acid to form colloidally stable dispersions.

[0094] A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. As encapsulating agent, a lipid is a substance that is insoluble or partially insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids and are encompassed by the compositions and methods of the present disclosure. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently.

[0095] In some aspects, LNPs may be designed to protect RNA molecules with unmodified and / or modified nitrogenous bases and various sizes (e.g., mRNA, modified mRNA [modRNA], saRNA,gRNA and / or circRNA) from extracellular RNases and / or may be engineered for systemic delivery of the RNA to target cells. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intravenously administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intramuscularly administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intradermally administered to a subject in need thereof. In some aspects, such LNPs may be particularly useful to deliver RNA molecules when RNA molecules are intranasally administered to a subject in need thereof.

[0096] In one aspect, the RNA in the RNA drug substance is at a concentration of < 1 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.05 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least or at least about 1 mg / mL. In another aspect, the RNA concentration is from or from about 0.05 mg / mL to about 0.5 mg / mL. In another aspect, the RNA is at a concentration of at least 10 mg / mL. In another aspect, the RNA is at a concentration of at least 50 mg / mL. In some aspects, the RNA is or is not at a concentration of at least, at most, exactly, between (inclusive or exclusive) any two of, or about 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, or more.

[0097] The present disclosure provides for DNA molecules encoding RNA molecules for use in an RNA drug substance and a lipid preparation mixture or compositions thereof comprising at least one RNA encoding, e.g., an antigen complexed with, encapsulated in, and / or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes and / or nanoliposomes. In some aspects, the composition comprises a lipid nanoparticle.

[0098] A lipid nanoparticle or LNP refers to particles of any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g., in an aqueous environment and / or in the presence of RNA. In some aspects, lipid nanoparticles are included in a formulation that may be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some aspects, the lipid nanoparticles of the present disclosure comprise a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids, or combinations thereof. In some aspects, the LNPs comprise at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and / or at least one polymer conjugated lipid (e.g., a polyethylene glycol (PEG)-modified lipid). In some aspects, 1, 2, 3, or more of the foregoing excipients may be excluded from the LNPs.

[0099] In some aspects, the LNPs comprise 20-60 mol% cationic (e.g., ionizable) lipid(s). For example,the LNPs may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise or do not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% cationic (e.g., ionizable) lipid(s). In some aspects, the LNPs comprise 45 to 55 mole percent (mol%) cationic (e.g., ionizable) lipid(s). For example, LNPs may comprise or not comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% cationic (e.g., ionizable) lipid(s).

[0100] In some aspects, the LNPs comprise 5-25 mol% neutral (e.g., non-cationic) lipid(s). For example, the LNPs may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% neutral (e.g., non-cationic) lipid(s). In some aspects, the LNPs comprise 5 to 15 mol% neutral (e.g., non-cationic) lipid(s). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% neutral (e.g., non-cationic) lipid(s).

[0101] In some aspects, the LNPs comprise 25-55 mol% structural lipid(s) (e.g., a steroid). For example, the LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, SO-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% structural lipid(s) (e.g., a steroid). In some aspects, the LNPs comprise 35 to 40 mol% structural lipid(s) (e.g., a steroid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 35, 36, 37, 38, 39, or 40 mol% structural lipid(s) (e.g., a steroid).

[0102] In some aspects, the LNPs comprise 0.5-15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). For example, the lipid nanoparticles (LNPs) may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol% polymer conjugated lipid(s) (e.g., a PEG-conjugated lipid). In some aspects, the LNPs are or are not at least, at most, exactly, or between (inclusive or exclusive) any two of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). In some aspects, the LNPs comprise 1 to 2 mol% polymer conjugated lipid(s) (e.g., a polyethylene glycol (PEG)-conjugated lipid). For example, LNPs may comprise at least, at most, exactly, or between (inclusive or exclusive) any two of 1, 1.5, or 2 mol% polymer conjugated lipid(s) (e.g., a PEG-conjugated lipid).

[0103] LNPs described herein can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety. For example, methods of preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term “colloid” as used herein relates to a type of mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal dispersion. Sometimes the term “colloid” refers only to the particles in the mixture and not the entire dispersion.

[0104] While methods for preparing a colloid containing an organic solvent are described herein, other methods having organic solvent-free characteristics may also be used according to the present disclosure.

[0105] In some aspects, an RNA-loaded LNP dispersion may be produced by inline mixing of an RNA solution or adjusted RNA solution described herein (e.g., an RNA drug substance) and a lipid preparation described herein (comprising, e.g., at least one cationic lipid and optionally one or more other lipid components, in an organic solvent) under conditions such that a sudden change in solubility of lipid component(s) is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some aspects, suitable buffering agents comprise tris, histidine, citrate, acetate, phosphate, and / or succinate. In some aspects, 1, 2, 3, or more of the foregoing buffering agents are excluded. The pH of a liquid formulation relates to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half a pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, properties of a cationic lipid are chosen such that nascent formation of particles occurs by association with an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which, for example, in some aspects, may result in greater encapsulation efficiency than is achieved in the absence of interactions between nucleic acids and at least one of the lipid components. In certain aspects, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

[0106] Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated byreference in their entirety for all purposes.

[0107] Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species, such as RNA species. In an LNP formulation, it is possible that each nucleic acid species is separately formulated as an individual LNP formulation. In that case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

[0108] The resulting dispersion following the formation of LNPs can be filtered as liquid feed in a single pass mode through a single pass tangential flow filtration (SPTFF) system and recovering the retentate (containing the LNPs) and permeate from the system in separate containers without recirculation through the SPTFF system, thereby filtering the liquid feed.

[0109] The following sequences are useful in the compositions and methods disclosed herein:

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[0120] Embodiments of the disclosure are further described in the following numbered embodiments: E1) A DNA molecule comprising a nucleic acid sequence having at least 85% identity to any one of SEQ ID NOs: 1-5.

[0121] E2) The DNA molecule of embodiment 1 , wherein the nucleic acid sequence has at least 90% identity to any one of SEQ ID NOs: 1-5.

[0122] E3) The DNA molecule of embodiment 1 , wherein the nucleic acid sequence has at least 95% identity to any one of SEQ ID NOs: 1-5.

[0123] E4) The DNA molecule of embodiment 1 , wherein the nucleic acid sequence has at least 98% identity to any one of SEQ ID NOs: 1-5.

[0124] E5) The DNA molecule of embodiment 1 , wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 1-5.

[0125] E6) A host cell comprising the DNA molecule of any one of embodiments 1-5.

[0126] E7) The DNA molecule of any one of embodiments 1-5, further comprising an expression cassette.

[0127] E8) The DNA molecule of embodiment 7, wherein the expression cassette comprises a transcription start site, a 5’ UTR, a gene of interest, a 3’ UTR, and a poly(A) tail.

[0128] E9) The DNA molecule of embodiment 8, wherein the gene of interest comprises a coding region for an antigen derived from a pathogen associated with an infectious disease.

[0129] E10) A method of transfecting a cell comprising contacting a cell with the DNA molecule of any one of embodiments 1-5 in vitro, such that the DNA molecule enters the cell.

[0130] EXAMPLES

[0131] 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.

[0132] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art.

[0133] EXAMPLE 1

[0134] PLASMID CONSTRUCTIONThe pBRD3 vector was constructed as a minimal plasmid entry vector (backbone) for use in RNA in vitro transcription (IVT) processes. The vector (lacking a gene of interest) was obtained and used to transform E. coli cells. The E. coli grew well, and analysis showed an intact plasmid with no sequence variants observed (and displaying good plasmid titer, plasmid fidelity, and plasmid topology). An mRNA cassette containing an RNA polymerase promoter, gene of interest (GOI), and a poly(A) tail was cloned into the pBRD3 entry vector and transformed into E. coli. Unfortunately, inappropriately low growth (no colonies) was observed after 16-20 hours of incubation, and troubleshooting efforts only led to very small colonies after more than 24 hours of incubation. These small colonies were used to inoculate liquid broth cultures, but again, little to no growth was observed even after 24 hours. After an extended incubation some cultures grew, but these contained mutations within the plasmid, frequent poly(A) tail truncations, and altered plasmid topology.

[0135] The pBRD3 plasmid was modified to improve performance. The kanamycin resistance cassette in pBRD3 was replaced with a stronger hybrid (NeoR / KanR) resistance marker, resulting in pBRD20. Introduction of multiple mRNA cassettes via standard cloning methods yielded robust bacterial growth both on agar plates and in broth cultures. Analysis of the plasmid DNA indicated optimal metrics (plasmid yield, topology, intact poly(A) tails, no sequence variants).

[0136] Following the success of PBRD20, smaller resistance cassettes were tested. pBRD20 was modified to obtain pBRD28, which included a strong kanamycin promoter and a modified kanamycin resistance cassette (containing three silent point mutations compared to bacterial kanR). Multiple mRNA cassettes were cloned into pBRD28 via standard methods and robust E. coli growth and high quality plasmid DNA was observed.

[0137] pBRD28 was then modified to remove the three silent point mutations and obtain pBRD40 / pMCT1. Multiple mRNA cassettes were cloned into pBRD40 / pMCT1 via standard methods and robust bacterial growth and plasmid DNA meeting internal quality metrics was observed. This plasmid DNA supported in vitro transcription (IVT) processes. pBRD40 / pMCT1 was then modified to generate pMCT2.1, producing a minimal plasmid by deleting approximately 350bp of additional sequence from two regions. pMCT2.1 was used as an entry vector to clone multiple mRNA cassettes using standard molecular biology techniques. This vector supported good E. coli growth on solid and liquid media and high quality plasmid DNA. This plasmid DNA supported IVT processes. The results are presented in Table 1.

[0138] Table 1: Plasmid construction

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[0149] 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 DNA molecule comprising a nucleic acid sequence having at least 85% identity to any one of SEQ ID NOs: 1-5.

2. The DNA molecule of claim 1 , wherein the nucleic acid sequence has at least 90% identity to any one of SEQ ID NOs: 1-5.

3. The DNA molecule of claim 1 , wherein the nucleic acid sequence has at least 95% identity to any one of SEQ ID NOs: 1-5.

4. The DNA molecule of claim 1 , wherein the nucleic acid sequence has at least 98% identity to any one of SEQ ID NOs: 1-5.

5. The DNA molecule of claim 1 , wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 1-5.

6. A host cell comprising the DNA molecule of any one of claims 1-5.

7. The DNA molecule of any one of claims 1-5, further comprising an expression cassette.

8. The DNA molecule of claim 7, wherein the expression cassette comprises a transcription start site, a 5’ UTR, a gene of interest, a 3’ UTR, and a poly(A) tail.

9. The DNA molecule of claim 8, wherein the gene of interest comprises a coding region for an antigen derived from a pathogen associated with an infectious disease.

10. A method of transfecting a cell comprising contacting a cell with the DNA molecule of any one of claims 1-5 in vitro, such that the DNA molecule enters the cell.

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