High purity RNA compositions and methods for preparation thereof

Abstract

To provide improved RNA compositions for use in therapeutic applications.SOLUTION: The RNA compositions are particularly suited for use in human therapeutic applications (e.g., in RNA therapeutics). The RNA compositions are made by improved processes, in particular, improved in vitro-transcription (IVT) processes. The invention also relates to methods for producing and purifying RNAs (e.g., therapeutic RNAs), and methods for using the RNA compositions and therapeutic applications thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 394,711, filed Sep. 14, 2016, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention The ability to design, synthesize and deliver nucleic acids, such as ribonucleic acid (RNA), e.g., messenger RNA (mRNA), within cells has led to advances in the fields of therapeutics, diagnostics, reagents and biological assays. Many advances have been made in the process of intracellular translation of nucleic acids and production of at least one encoded peptide or polypeptide of interest.

[0003] mRNA has great therapeutic potential in that mRNA therapeutics can express essentially any desired protein transiently while avoiding the adverse effects of viral and DNA-based nucleic acid delivery approaches. However, mammalian cells, particularly human cells, contain sensors of nucleic acids, including RNA, as part of the innate immune system, and it is desirable to avoid such sensing and immune responses when developing mRNA therapeutics.

[0004] Theoretically, mRNA produced by chemical synthesis holds promise as an mRNA therapeutic, but the majority of research in this important therapeutic area to date has focused on in vitro transcribed (IVT) mRNA, as this enzymatic process facilitates the generation of long RNAs, approximately 1-2 or more kB, the standard length for most mRNA molecules.

[0005] Early studies showed that incorporation of modified nucleosides, especially pseudouridine, reduced innate immune activation and increased mRNA translation, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remained (Kariko et al. (2005) Immunity 23(2):165-75). Progress was made toward identifying contaminants in nucleoside-modified IVT RNA, identifying double-stranded RNA (dsRNA) as at least partially responsible for innate immune activation. Removal of such contaminants by high performance liquid chromatography (HPLC) resulted in reduced IFN and proinflammatory cytokine levels and thus higher expression levels in primary cells (Kariko et al. (2011) Nuc. Acids Res. 39:e142). Notably, unmodified mRNA translated better after HPLC purification but still induced high levels of cytokine secretion.

[0006] WO2013 / 102203 describes an RNAse III treatment method used to remove dsRNA from IVT mRNA for repeated or continuous transfection into human or animal cells, in particular for reprogramming cells from one differentiation state to another. The method aims to result in a preparation with reduced levels of dsRNA and increased levels of intact ssRNA, as evidenced by high levels of reprogramming factors and lower toxicity to cells. However, such a method is not compatible for use in the preparation of mRNA for therapeutic applications, in particular for human therapeutic applications. RNAse III is known to digest ssRNA as well as dsRNA, and in attempting to remove dsRNA contaminants, the integrity of the desired ssRNA product is necessarily compromised. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2013 / 102203 issue [Non-patent literature]

[0008] [Non-Patent Document 1] Kariko et al. (2005) Immunity 23(2):165~75 [Non-Patent Document 2] Kariko et al.(2011)Nuc.Acids Res.39:e142 Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need to better understand the nature of contaminants in IVT-generated mRNA preparations in order to better control the levels and nature of contaminants in IVT preparations. Additionally, a need exists for improved methods for preparing mRNA for therapeutic use and for high purity compositions produced according to such methods. [Means for solving the problem]

[0010] The present invention comprises, at least in part, the discovery of a novel method for in vitro RNA synthesis and related products. The RNA transcripts produced by the methods described herein have enhanced properties that result in qualitatively and quantitatively superior compositions that contain the RNA transcripts. The RNA transcripts produced by the methods described herein have enhanced properties that are particularly important for mRNA, lncRNA, and other therapeutic and diagnostic RNA applications, such as improved immune silencing and better safety profiles.

[0011] In particular, the IVT RNA composition of the present invention is substantially free of certain undesirable contaminants that are routinely associated with the IVT process.In particular, however, the method of the present invention achieves an mRNA composition suitable for therapeutic use by controlling the nature and level of contaminants produced in the IVT reaction, i.e., contaminants are not produced in the initial reaction, in contrast to methods described in the art that attempt to remove the contaminants as they are produced.Without being bound by theory, it is believed that preventing the production of undesirable contaminants in the IVT reaction from the outset provides an improved composition with higher purity and potency, which can be measured, for example, in terms of increased translation from full-length intact mRNA in the composition.

[0012] A composition comprising an in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition being substantially free of reverse complement transcripts, is provided in some aspects of the present invention. In some embodiments, less than about 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.55% of the total mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 1.0% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.5% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.25% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.1% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.05% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.01% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.005% of the mass of the RNA in the composition is a reverse complement transcript. In some embodiments, less than about 0.001% of the mass of the RNA in the composition is a reverse complement transcript. In exemplary embodiments, the mass of the RNA in the composition is determined by LC, J2 Elisa, RNase III, gel electrophoresis with radiolabeled NTPs, LCMS+ / -nuclease or chemical digestion, NMR with labeled NTPs, chemically / isotopically / radioactively / etc. labeled NTPs, cells, biochemical means, RIG-I ATPase activity or MS or gel electrophoresis, or other methods known in the art suitable for detection and / or quantification of RNA in an RNA-containing composition.

[0013] In some embodiments, the reverse complement transcript is fully complementary to a region of an RNA that is a desired or intended IVT transcript (e.g., an mRNA, lncRNA, or other RNA greater than 50 nucleotides in length intended for therapeutic use). A product that is fully complementary to an RNA transcript would be considered to have 100% complementarity (e.g., over the length of the reverse complement transcript). In other embodiments, the reverse complement transcript is partially complementary to a region of the RNA transcript. In some embodiments, the reverse complement transcript is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a region of the RNA transcript. In still other embodiments, the reverse complement product is 70%-90%, 75%-90%, 80%-90%, 85%-90%, 90%-95%, 91%-95%, 92%-95%, 93%-95%, 94%-95%, 95%-99%, 96%-99%, 97%-99%, or 98%-99% complementary to a region of the RNA transcript.

[0014] Those skilled in the art will understand that unintended or undesired reverse complement transcripts generated in an IVT reaction may not only have complementarity to an RNA transcript that is the intended or desired product of the IVT reaction (e.g., an mRNA, IncRNA, or other RNA greater than 50 nucleotides in length intended for therapeutic use), but may also have complementarity to the strand of the DNA template from which the intended or desired RNA transcript is generated. Without wishing to be bound by theory, it is believed that the possibility exists that certain unintended or undesired transcripts contaminating the IVT RNA composition that are reduced or eliminated according to the novel processes of the present invention may be transcribed from an intended or desired RNA transcript, and that certain unintended or undesired transcripts contaminating the IVT RNA composition may be transcribed from the DNA template used in the IVT reaction. The latter assumption, while possible, does not appear to be supported by the data presented herein, which show that reverse complement transcripts are primarily complementary to the 5'UTR and / or polyA tail of the mRNA transcript, and that reverse complement transcripts complementary to portions of the DNA template (e.g., sequence elements) that are not present in the transcribed mRNA are significantly reduced, and in some cases undetectable.

[0015] In another aspect, the invention is a composition comprising an in vitro transcribed (IVT) RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, wherein the composition is substantially free of cytokine-induced RNA contaminants. In some embodiments, less than about 0.5% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, less than about 0.25% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, less than about 0.1% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, less than about 0.05% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, less than about 0.01% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, less than about 0.005% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, less than about 0.001% by mass of the RNA in the composition is a cytokine-induced RNA contaminant. In some embodiments, the mass of RNA in the composition is determined by LC or MS or gel electrophoresis, or other methods known in the art.

[0016] In some embodiments, the invention features a composition including an in vitro transcribed (IVT) RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, wherein the composition has reduced levels of cytokine-induced RNA contaminants and / or reverse complement transcripts.

[0017] In some embodiments of the compositions of the invention described herein, the total mass of cytokine-induced RNA contaminants and / or reverse complement transcripts is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.55%, 0.5%, 0.45%, 0.4%, 0.35%, 0.3%, 0.0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the total mass of RNA in the composition. In some embodiments, the ratio of cytokine-induced RNA contaminants to RNA transcripts (e.g., intended or desired RNA transcripts) in the composition is 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, or 1:99. In other embodiments, the ratio of reverse complement transcript to RNA transcript (e.g., intended or desired RNA transcript) in the composition is 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, or 1:99.

[0018] The size of the contaminants can vary. In some embodiments, the length of the cytokine-induced RNA contaminants and / or RNA transcripts is greater than 2 nucleotides up to the length of a full-length transcript (e.g., the intended or desired transcript, e.g., an mRNA transcript). In other embodiments, the length of the cytokine-induced RNA contaminants and / or RNA transcripts is greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, respectively, up to the length of a full-length transcript. In other embodiments, the length of the cytokine-induced RNA contaminants and / or RNA transcripts is 2-500 nucleotides, 10-500 nucleotides, 15-500 nucleotides, 20-500 nucleotides, 30-500 nucleotides, 40-500 nucleotides, 50-500 nucleotides, 100-500 nucleotides, 200-500 nucleotides, 300-500 nucleotides, 400-500 nucleotides, 2-200 nucleotides, 10-200 nucleotides, The length of the nucleic acid sequence may be 15-200 nucleotides, 20-200 nucleotides, 30-200 nucleotides, 40-200 nucleotides, 50-200 nucleotides, 100-200 nucleotides, 200-300 nucleotides, 300-400 nucleotides, 2-100 nucleotides, 10-100 nucleotides, 15-100 nucleotides, 20-100 nucleotides, 30-100 nucleotides, 40-100 nucleotides, or 50-100 nucleotides.

[0019] Those skilled in the art will appreciate that RNA contaminants of certain structures and / or lengths, e.g., RNA contaminants at least 15 or at least 20 or at least 25 nucleotides in length, particularly RNA contaminants that are double-stranded in nature (dsRNA), are highly likely to stimulate undesirable or unwanted immune responses. Removal of such contaminants is possible using certain art-recognized methods (e.g., enzymatic and / or purification processes or method steps). However, each such additional purification process or step in the production of, e.g., mRNA, lncRNA, or other RNAs greater than 50 nucleotides in length intended for therapeutic use, induces the possibility of reduced fidelity of the intended product, e.g., by subjecting the direct IVT reaction product to (1) enzymatic conditions (e.g., RNAse treatment to generate fragments of RNA) and / or (2) high temperature non-physiological solvent conditions (e.g., HPLC or RP chromatographic conditions) that may impair the quality of the RNA product in the process of degrading or removing the contaminant.

[0020] In certain aspects of the present invention, the undesired or undesired contaminant is, for example, a population of RNA species with a distribution of sizes and masses within a certain range.For example, a certain type of contaminant may have less than 5% of any individual species of contaminant (i.e., RNA species of the same sequence, same length, etc.).In other embodiments, the contaminant may have less than 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.001%, 0.0005%, or 0.0001% of any individual species.

[0021] In some embodiments, the reverse complement transcript is an RNA (dsRNA, ssRNA or ds-ssRNA hybrid with ds and ss portions) that includes a strand that includes a sequence that is the reverse complement of the IVT RNA or RNA (dsRNA, ssRNA or ds-ssRNA). In some embodiments where the polyA tail is encoded within a polyA:T tract in the DNA template, the reverse complement product includes a strand that includes a polyU sequence, or other methods commonly used in the art for inserting a polyA tail into RNA. The polyU sequence is, for example, pppU(U)n, where n is 1 or greater. In some embodiments, n is 1-100 (for an encoded polyA tail where the target RNA has a 100 nt polyA tail). In other embodiments, n is greater than 30 or 30-200. In an exemplary embodiment, the reverse complement product starts with a 5' triphosphate (5'-PPP). In other embodiments, the reverse complement product begins with a 5' diphosphate (5'-PP) or a 5' monophosphate (5'-P).

[0022] In some embodiments, the reverse complement transcript comprises the reverse complement of the 5' end of the IVT RNA and / or the reverse complement of the 3' end of the IVT RNA. In some embodiments, the reverse complement of the 5' end of the IVT RNA comprises a sequence complementary to all or a portion of the 5' UTR of the IVT RNA. In other embodiments, the reverse complement of the 3' end of the IVT RNA comprises a sequence complementary to all or a portion of the polyA tail of the IVT RNA. In yet other embodiments, the reverse complement of the 3' end is the reverse complement of a tailless RNA. In yet other embodiments, the reverse complement transcript comprises a sequence complementary to all or a portion of the 5' end, 3' end, open reading frame and / or polyA tail of the RNA, or any combination thereof.

[0023] In exemplary aspects of the invention, the cytokine-induced RNA contaminant is RNA (dsRNA, ssRNA or ds-ssRNA). In some embodiments, the cytokine-induced RNA contaminant is an IVT RNA that includes a strand containing a polyU sequence or a strand that includes a reverse sequence that is the reverse complement of the dsRNA or ssRNA in some embodiments.

[0024] In some embodiments, the strand that contains a sequence that is the reverse complement of the IVT RNA or that contains a polyU sequence starts with a 5' triphosphate (5'-PPP). In some embodiments, the polyU sequence is more than 20 nucleotides in length. In some embodiments, the polyU sequence is more than 30 nucleotides in length. In other embodiments, the polyU sequence is single-stranded. In yet other embodiments, the polyU sequence is double-stranded.

[0025] In some embodiments, the cytokine-induced RNA contaminant comprises a reverse complement of the 5' end of the IVT RNA and / or a reverse complement of the 3' end of the IVT RNA. In some embodiments, the reverse complement of the 5' end of the IVT RNA comprises a sequence complementary to all or a portion of the 5' UTR of the IVT RNA. In other embodiments, the reverse complement of the 3' end of the IVT RNA comprises a sequence complementary to all or a portion of the poly-A tail of the IVT RNA. In some embodiments, the reverse complement comprises a sequence complementary to the first 10-15 nucleotides of the 5' UTR. In some embodiments, the reverse complement comprises a sequence complementary to the first 10-20 nucleotides of the 5' UTR. In some embodiments, the reverse complement comprises a sequence complementary to the first 10-30 nucleotides of the 5' UTR. In some embodiments, the reverse complement comprises a sequence complementary to the first 10-40 nucleotides of the 5' UTR. In yet other embodiments, the cytokine-induced RNA contaminants include sequences complementary to all or part of the 5' end, 3' end, open reading frame and / or polyA tail of an RNA, or any combination thereof.

[0026] In some embodiments, the cytokine-induced RNA contaminants are single-stranded triphosphate reverse complements of 20 nucleotides or more. In other embodiments, the cytokine-induced RNA contaminants are single-stranded triphosphate reverse complements of 25 nucleotides or more. In other embodiments, the cytokine-induced RNA contaminants are single-stranded triphosphate reverse complements of 30 nucleotides or more. In some embodiments, the single-stranded triphosphate reverse complements are 20-200 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complements are 20-100 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complements are 20-50 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complements are 25-200 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complements are 25-100 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complements are 25-50 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complements are 30-200 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complement is between 30 and 100 nucleotides in length. In some embodiments, the single-stranded triphosphate reverse complement is between 30 and 50 nucleotides in length.

[0027] In other embodiments, the cytokine-induced RNA contaminants are single-stranded reverse complements having terminal triphosphate-A, triphosphate-C, or triphosphate-U.

[0028] In other embodiments, the cytokine-induced RNA contaminants are double-stranded triphosphate reverse complements of 20 or more nucleotides. In some embodiments, the double-stranded triphosphate reverse complements have between 20 and 200 nucleotides. In still other embodiments, the cytokine-induced RNA contaminants are double-stranded triphosphate reverse complements that are completely double-stranded (no single-stranded regions). In other embodiments, the cytokine-induced RNA contaminants are double-stranded triphosphate reverse complements that include single-stranded overhangs.

[0029] In some aspects of the invention, the dsRNA comprises a strand, in some embodiments 20-100 nucleotides in length. In other embodiments, the dsRNA is a duplex between about 20 and about 50 bp in length. In still other embodiments, the dsRNA comprises a strand between 1-1,000, 5-1,000, 10-1,000, 100-1,000, 500-1,000, 1-10, 1-20, 1-50, 1-100, 5-10, 5-20, 5-30, 5-50, 5-100, 5-200, 5-300, 5-400, 10-20, 10-30, 10 In yet other embodiments, the dsRNA comprises a strand ranging from 1 nucleotide to the full length transcript length.

[0030] In other embodiments, less than about 0.5% of the mass of RNA in the composition is dsRNA greater than 40 base pairs in size.

[0031] The purity of the product can be evaluated using known analytical methods and assays. In an exemplary embodiment of the present invention, the amount of reverse complement transcript or cytokine-induced RNA contamination is determined by high performance liquid chromatography (e.g., reverse phase chromatography, size exclusion chromatography), Bioanalyzer chip-based electrophoresis system, ELISA, flow cytometry, acrylamide gel, reconstitution or alternative assay. This assay can be performed with or without nuclease treatment of the RNA preparation (P1, RNaseIII, RNaseH, etc.). Electrophoresis / chromatography / mass spectrometry of nuclease digestion products can also be performed.

[0032] In some embodiments, the mass of the RNA is determined by mass spectrometry, such as LC-MS, MALDI-TOF (matrix-assisted laser desorption ionization time of flight).

[0033] In some embodiments, the composition includes a contaminating transcript having a length shorter than the full-length transcript, e.g., at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides less than the full-length. The contaminating transcript may include a reverse or forward transcript (transcript) having a length shorter than the full-length transcript, e.g., at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides less than the full-length. Exemplary forward transcripts include, for example, abortive transcripts. In certain embodiments, the composition includes a triphosphate polyU reverse complement of less than 30 nucleotides. In some embodiments, the composition includes a triphosphate polyU reverse complement of any length hybridized to the full-length transcript. In other embodiments, the composition includes a single-stranded triphosphate forward transcript. In other embodiments, the composition includes a single-stranded RNA with a terminal triphosphate-G. In other embodiments, the composition comprises single- or double-stranded RNA of fewer than 12 nucleotides or base pairs (including forward or reverse complement transcripts). In any of these embodiments, the composition may comprise less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of any one of these less than full-length transcripts or combinations thereof.

[0034] In other embodiments, the RNA is produced by a process or (a) forming a reaction mixture comprising a DNA template, NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and optionally guanosine diphosphate (GDP), and (a buffer containing a T7 cofactor, e.g., magnesium); (b) incubating the reaction mixture under conditions such that the RNA is transcribed; and wherein the concentration of at least one of GTP, CTP, ATP, and UTP is at least 2-fold greater than the concentration of any one or more of ATP, CTP, or UTP, or the reaction further comprises a nucleotide diphosphate (NDP) or a nucleotide analog, wherein the concentration of the NDP or nucleotide analog is at least 2-fold greater than the concentration of any one or more of ATP, CTP, or UTP. In some embodiments, the ratio of the concentration of GTP to the concentration of any one of ATP, CTP, or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, or at least 6:1.

[0035] In some embodiments, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP is 2:1, 4:1, and 4:1, respectively. In other embodiments, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP is 3:1, 6:1, and 6:1, respectively. The reaction mixture may include GTP and GDP, and the ratio of the concentration of GTP+GDP to the concentration of any one of ATP, CTP, or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, or at least 6:1. In some embodiments, the ratio of the concentration of GTP+GDP to the concentration of ATP, CTP, and UTP is 3:1, 6:1, and 6:1, respectively.

[0036] In yet other embodiments, the RNA is produced by a process or (a) forming a reaction mixture comprising a DNA template, adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and optionally guanosine diphosphate (GDP), and a magnesium-containing buffer; (b) incubating the reaction mixture under conditions such that the RNA is transcribed; and wherein the effective concentration of phosphate in the reaction is at least 150 mM phosphate, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, at least 200 mM, at least 210 mM, or at least 220 mM. The effective concentration of phosphate in the reaction may be 180 mM. In some embodiments, the effective concentration of phosphate in the reaction is 195 mM. In other embodiments, the effective concentration of phosphate in the reaction is 225 mM.

[0037] In other embodiments, the RNA is produced by a process or (a) forming a reaction mixture comprising a DNA template, adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and optionally guanosine diphosphate (GDP), and a magnesium-containing buffer; (b) incubating the reaction mixture under conditions such that the RNA is transcribed; and Wherein the magnesium-containing buffer comprises Mg2+, and the molar ratio of the concentration of ATP+CTP+UTP+GTP and optionally GDP to the concentration of Mg2+ is at least 1.0, at least 1.25, at least 1.5, at least 1.75, at least 1.85, at least 3 or more. The molar ratio of the concentration of ATP+CTP+UTP+GTP and optionally GDP to the concentration of Mg2+ may be 1.5. In some embodiments, the molar ratio of the concentration of ATP+CTP+UTP+GTP and optionally GDP to the concentration of Mg2+ is 1.88. In some embodiments, the molar ratio of the concentration of ATP+CTP+UTP+GTP and optionally GDP to the concentration of Mg2+ is 3.

[0038] In some embodiments, the composition is produced by a process that does not include a dsRNase (e.g., RNase III) treatment step. In other embodiments, the composition is produced by a process that does not include a reverse phase (RP) chromatography purification step. In yet other embodiments, the composition is produced by a process that does not include a high performance liquid chromatography (HPLC) purification step.

[0039] In some embodiments, the RNA is a modified mRNA. In other embodiments, the RNA is an unmodified RNA. In other embodiments, the RNA is an lncRNA. In yet other embodiments, the RNA is more than 50 nucleotides in length. The RNA may include UTP, where the UTP is modified UTP.

[0040] In some embodiments, the amount of a reverse complement transcript or a cytokine-induced species is (a) generating a composition comprising a model RNA from a DNA template encoding the model RNA under the same IVT conditions used to generate the IVT RNA; and (b) determining the amount of the reverse complement transcript or cytokine-induced species in a composition comprising the model RNA by LC-MS; is indirectly determined by a process that includes Here, the amount of reverse complement transcript or cytokine induced species by LC-MS in a composition comprising a model RNA indicates the amount of reverse complement transcript or cytokine induced species in a composition comprising an IVT RNA.

[0041] In another aspect, the invention is an in vitro transcribed (IVT) RNA composition, wherein the RNA has not been subjected to RNase III treatment and / or RP purification.

[0042] In yet another aspect, the invention is a composition comprising an in vitro transcribed (IVT) single-stranded RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, wherein greater than 98% of the RNA is single-stranded, and the single-stranded RNA comprises transcripts of different lengths. In some embodiments, the single-stranded RNA comprising transcripts of different lengths comprises full-length transcripts and abortive transcripts. In some embodiments, 80-98% of the single-stranded non-full-length transcripts comprise abortive transcripts. In yet other embodiments, 95-98% of the single-stranded non-full-length transcripts comprise abortive transcripts.

[0043] A unit of use composition is provided in another aspect of the invention, the unit of use composition being an in vitro transcribed (IVT) single stranded RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, wherein the composition is free of residual organic solvents.

[0044] In another aspect, the present invention is a composition comprising an in vitro transcribed (IVT) single-stranded RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, wherein the composition is non-immunogenic, and the single-stranded RNA comprises transcripts of different lengths. In some embodiments, the single-stranded RNA comprises transcripts of different lengths comprises full-length transcripts and fragment transcripts, such as abortive transcripts. Fragment transcripts include, for example, non-full-length sense RNAs, truncated or prematurely terminated transcripts, and abortive transcripts, which are typically less than the first 25 nucleotides of the transcript.

[0045] In another aspect, the invention is a method of preparing RNA, the method comprising: (a) forming a reaction mixture comprising a DNA template, NTPs, including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and optionally guanosine diphosphate (GDP), and a buffer, e.g., a magnesium-containing buffer; and (b) incubating the reaction mixture under conditions such that the RNA is transcribed; wherein the concentration of at least one of GTP, CTP, ATP, and UTP is at least 2-fold greater than the concentration of any one or more of ATP, CTP, or UTP, or the reaction further comprises a nucleotide diphosphate (NDP) or a nucleotide analog, and the concentration of the NDP or nucleotide analog is at least 2-fold greater than the concentration of any one or more of ATP, CTP, or UTP. In some embodiments, the ratio of the concentration of GTP to the concentration of any one of ATP, CTP, or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, or at least 6:1 to produce RNA.

[0046] In some embodiments, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP is 2:1, 4:1, and 4:1, respectively. In other embodiments, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP is 3:1, 6:1, and 6:1, respectively. In yet other embodiments, the reaction mixture comprises GTP and GDP, and the ratio of the concentration of GTP+GDP to the concentration of any one of ATP, CTP, or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, or at least 6:1. In other embodiments, the ratio of the concentration of GTP+GDP to the concentration of ATP, CTP, and UTP is 3:1, 6:1, and 6:1, respectively.

[0047] Any of the compositions described herein may be a reaction mixture, e.g., a mixture of an IVT reaction that has not been purified by other methods such as RP chromatography, In other embodiments, the composition is a final product ready for therapeutic administration to a subject.

[0048] Each of the limitations of the present invention may encompass various embodiments of the present invention. Thus, each of the limitations of the present invention including any one element or combination of elements is anticipated to be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways.

[0049] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like numeral. For clarity, not every component may be shown in every figure. In the drawings: [Brief description of the drawings]

[0050] [Figure 1] 1 is a graph showing the results of an IFN-β assay screening hEPO chemical variants, nLuc and vehicle control, and short model RNA-1 in BJ fibroblasts. [Diagram 2] LCMS analysis of short model transcripts is shown. The models indicate that abortive species are present in all three chemicals. The top trace shows unmodified short model RNA-1, the middle trace shows short model RNA with all uridines modified to pseudouridines and all cytidines modified with 5'O-methyl, and the bottom trace shows short model RNA1 with some uridine and cytidine residues modified. [Diagram 3]

[0036] Figure 2 shows the results of LCMS analysis of model transcripts. The model shows the impurity profile of model RNA-4 and hEPO prepared by IVT. [Figure 4] It is shown that RNA-templated RNA transcription can be performed using T7 in the absence of a DNA template that gives rise to an immunostimulatory product upon processing. [Figure 5A] Figures 5A and 5B show the effect of reversed phase (RP) and IVT with excess GTP on the amount of RNase III substrate. Both the Alpha Process and RP purification reduce RIII substrate. An additive effect is shown by combining the two. Figure 5A shows capillary electrophoresis analysis of RNase III-treated hEPO G5 material. [Figure 5B] FIG. 5B shows capillary electrophoresis analysis of RNase III-treated hEPO G0 material. [Figure 6] Transfection data from hEPO protein expression and IFN-β are shown. [Figure 7] Capillary electrophoresis analysis of short transcripts transcribed using different processes and treated with RNase III. The data show the effect of treating a model RNA with RNase III. [Figure 8A] The results of the RP-IP purity method are shown in Figure 8. Figure 8A shows model RNA-4 subjected to RNase III treatment after IVT using the equimolar method. [Figure 8B] The results of the RP-IP purity method are shown in Figure 8B. Figure 8B shows model RNA-4 subjected to RNase III treatment after IVT with excess GTP. [Figure 9] These are the RP fractions of hEPO that were treated with RNAse III and the RP fractions of hEPO that were not treated with RNAse III. [Figure 10A] Figures 10A-10D show RNase III fragment analyzer data for hEPO fractions after equimolar (Figures 10A, 10B, and 10C) reactions. hEPO was modified so that its uridine bases were 1-methylpseudouridine. Treatment with RNase III did not produce an appreciable difference in purity using the method with excess GTP. There is significant substrate at equimolar. [Figure 10B] FIG. 10B is a continuation of FIG. 10A. [Figure 10C] FIG. 10C is a continuation of FIG. 10A. [Figure 10D] FIG. 10D shows in vitro IFNβ analysis of hEPO EQ G5, untreated or after RNase III treatment under equimolar conditions. [Figure 11A] Figures 11A-11D show capillary electrophoresis analysis of RP fraction hEPO alpha + / - RIII treatment. Figures 11A, 11B and 11C show the effect of IVT with excess GTP in the reaction. [Figure 11B] FIG. 11B is a continuation of FIG. 11A. [Figure 11C] FIG. 11C is a continuation of FIG. 11A. [Figure 11D] FIG. 11D shows IVT with excess GTP, which did not result in an IFN response. [Figure 12] The results of a J2 anti-dsRNA ELISA assay are shown. [Figure 13] dsRNA is removed by RNase III treatment. [Figure 14] We present the results of an IVT characterization study, showing that IVT with excess GTP is less sensitive to cold-induced cytokine spikes. [Figure 15] 1 shows the results of nuclease P1 in an IVT characterization study. [Figure 16A] FIG. 16A shows impurity analysis by LCMS of RNA-based IVT in different chemistries using G5 in an equimolar process (FIG. 16A). [Figure 16B] FIG. 16B shows impurity analysis by LCMS of RNA-based IVT in different chemistries using G5 in the Alpha process (FIG. 16B). [Figure 17] IFN-β in BJ fibroblasts under different IVT conditions. [Figure 18] 1 shows that dsRNA cannot be capped by vaccinia. [Figure 19] The effect of CIP treatment on different dsRNA species is shown. [Figure 20] FA purity data from in vivo experiments are shown. [Figure 21] 1 shows IFN-β induction in BJ fibroblasts. [Figure 22] 1 shows in vivo expression of hEPO. [Figure 23A] 23A-23D show cytokine Luminex data from in vivo experiments. [Figure 23B] FIG. 23B is a continuation of FIG. 23A. [Figure 23C] FIG. 23C is a continuation of FIG. 23A. [Figure 23D]FIG. 23D is a continuation of FIG. 23A. [Figure 24] 1 shows the frequency of in vivo B cell activation. [Diagram 25] 1 shows the results of an IFN-β assay analyzing short dsRNA, which is an in vitro assay of short 5′ triphosphorylated oligos. [Figure 26] 1 shows the results of an IFN-β assay analyzing 20mer and poly U / A dsRNA. [Figure 27] 1 shows an analysis of 3' overhangs on IFN-β response. [Figure 28] 1 shows the results of a cytokine assay testing dsRNA standards with 5' overhangs, full duplexes, and 3' overhangs of various lengths. [Figure 29] 1 is a graph showing in vitro analysis of polyU species. [Diagram 30] 1 is a graph showing in vitro analysis of ssRNA oligo standards. [Diagram 31] 1 is a graph showing in vitro analysis of dsRNA oligo standards with different 5' functionality. [Diagram 32] 1 is a graph demonstrating that phosphatases cannot dephosphorylate dsRNA. [Diagram 33] 1 is a graph demonstrating ssRNA impurity dose response (IFNβ in BJ fibroblasts). [Diagram 34] 1 is a graph showing dsRNA impurity dose response (IFNβ in BJ fibroblasts). [Diagram 35] FIG. 13 is a graph showing IFNβ response of modified 5′ nucleotides in forward oligo standards. [Diagram 36] FIG. 13 is a graph showing IFNβ response to modified 5′ nucleotides on reverse complement oligo standards. [Figure 37] 1 is a graph showing IFNβ response of 5′ hydroxyl functionalized dsRNA. [Figure 38] 1 is a graph demonstrating that the Alpha Process produces more OH(clean) than the equimolar process. [Figure 39] Graph showing calculated dsRNA per ug of mRNA. [Diagram 40] FIG. 1 is a schematic diagram showing the traditional in vitro transcription (IVT) process and the types of impurities formed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] To enhance the methods of producing protein-encoding polymers, new methods of generating RNA have been developed. It has been discovered that modifications may be made to the in vitro transcription (IVT) process to produce RNA preparations with properties significantly different from those of RNA produced using traditional in vitro transcription processes. RNA preparations produced according to the methods of the invention (also referred to herein as IVT RNA compositions) have properties that allow for the production of qualitatively and quantitatively superior compositions containing said RNA transcripts. Even when combined with extensive purification processes, RNA produced using traditional IVT methods is qualitatively and quantitatively different from the RNA preparations of the invention. For example, the RNA preparations of the invention (and compositions comprising same) are less immunogenic than RNA preparations (and compositions comprising same) produced using traditional IVT. The RNA preparations produced according to the methods of the invention (also referred to herein as IVT RNA compositions) further have properties that allow for the production of qualitatively and quantitatively superior protein production, e.g., upon translation. For example, proteins produced from the RNA preparations of the invention are less immunogenic than RNA preparations produced using traditional IVT.

[0052] Furthermore, increased protein expression levels with higher purity result from the RNA preparations described herein. Without being bound to a mechanism, it is believed that the substantial protein expression levels are a result of high integrity of the mRNA in the purified sample. Although some purification procedures may effectively remove a certain level of contaminants by degrading those contaminants, the integrity of the pharmaceutical product is adversely affected. For example, RNAse digestion of mRNA samples is claimed in the prior art to be useful for removing RNA contaminants. However, RNAse digestion also reduces the integrity of the mRNA by degrading a portion of the full-length transcripts generated by the IVT reaction. In contrast to the prior art IVT / purification processes, the integrity of the mRNA using the method of the present invention is very high because the method produces little or no double-stranded transcripts that would require removal using procedures such as RNAse digestion.

[0053] The RNA produced by the process described herein is any RNA that is more than 30 nucleotides long and can be used for therapeutic or diagnostic purposes.In some embodiments, the RNA is more than 40, 50, 60, 75, 100, 200, 300, 400, 500, or 1,000 nucleotides long.In some embodiments, the RNA is more than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, or 12,000 nucleotides long.In some embodiments, the RNA is mRNA. In some embodiments, the RNA is about 500 to about 4000 nucleotides in length, 1000 to about 2000 nucleotides in length, 750 to about 1800 nucleotides in length, about 1500 to about 3000 nucleotides in length, about 4000 to about 7000 nucleotides in length, or about 6000 to about 12000 nucleotides in length. The mRNA may be modified or unmodified. In other embodiments, the RNA is one or more of the following: mRNA, modified mRNA, unmodified RNA, lncRNA, self-replicating RNA, circular RNA, CRISPR guide RNA.

[0054] Traditional IVT reactions are performed by incubating a DNA template with RNA polymerase and equimolar amounts of nucleotide triphosphates including GTP, ATP, CTP, and UTP in a transcription buffer. From this reaction, an RNA transcript with a 5'-terminal guanosine triphosphate is generated. These reactions also result in the generation of numerous impurities such as double-stranded and single-stranded RNA that are immunostimulatory and may have additive effects. The methods of the invention that prevent the formation of reverse complements prevent innate immune recognition of both species. In some embodiments, the methods of the invention result in the production of RNA with significantly lower T cell activity than RNA preparations produced using prior art methods with equimolar NTPs. The prior art attempts to remove these undesirable components using a series of subsequent purification steps. Such purification methods are undesirable because they involve additional time and resources and result in residual organic solvent contamination in the final product (undesirable for pharmaceutical products). Scaling up processes such as reverse phase chromatography (RP) is labor- and capital-intensive, utilizing, for example, explosion-proof equipment, HPLC columns, and purification systems rated for high pressure, high temperature, flammable solvents, etc. The throughput of this scale and large-scale manufacturing is limited by these factors. Subsequent purification is also required to remove the alkylammonium ion pair utilized in the RP process. In contrast, the method described herein further improves currently utilized methods (e.g., RP). The lower the impurity load, the higher the purification recovery of full-length RNA free of cytokine-induced contaminants, e.g., the higher the quality of the material to begin with. When using RNase III as a preparative purification, an additional advantage of the modified IVT process of the present invention is that there is less RNase III substrate, so there are fewer inactive / irrelevant cleavage products (that degrade but do not translate) generated by RNase III treatment. With only traces of dsRNA / RNase III substrate, there is more final intact RNA product (intact cap / ORF / polyA) that can translate protein, even if the cytokine is silent.This reduces the burden on subsequent purification.

[0055] Surprisingly, according to an embodiment of the present invention, it has been discovered that the manipulation of one or more reaction parameters in an IVT reaction produces RNA preparations of highly functional RNA without one or more undesirable contaminants produced using prior art processes. One parameter in an IVT reaction that can be manipulated is the relative amount (e.g., different nucleotide amounts or concentrations) of a nucleotide or nucleotide analog compared to one or more other nucleotides or nucleotide analogs in the reaction mixture. For example, an IVT reaction may include an excess of nucleotides, such as monophosphate nucleotides, diphosphate nucleotides or triphosphate nucleotides, and / or an excess of nucleotide analogs and / or nucleoside analogs. The method of the present invention produces a high yield of product that is significantly purer than the product produced by traditional IVT methods.

[0056] A nucleotide analog is a compound that has the general structure of a nucleotide or is structurally similar to a nucleotide or a portion thereof. In particular, a nucleotide analog is, for example, a nucleotide that contains an analog of the nucleic acid portion, sugar portion, and / or phosphate group of the nucleotide. Nucleotides include, for example, nucleotide monophosphate, nucleotide diphosphate, and nucleotide triphosphate. A nucleotide analog as used herein is structurally similar to a nucleotide or a portion thereof, but does not have the typical nucleotide structure (nucleobase-ribose-phosphate). A nucleoside analog is a compound that has the general structure of a nucleoside or is structurally similar to a nucleoside or a portion thereof. In particular, a nucleoside analog is, for example, a nucleoside that contains an analog of the nucleic acid and / or sugar portion of the nucleoside.

[0057] Nucleoside triphosphate, as used herein, refers to a molecule that contains a nucleic acid base (i.e., a nucleoside) bound to a ribose and three phosphates (i.e., a nucleotide). Nucleotide diphosphate refers to the same molecule, but with two phosphate moieties. Nucleotide monophosphate refers to the same molecule, but with one phosphate moiety. Nucleotide monophosphate, nucleotide diphosphate, and triphosphate may be referred to herein as NMP, NDP, and NTP, respectively. N in NMP, NDP, and NTP refers to any nucleotide, including naturally occurring, synthetic, and modified nucleotides. Thus, the terms NDP and NTP refer to nucleotide diphosphate and nucleotide triphosphate, respectively, with any nucleotide therein, whether naturally occurring, synthetic, or modified.

[0058] Natural nucleotide diphosphates include at least adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and uridine diphosphate (UDP). Natural nucleotide triphosphates include at least adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), 5-methyluridine triphosphate (m5UTP), and uridine triphosphate (UTP). In some embodiments, the NDP and / or NTP are modified. For example, the modified NDP or NTP may have a handle to allow for easy purification and isolation.

[0059] Nucleotide triphosphates are added to RNA chains by polymerases such as T7 polymerase. In contrast, nucleotide diphosphates and monophosphates may initiate the reaction (e.g., serve as the first transcribed monomers), but are not incorporated into the chain by T7 polymerase (e.g., are not incorporated elsewhere in the chain). In some cases, a nucleotide diphosphate such as GDP may be incorporated as the first monomer. For example, if T7 starts with GDP and produces 5'GDP, functional RNA may be produced. The 5'GDP-initiated RNA is still a substrate for vaccinia capping enzyme. If excess NMPs such as GMP are used in the reaction, the purity may be increased by ligating the cap, since transcripts with 5'PO4 are substrates for ligase(s) (e.g., DNA / RNA ligase(s)).

[0060] Nucleotide analogues useful in the present invention are structurally similar to nucleotides or portions thereof, but are not polymerizable, for example, by T7. Nucleotide / nucleoside analogues (including C, T, A, U, G, dC, dT, dA, dU, or dG analogues) used herein include, for example, antiviral nucleotide analogues, phosphate analogues (soluble or immobilized, hydrolyzable or non-hydrolyzable), dinucleotides, trinucleotides, tetranucleotides, such as cap analogues, or precursors / substrates for enzymatic capping (vaccinia, or ligase), nucleotides labeled with functional groups to facilitate ligation / conjugation of caps or 5' portions (IRES), nucleotides labeled with 5'PO4 to facilitate ligation of caps or 5' portions, or nucleotides labeled with functional groups / protecting groups that may be chemically or enzymatically cleavable. Antiviral nucleotide / nucleoside analogues include, but are not limited to, ganciclovir, entecavir, telbivudine, vidarabine, and cidofovir.

[0061] IVT reaction conditions In an exemplary embodiment, the method of the present invention involves the production of RNA by an IVT reaction. IVT is an art-recognized method used to generate synthetic polynucleotides in vitro. In vitro transcribed (IVT) RNA can be engineered to transiently express proteins by structurally similar to natural RNA. However, this drug class has its own challenges, especially related to the control of the translation efficiency and immunogenicity of IVT RNA. In particular, IVT RNA produces undesirable natural immune effects, and highly stringent purification procedures by HPLC are typically applied as an additional final RNA production step. Removal of small amounts of short double-stranded RNA fragments is crucial to achieve this further reduction in immune response.

[0062] Typical reactions used in the prior art provide products with high fidelity and reasonably high yields. However, the products contain baseline levels of contaminants, only some of which can be removed using routine purification methods. An IVT reaction typically includes: an RNA polymerase, e.g., T7 RNA polymerase, at a final concentration of e.g., 1000-12000 U / mL, e.g., 7000 U / mL; a DNA template, e.g., at a final concentration of 10-70 nM, e.g., 40 nM; nucleotides (NTPs), at a final concentration of e.g., 0.5-10 mM, e.g., 7.5 mM each; magnesium, at a final concentration of e.g., 12-60 mM, e.g., magnesium acetate, at 40 mM; a buffer, e.g., HEPES or Tris, at a pH of e.g., 7-8.5, e.g., 40 mM Tris HCl, pH 8. In some embodiments, 5 mM dithiothreitol (DTT) and / or 1 mM spermidine may be included. In some embodiments, an RNase inhibitor is included in the IVT reaction to ensure that RNase-induced degradation is prevented during the transcription reaction. For example, mouse RNase inhibitor may be utilized at a final concentration of 1000 U / mL. In some embodiments, pyrophosphatase is included in the IVT reaction to cleave the inorganic pyrophosphate generated after each nucleotide is incorporated into two units of inorganic phosphate. This ensures that the magnesium remains in solution and does not precipitate as magnesium pyrophosphate. For example, E. coli inorganic pyrophosphatase may be utilized at a final concentration of 1 U / mL.

[0063] A typical in vitro transcription reaction includes the following: 1.0μg template cDNA 2.0 μl of 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT or TCEP, 10 mM spermidine) 3 Custom NTPs (25 mM each) 7.2 μl 4 RNase inhibitor 20U 5 T7 RNA polymerase 3000 U 6 dH2O up to 20.0 μl, and 7 Incubate at 37℃ for 1 to 5 hours.

[0064] The crude IVT mixture may be stored at 4°C for 4-12 hours. The original template is then digested using 1 unit of RNase-free DNase. After incubation at 37°C for 15 minutes, the RNA is purified using a purification technique such as dT resin, reverse-phase HPLC, or Ambion's MEGACLEAR™ Kit (Austin, TX) according to the manufacturer's instructions. The exemplary IVT reaction is not limiting with respect to the components or amounts of components used.

[0065] As with traditional methods, the RNA of the present invention may be produced by forming a reaction mixture containing a DNA template and one or more NTPs, such as ATP, CTP, UTP, GTP (or corresponding analogs of the aforementioned components), and a buffer. The reaction is then incubated under conditions such that the RNA is transcribed. However, the method of the present invention includes the surprising finding that the presence of an excess of one or more nucleotides and / or nucleotide analogs can significantly affect the final product. The method of the present invention can be used to produce high quality products, lacking unintended or undesired impurities and without affecting the effectiveness of the reaction.

[0066] The IVT method of the present invention includes changing the amount (e.g., molar amount or quantity) of nucleotide and / or nucleotide analog in the reaction mixture. In some embodiments, one or more nucleotides and / or one or more nucleotide analogs may be added to the reaction mixture in excess. Excess nucleotide and / or nucleotide analog is any amount that is greater than the amount of one or more other nucleotides, such as NTPs, in the reaction mixture. For example, excess nucleotide and / or nucleotide analog may be an amount that is greater than the amount of each or at least one of the other individual NTPs in the reaction mixture, or may refer to an amount of other NTPs that is greater than an equimolar amount.

[0067] In embodiments where the nucleotide and / or nucleotide analogue included in the reaction mixture is a NTP, the NTP may be present at a higher concentration than all three other NTPs included in the reaction mixture. The other three NTPs may be at equimolar concentrations with respect to each other. Alternatively, one or more of the other three NTPs may be at a different concentration than the other one or more NTPs.

[0068] In some embodiments, the excess of the selected NTP is 2x or 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even more than the amount of any one or more of the other individual NTPs in the reaction mixture. In other embodiments, the excess of the selected NTP is 2x or 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even more than the total amount of the other individual NTPs in the reaction mixture. In an exemplary embodiment, the NTP is in molar excess over the other NTPs in the reaction mixture. For example, an excess of NTPs may be added in a molar ratio of, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or greater of one or more other NTPs in the reaction mixture. In other embodiments, the excess selected NTP is at a concentration of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100 mM, 120 mM, 150 mM, or an amount even greater than the amount of any one or more of the other individual NTPs in the reaction mixture, or in the range of 60-100 mM or 4.5-100 mM. In other embodiments, the excess of the selected NTP is at a concentration of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100 mM, 120 mM, 150 mM, or any one or more amounts greater than the sum of the other NTPs in the reaction mixture.

[0069] In some instances, the excess NTP in the reaction mixture is NTP-1, and the other NTPs in the reaction mixture are NTP-2, NTP-3, and NTP-4. In some embodiments, NTP-1 is present in the reaction mixture in a higher concentration than NTP-2, NTP-3, and NTP-4, and NTP-2, NTP-3, and NTP-4 are each in equimolar amounts. In some embodiments, the ratio of NTP-1:NTP-2:NTP-3:NTP-4 is at least 2:1:1:1, at least 3:1:1:1, at least 4:1:1:1, at least 5:1:1:1, at least 6:1:1:1, at least 7:1:1:1, at least 8:1:1:1, at least 9:1:1:1, at least 10:1:1:1, at least 11:1:1:1, at least 12:1:1:1, at least 13:1:1:1, at least 14:1:1:1, at least 15:1:1:1, at least 16:1:1:1, at least 17:1:1:1, at least 18:1:1:1, at least 19:1:1:1, each with a potential capping limit of 20 for NTP-1. In some embodiments, the ratio of NTP-1:NTP-2+NTP-3+NTP-4 is at least 3:3, at least 5:3, at least 6:3, at least 7:3, at least 8:3, at least 9:3, at least 10:3, or at least 15:3, with a potential upper limit of 20:3, respectively.

[0070] In other embodiments, NTP-1 is present in the reaction mixture at a higher concentration than NTP-2, NTP-3, and NTP-4, NTP-2 and NTP-3 are each in equimolar amounts, and NTP-4 is present in the reaction mixture at a higher concentration than NTP-2 and NTP-3 and at a lower concentration than NTP-1. For example, in some embodiments, the ratio of NTP-1:NTP-4:NTP-2:NTP-3 is at least 3:2:1:1, at least 4:3:1:1, at least 4:2:1:1, at least 5:3:1:1, at least 5:3:2:2, at least 6:4:2:2, at least 8:4:2:2, at least 9:2:1:1, at least 10:2:1:1, at least 11:2:1:1, at least 12:2:1:1, at least 13:2:1:1, at least 14:2:1:1, at least 15:2:1:1, at least 16:2:1:1, at least 17:2:1:1, at least 18:2:1:1, at least 19:2:1:1, each with a potential upper limit of 20 for NTP-1.

[0071] In other embodiments, NTP-1 is present in the reaction mixture at a concentration greater than NTP-2, NTP-3, and NTP-4, NTP-2 and NTP-3 are each equimolar, and NTP-4 is present in the reaction mixture at a concentration less than NTP-1, NTP-2, and NTP-3. For example, in some embodiments, the ratio of NTP-1:NTP-3:NTP-2:NTP-4 is at least 3:2:2:1, at least 4:3:3:1, at least 4:2:2:1, at least 5:3:3:1, at least 5:3:3:2, at least 6:4:4:2, at least 8:4:4:2, at least 9:2:2:1, at least 10:2:2:1, at least 11:2:2:1, at least 12:2:2:1, at least 13:2:2:1, at least 14:2:2:1, at least 15:2:2:1, at least 16:2:2:1, at least 17:2:2:1, at least 18:2:2:1, at least 19:2:2:1, each with an upper potential cap of 20 for NTP-1.

[0072] In some embodiments, NTP-1 is GTP, ATP, UTP, or CTP. In some embodiments, NTP-2 is GTP, ATP, UTP, or CTP. In some embodiments, NTP-3 is GTP, ATP, UTP, or CTP. In some embodiments, NTP-4 is GTP, ATP, UTP, or CTP.

[0073] In some embodiments, the NTP is GTP and is present in the mixture in a ratio of at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, or at least 15:1 relative to the concentration of any one of ATP, CTP, or UTP. The ratio of GTP to other NTPs may be about 2:1 to about 3:1, about 2.5:1 to about 3.5:1, about 3:1 to about 4:1, about 3.5:1 to about 4.5:1, about 4:1 to about 5:1, about 4.5:1 to about 5.5:1, about 5:1 to about 6:1, about 5.5:1 to about 6.5:1, about 6:1 to about 7:1, about 6.5:1 to about 7.5:1, about 7:1 to about 8:1, about 7.5:1 to about 8.5:1, about 8:1 to about 9:1, about 8.5:1 to about 9.5:1, and about 9:1 to about 10:1. In one embodiment, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP may be 2:1, 4:1, and 4:1, respectively. In another embodiment, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP may be 3:1, 6:1, and 6:1, respectively.

[0074] In embodiments where the nucleotides and / or nucleotide analogs included in the reaction mixture are NDPs or nucleotide analogs, the NDPs or nucleotide analogs may be present at a higher concentration than all four NTPs included in the reaction mixture. The four NTPs may be at equimolar concentrations with respect to each other. Alternatively, one or more of the four NTPs may be at a different concentration than one or more of the other NTPs.

[0075] In other embodiments, the excess of selected NDP or nucleotide analogue is 2x or 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even more than the amount of any one or more of the individual NTPs in the reaction mixture. In other embodiments, the excess of selected NDP or nucleotide analogue is 2x or 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even more than the total amount of the individual NTPs in the reaction mixture. In an exemplary embodiment, the NDP or nucleotide analogue is in molar excess over the other NTPs in the reaction mixture. For example, an excess of NDP or nucleotide analogue may be added in a molar ratio of, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or greater, of one or more NTPs in the reaction mixture. In other embodiments, the excess of selected NTP or nucleotide analogue is at a concentration of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100 mM, 120 mM, 150 mM, or even higher than the amount of any one or more individual NTPs in the reaction mixture, or in the range of 60-100 mM or 4.5-100 mM. In other embodiments, the excess of selected NTP or nucleotide analogue is at a concentration of 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 100 mM, 120 mM, 150 mM, or even in excess of any one or more of the total NTPs in the reaction mixture.

[0076] In some examples, the NTPs in the reaction mixture are NTP-1, NTP-2, NTP-3, and NTP-4. In some embodiments, the NDP or nucleotide analog is present in the reaction mixture at a higher concentration than NTP-1, NTP-2, NTP-3, and NTP-4, where NTP-1, NTP-2, NTP-3, and NTP-4 are each in equimolar amounts. In some embodiments, the ratio of NDP or nucleotide analog:NTP-1+NTP+2:NTP-3+NTP-4 is at least 4:4, at least 5:4, at least 6:4, at least 7:4, at least 8:4, at least 9:4, at least 10:4, or at least 15:4, with a potential upper cap of 20:4, respectively.

[0077] In some embodiments, an excess of NDP or nucleotide analogue is combined with an equal or greater concentration of one of the four NTPs.

[0078] In other embodiments, the NDP or nucleotide analog is present in the reaction mixture at a higher concentration than NTP-1, NTP-2, NTP-3, and NTP-4, and NTP-1, NTP-2, and NTP-3 are each in equimolar amounts, and NTP-4 is present in the reaction mixture at a lower or greater concentration than NTP-1, NTP-2, and NTP-3. For example, in some embodiments, the ratio of NDP or nucleotide analog:NTP-1:NTP-3:NTP-2:NTP-4 is at least 3:2:2:2:1, at least 4:3:3:3:1, at least 4:2:2:2:1, at least 5:3:3:3:1, at least 5:3:3:3:2, at least 6:4:4:4:2, at least 8:4:4:4:2, at least 9:2:2:2:1, at least 10:2:2 :2:1, at least 11:2:2:2:1, at least 12:2:2:2:1, at least 13:2:2:2:1, at least 14:2:2:2:1, at least 15:2:2:2:1, at least 16:2:2:2:1, at least 17:2:2:2:1, at least 18:2:2:2:1, at least 19:2:2:2:1, with an upper potential cap of 20 NDPs or nucleotide analogs, respectively.

[0079] In other embodiments, the NDP or nucleotide analogue is present in the reaction mixture at a higher concentration than NTP-1, NTP-2, NTP-3, and NTP-4, where NTP-2 and NTP-3 are each in equimolar amounts, and NTP-1 and / or NTP-4 are present in the reaction mixture at a concentration less than or greater than NTP-2 and NTP-3. In other embodiments, the NDP or nucleotide analogue is present in the reaction mixture at a higher concentration than NTP-1, NTP-2, NTP-3, and NTP-4, where NTP-1, NTP-2, NTP-3, and NTP-4 are each present in different amounts from one another.

[0080] In some embodiments, the upper limit of excess nucleotide or nucleotide analog in the reaction mixture is dictated by solubility limits.

[0081] In some embodiments, the NTP is a salt NTP. For example, the NTP may be ammonium NTP, tris NTP, lithium NTP, potassium NTP, or sodium NTP.

[0082] In one embodiment of the present invention, the IVT method can include adding a combination of NTP and NDP to the reaction mixture.The combination of NTP and NDP can be added in excess to the reaction mixture.The excess of the combination of NTP and NDP is an amount that is greater than the amount of at least one of the other NTPs or one or more of all other NTPs in the reaction mixture.For example, the excess NTP and NDP can be a total amount that is greater than the amount of at least one of the other NTPs in the reaction mixture.

[0083] Thus, in some embodiments, an IVT reaction may contain an equimolar amount of nucleotide triphosphate relative to at least one of the other nucleotide triphosphates when used in combination with the corresponding nucleotide diphosphate, or may contain a nucleotide triphosphate that is less than in excess, so long as the total amount of that nucleotide is present in the reaction in excess. The corresponding nucleotide diphosphate refers to a nucleotide diphosphate that has the same base as the nucleotide triphosphate. For example, the nucleotide triphosphate may be GTP, and the nucleotide diphosphate may be GDP.

[0084] In some embodiments, the NTP and NDP are equimolar in combination. In another embodiment, the amount of NTP is greater than the amount of NDP in the combination added to the reaction mixture. The amount of NDP may be greater than the amount of NTP in the combination added to the reaction mixture. In some embodiments, the combined mixture of excess NTP and NDP is 2 or fold (x), 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even greater than the amount of other individual NTPs in the reaction mixture. In each embodiment, the other individual NTPs may be present in the reaction mixture in the same (equimolar) or different amounts. The fold difference described herein refers to a comparison with at least one, at least two, or all three of the other NTPs in the reaction mixture.

[0085] In other embodiments, the NTP is 2x or 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even more than the amount of NDP in the reaction mixture. In still other embodiments, the NDP is 2x or 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x-100x, 10x-90x, 10x-80x, 10x-70x, or even more than the amount of NTP in the reaction mixture.

[0086] In each embodiment described herein, the NTP and NDP can be, for example, GTP and GDP, respectively, and can be present in the mixture at a concentration of at least 6 times or fold (x), 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 15x to 100x, 10x to 90x, 10x to 80x, 10x to 70x, or even greater than the amount of any one of ATP, CTP, or UTP in the reaction mixture. In an exemplary embodiment, the combination of NTP and NDP is in molar excess relative to other individual NTPs in the reaction mixture. For example, the combination mixture of NTP and NDP can be added to the reaction mixture in a molar ratio of, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or more. The ratio of GTP and GDP to other NTPs may be about 2:1 to about 3:1, about 2.5:1 to about 3.5:1, about 3:1 to about 4:1, about 3.5:1 to about 4.5:1, about 4:1 to about 5:1, about 4.5:1 to about 5.5:1, and about 5:1 to about 6:1. In one embodiment, the ratio of the concentration of GTP to the concentrations of ATP, CTP, and UTP may be 3:1, 6:1, and 6:1, respectively.

[0087] In other embodiments, the ratio of NTP to NDP, and in some embodiments, GTP to GDP, is considered relative to the ratio of purine nucleotides to pyrimidine nucleotides (Pu:Py) in the reaction mixture. In some embodiments, the ratio of GTP:GDP to Pu:Py is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or more in the reaction mixture.

[0088] In some embodiments, the buffer comprises phosphate. The effective concentration of phosphate is at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, at least 200 mM, at least 210 mM, at least 220 mM, or at least 230 mM phosphate. In one embodiment, the effective concentration of phosphate is 180 mM. In another embodiment, the effective concentration of phosphate is 195 mM.

[0089] In another embodiment, the buffer contains magnesium. The buffer has a Mg content of at least 1.0, at least 1.1, at least 1.2, at least 1.25, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.75, at least 1.8, and at least 1.85 or 3. 2+ In another embodiment, the molar ratio of the concentration of ATP+CTP+UTP+GTP and optionally GDP to the molar concentration of ATP+CTP+UTP+GTP and optionally GDP is 1.0, 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.85, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 or any range of these variables. In one embodiment, the ratio is 1.5. In another embodiment, the molar ratio is 1.88. In one embodiment, the ratio is 3.

[0090] In exemplary aspects of the invention, the IVT reaction (reaction mixture) includes an RNA polymerase, such as T7, SP6, T3, etc. In some embodiments, the polymerase, such as T7 polymerase, is included at a concentration of greater than 5 U / μl, greater than 10 U / μl, greater than 20 U / μl, greater than 50 U / μl, or greater than 100 U / μl. In some embodiments, the concentration of the polymerase, such as T7 polymerase, ranges from about 1 to about 250 U per μl of reaction mixture, such as about 1 to about 100 U / μl, or about 100 to about 250 U / μl. In some embodiments, the T7 polymerase concentration ranges from about 30 to about 60 U / μl, about 60 to about 80 U / μl, about 80 to about 100 U / μl, about 100 to about 150 U / μl, or about 150 to about 200 U / μl. In some embodiments, the polymerase, e.g., T7 polymerase, is included at a concentration of 7, 14, 25, 50, 75, or 140.

[0091] As used herein, DNA template refers to a polynucleotide template for RNA polymerase. The DNA template useful in accordance with the methods described herein, in some embodiments, comprises a gene of interest, for example, encoding a polypeptide of interest. In some embodiments, the DNA template comprises a sequence encoding an RNA polymerase promoter, for example, a T7 promoter, located 5' to the gene of interest and operably linked thereto, and optionally, a polyA tail located 3' to the gene of interest.

[0092] Any RNA polymerase known in the art can be used in the method of the present invention.RNA polymerase includes, but is not limited to, phage RNA polymerase, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or mutant polymerase, such as, but is not limited to, polymerase that can incorporate modified nucleic acid.As a non-limiting example, RNA polymerase can be modified to show increased ability to incorporate 2' modified nucleotide triphosphate compared to unmodified RNA polymerase.

[0093] As used herein, "gene of interest" refers to a polynucleotide that codes for a polypeptide or protein of interest.Depending on the context, gene of interest refers to deoxyribonucleic acid, e.g., a gene of interest in a DNA template that can be transcribed into an RNA transcript, or ribonucleic acid, e.g., a gene of interest in an RNA transcript that can be translated in vitro, in vivo, in situ or ex vivo to produce the encoded polypeptide of interest.Polypeptides of interest include, but are not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, and the like.

[0094] "RNA transcript" refers to a ribonucleic acid produced by an IVT reaction using a DNA template and an RNA polymerase. In some embodiments, the RNA transcript is an mRNA, which typically includes the coding sequence of the gene of interest and a polyA tail. The RNA transcript includes an mRNA. The RNA transcript may include modifications, such as modified nucleotides. As used herein, the term RNA transcript includes and is interchangeable with mRNA, modified mRNA "mmRNA" or modified mRNA, and primary constructs.

[0095] purity The RNA produced according to the methods of the present invention is surprisingly pure and has high integrity. It has fewer contaminants than RNA preparations produced according to traditional IVT methods. In some embodiments, it has fewer immune stimulatory contaminants than RNA preparations produced according to traditional IVT methods. The contaminants are RNA fragments generated by reactions other than the desired RNA. In some embodiments, the RNA fragment contaminants are reverse complement transcripts and / or cytokine-induced RNA contaminants. In other embodiments, the RNA fragment contaminants are double-stranded RNA or immunogenic contaminants.

[0096] In some embodiments, the RNA preparation of the present invention is less contaminant than the RNA preparation produced according to traditional IVT methods. In some embodiments, the RNA preparation of the present invention is at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% less contaminant than the RNA preparation produced according to traditional IVT methods. In other embodiments, the RNA preparation of the present invention is substantially free of contaminants. In other embodiments, the RNA preparation of the present invention is 100% free of contaminants.

[0097] Thus, the invention in some aspects includes the preparation of IVT RNA that is substantially free of reverse complement transcripts without the need for further purification steps. As used herein, the term "reverse complement transcript" refers to an RNA molecule resulting from RNA-templated transcription. The reverse complement transcript may be an RNA-templated transcript in some embodiments. Without being bound by theory, it is believed that the reverse complement product is primarily or entirely an RNA-templated transcript. If the reverse complement product is composed of a DNA-templated transcript, the product will contain nucleotide sequences that are complementary to, for example, the T7 promoter region from the DNA template. Reverse complement products characterized to date do not primarily contain sequences that are complementary to, for example, the T7 promoter region. In some embodiments, the reverse complement transcript is a double-stranded RNA (dsRNA), which may contain a single strand that encodes a sequence that is the reverse complement of at least a portion of the IVT RNA. In other embodiments, the reverse complement product may be a dsRNA with a single strand that contains a poly-U sequence. Either the reverse complement strand encoding the polypeptide of interest or the strand encoding the polyU sequence may begin with a 5' triphosphate (5'ppp). The RNA templated transcript may comprise the reverse complement of the 5' end of the IVT RNA and / or the reverse complement of the 3' end of the IVT RNA. Additionally, the reverse complement of the 5' end of the IVT RNA may be complementary to all or part of the 5' UTR of the IVT RNA. The reverse complement may comprise a sequence complementary to the first 10-15, the first 5-15, the first 5-20, the first 10-20, the first 15-25 nucleotides of the 5' UTR. Similarly, the reverse complement of the 3' end of the IVT RNA may be complementary to all or part of the polyA tail of the IVT RNA. The reverse complement transcript may be templated from anywhere on the RNA and therefore may be of any size or complementary to any position on the template. For example, the reverse complement product can be a 5mer, 10mer, 15mer, 20mer, 25mer, 40mer, 50mer, 60mer, 70mer, 100mer, 200mer, etc., up to the full length of the intended or desired product.

[0098] The present invention features a composition comprising an IVT RNA and a pharma- ceutically acceptable excipient that is substantially free of reverse complement transcripts. In some embodiments, in an IVT, the RNA that is substantially free of reverse complement transcripts comprises reverse complement transcripts that constitute less than about 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.55%, 0.5%, 0.45%, 0.4%, 0.35%, 0.3%, 0.025%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the total RNA mass. The mass of the RNA composition may be determined by any means known in the art. Examples of methods for determining the mass of RNA include liquid chromatography and mass spectrometry.

[0099] Without being bound by theory, in some embodiments of the invention, the contaminants are believed to be single-stranded reverse complements bound to a population of IVT RNAs that form double-stranded structures in the context of longer RNAs. T7 may template the abortive (sense strand) nascent RNA as well as the full-length product nascent RNA. The RNA template transcript (antisense), once transcribed (primarily initiated at pppC, pppU, and pppA), likely remains associated with the nascent sense RNA. Alternatively, if such constructs form, they may be essentially silent. The combination of the two strands is effectively a dsRNA with 5'ppp. The presence of 5'ppp on one or more hybridized strands renders the structure immunostimulatory. Even when the antisense RNA template transcript is dissociated from the RNA, the presence of ssRNA with pppC, pppU, and pppA is still cytokine-inducing. Since the method of the present invention produces RNA that is devoid of dsRNA as seen in the J2 ELISA and RNase III treatment, the product will not adopt large full-length RNA structures, and the RNA will likely fold similarly if transcribed in equimolar amounts or in the IVT process of the present invention.

[0100] In some embodiments, the RNA preparations of the invention are substantially free of cytokine-induced RNA contaminants. As used herein, the term "cytokine-induced contaminants" refers to RNA molecules that induce cytokine production, e.g., type I interferon (IFNα / β induction), as determined, e.g., in a cell-based cytokine induction assay, e.g., as determined in a BJ fibroblast / IFNβ assay and / or a Luminex assay, as described in the Examples herein. In exemplary aspects of the invention, the term "cytokine-induced" contaminants refers to RNA molecules that induce cytokine induction and are substantially double-stranded in nature.

[0101] Without being bound by theory, double-stranded RNA molecules resulting from aberrant polymerase transcription, such as transcription templated from the desired RNA generated in an IVT reaction, are believed to induce cytokines through activation of innate immune responses that resemble natural antiviral immune responses, including two types of pathogen recognition receptors (PRRs): Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), such as Toll-like receptor 3 (TLR3), and RNA helicases, such as RIG-I and MDA5. Examples of other cytokine-inducing molecules include RNase III substrates. As used herein, RNase III substrates refer to double-stranded RNA molecules that are susceptible to cleavage by RNase III enzymes.

[0102] In some embodiments, the cytokine-induced RNA contaminant may be a double-stranded RNA with a reverse sequence or polyU sequence complementary to the IVT RNA. The reverse complement or polyU sequence of the IVT RNA may begin at 5'ppp.

[0103] The cytokine-induced RNA contaminant may comprise a reverse complement of the 5' end of the IVT RNA and / or may comprise a reverse complement of the 3' end of the IVT RNA. Furthermore, the reverse complement of the 5' end of the IVT RNA may be complementary to all or part of the 5' UTR of the IVT RNA. The reverse complement may comprise a sequence complementary to the first 10-15, the first 5-15, the first 5-20, the first 10-20, the first 15-25 nucleotides of the 5' UTR. In some embodiments, the reverse complement may comprise a complementary sequence within the 5'UTR in the range of 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-800, 1-900, 1-1000, 1-2000, 1-2500, or 1-3000 nucleotides in length. In other embodiments, the reverse complement may comprise a complementary sequence within the 5'UTR in the range of 10 to 20, 10 to 30, 10 to 40, 10 to 50, 1 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 200, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, 10 to 1000, 10 to 2000, 10 to 2500, or 10 to 3000 nucleotides in length. In other embodiments, the reverse complement may comprise a sequence complementary to a range of 20-25, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-200, 20-300, 20-400, 20-500, 20-600, 20-700, 20-800, 20-900, 20-1000, 20-2000, 20-2500, or 20-3000 nucleotides in length within the 5'UTR. Similarly, the reverse complement of the 3' end of the IVT RNA may be complementary to all or part of the polyA tail of the IVT RNA. The reverse complement may comprise a sequence complementary to the first 10-15, the first 5-15, the first 5-20, the first 10-20, or the first 15-25 nucleotides of the 3'UTR.In some embodiments, the reverse complement may comprise a sequence complementary to a range of 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-800, 1-900, 1-1000, 1-2000, 1-2500, 1-3000, or 1 to the full length or maximum size RNA nucleotide length within the 3'UTR. In other embodiments, the reverse complement may comprise a sequence complementary to a range of 10 to 20, 10 to 30, 10 to 40, 10 to 50, 1 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 200, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, 10 to 1000, 10 to 2000, 10 to 2500, or 10 to 3000 nucleotides in length within the 3'UTR. In other embodiments, the reverse complement may comprise a sequence complementary to a range of 20 to 25, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 20 to 100, 20 to 200, 20 to 300, 20 to 400, 20 to 500, 20 to 600, 20 to 700, 20 to 800, 20 to 900, 20 to 1000, 20 to 2000, 20 to 2500, or 20 to 3000 nucleotides in length within the 3'UTR.

[0104] The present disclosure encompasses compositions that are substantially free of cytokine-induced RNA contaminants, comprising an IVT RNA and a pharma- ceutically acceptable excipient. In some embodiments, the cytokine-induced RNA contaminants comprise less than 0.5%, 0.45%, 0.4%, 0.35%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the amount of RNA. The mass of the RNA composition may be determined by any means known in the art. Examples include liquid chromatography and mass spectrometry.

[0105] The dsRNA of the cytokine-induced RNA contaminant and / or contaminant, such as a reverse complement transcript, may be 20-50 nucleotides in length. In other embodiments, the dsRNA may be 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, The dsRNA may be 200-225, 225-250, 250-275, 275-300, 300-325, 325-350, 350-375, 375-400, 400-425, 425-450, 450-475, 475-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, and 950-1000 nucleotides in length. In some embodiments, the mass of the dsRNA is greater than 40 base pairs and constitutes less than about 0.5% of the RNA composition.

[0106] The contaminant strand may have a 5'ppp end. In some embodiments, the contaminant strand may have lower amounts of pppA, pppC, and pppU compared to equimolar process produced RNA. In another embodiment, the contaminant strand may have a lower ratio of pppA:pppG, pppC:pppG, and / or pppU:pppG compared to equimolar process. The pppNTP may be detected by LC-MS after total nuclease digestion, e.g., Nuclease P1 treatment. Nuclease P1 digests RNA and DNA to single nucleotides. The only triphosphate species that should be present is for the initiating nucleotide. If no RNA-templated transcript is formed, then the 5'PPPG is the only target site for initiation, and thus the only triphosphate that should be present. The presence and amount of 5'pppA, 5'pppC, and / or 5'pppU detected by LC / MS after Nuclease P1 digestion indicates an RNA-templated RNA transcript.

[0107] In addition to being low in impurities, especially double-stranded impurities, IVT RNA compositions have a high percentage of full-length functional RNA transcripts relative to other RNA species in the composition, especially when compared to conventional purified RNA compositions produced using IVT methods in combination with purification steps, such as reverse phase chromatography or RNAse III treatment. In some embodiments, more than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% of the mass of the RNA comprises single-stranded full-length transcripts. In addition to single-stranded full-length transcripts, IVT RNA compositions may contain other single-stranded RNA species, such as single-stranded partial RNA transcripts in sense orientation, including abortive transcripts. However, IVT RNA compositions are substantially free of RNAse III insensitive fragments.

[0108] The RNA compositions described herein may contain other components other than full-length RNA transcripts, such as truncated and / or run-on transcripts. For example, the RNA may contain transcripts of different lengths, such as transcripts that are shorter or longer than the full-length transcript. Thus, in some embodiments, the RNA preparations of the invention contain truncated and / or abortive transcripts. RNA polymerase binds to a DNA promoter and synthesizes short mRNA transcripts. As used herein, the term "truncated transcript" refers to a transcript that has identity to the IVT RNA but is insufficient in length and lacks all elements necessary to encode a polypeptide of interest (e.g., polyA). In certain cases, truncated transcripts are released before the transcription complex leaves the promoter, referred to as abortive transcripts. As used herein, the term "abortive transcript" refers to a transcript that has identity to the IVT RNA but is insufficient in length and lacks all elements necessary to encode a polypeptide of interest (e.g., polyA), generally having a length of 15 nucleotides or less. In exemplary aspects of the invention, truncated and / or abortive transcripts are present and are not cytokine-inducible. In certain embodiments, truncated and / or abortive transcripts are removed from the sample. In some embodiments, truncated transcripts have a length of 100 nucleotides or less.

[0109] It has been determined that the method of the present invention also produces compositions with reduced 3' heterogeneity or 3' end heterogeneity, also referred to herein as increased 3' homogeneity or 3' end homogeneity. The inventors have determined that traditional equimolar IVT reaction conditions can result in transcripts that terminate at different 3' residues (e.g., transcripts that are not uniformly terminated). The assay described in the Examples was developed to detect 3' end heterogeneity resulting from traditional IVT reactions (assays that distinguish between non-A nucleobases occurring at the 3' end of a particular test transcript). In particular, the method of the present invention produces transcripts with a lower degree of 3' end heterogeneity (or more uniform 3' ends). For example, transcripts produced according to a traditional IVT reaction (e.g., an equimolar reaction) may produce a composition in which more than 50% (optionally more than 60%, more than 70%, more than 75%, more than 80%) of the transcripts have different ends, whereas transcripts produced according to an IVT reaction of the invention (e.g., an alpha reaction) may produce a composition in which less than 50% of the transcripts have the same end, i.e., terminate at the same nucleobase (e.g., relative to the DNA template) (optionally less than 40%, less than 30%, less than 25%, less than 20% or less) have different ends.

[0110] The truncated transcripts in the population of single-stranded partial RNA transcripts may include a range of sizes.For example, in some embodiments, at least 80% of the population of truncated transcripts has a length of 100 nucleotides or less.In other embodiments, at least 50%, 60%, 70%, 85%, 90%, 95%, 98% or 100% of the population of truncated transcripts has a length of 100 nucleotides or less.

[0111] The single-stranded RNA population in the IVT RNA composition described herein typically does not contain or is substantially free of RNAse III insensitive fragments. As used herein, "RNAse III insensitive fragments" refers to single-stranded transcripts that have identity (sense orientation) to IVT RNA but are of insufficient length and lack all elements required to code a polypeptide of interest (having less nucleotides than the full-length transcript), where the fragments are generated enzymatically, particularly by RNAse III cleavage. The generation of RNAse III insensitive fragments can occur, for example, in the traditional IVT process (as shown in Figure 40) in combination with RNAse III digestion.

[0112] As shown in Figure 40, the first step in the conventional IVT / RNAse III purification process is the addition of Mg 2+ The IVT method involves a transcription reaction utilizing a linear dsDNA template in the presence of NTPs and RNA polymerase at equimolar concentrations. This reaction produces a mixed population of single-stranded truncated / abortive transcripts, full-length RNA transcripts, run-on transcripts, and reverse complement impurities. The reverse complement impurities may bind to some of the single-stranded RNA or other impurities, such as truncated transcripts, to produce double-stranded RNA and / or RNA with both double-stranded and single-stranded regions. It has been assumed in the art that RNAse III can be used to degrade double-stranded RNA from IVT compositions, thereby effectively removing it from the composition. However, RNAse III can also degrade double-stranded regions of full-length RNA transcripts and / or run-on transcripts (e.g., double-stranded regions resulting from binding of the reverse complement within the polyA tail region), leaving single-stranded fragments of shorter length than the full-length RNA transcripts. These single-stranded fragments are RNAse III insensitive fragments as described herein. As a result of this RNAse degradation, a significant amount of the full-length transcripts generated during the IVT process is lost, thereby resulting in a significant loss of product integrity. These compositions have a significantly reduced ability to express proteins when delivered to cells or subjects.

[0113] The RNAse III insensitive fragments generated following RNAse III treatment of products generated according to a method such as that shown in Figure 40 may comprise a range of sizes. For example, in some embodiments, at least 80% of the population of abortive transcripts have a length greater than 100 nucleotides. In other embodiments, at least 50%, 60%, 70%, 85%, 90%, 95%, 98% or 100% of the population of RNAse III insensitive fragments have a length greater than 100 nucleotides.

[0114] Without being bound by theory, it is believed that the removal of certain species or contaminants, such as dsRNA species or contaminants, is important in the preparation of IVT RNA compositions for therapeutic use. In contrast, the presence of residual truncated and / or abortive transcripts in the IVT RNA composition is not believed to be necessary; such species are not believed to induce undesirable cytokines and / or innate immune responses against the IVT RNA. In other embodiments, the RNA preparations of the invention are substantially free of truncated or abortive transcripts.

[0115] Although truncated / abortive transcripts may be present in the IVT RNA composition or the present invention, RNAse III insensitive fragments are not present in the IVT RNA composition because the composition is not treated with RNAse III. Both truncated transcripts and RNAse III insensitive fragments have a variety of sizes or lengths, but the average length of the truncated transcripts is shorter than the average length of the RNAse III insensitive fragments. Thus, when the composition comprises a population of single-stranded partial RNA transcripts in the sense orientation, if more than 80% of the population of single-stranded partial RNA transcripts in the sense orientation have a nucleotide length of 100 nucleotides or less. In some embodiments, more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the population of single-stranded partial RNA transcripts in the sense orientation have a nucleotide length of 100 nucleotides or less. In other embodiments, more than 50%, 55%, 60%, 65%, 70%, 75%, 85%, or 88% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

[0116] In some embodiments, the RNA preparation is a pharmaceutical composition that includes a pharma- ceutically acceptable carrier. In other embodiments, the RNA preparation is a reaction product (e.g., an IVT reaction product) that has not yet been subjected to further purification techniques. The RNA preparation may contain a number of other components in addition to RNA. However, the reaction product is substantially free of reverse complement transcripts and / or cytokine-induced RNA contaminants.

[0117] Assay The amount of contaminants, including reverse complement transcripts and / or cytokine-induced RNA contaminants, may be determined by methods known in the art. Many methods are known in the art for determining the purity of a nucleic acid sample. Exemplary methods include, but are not limited to, high performance liquid chromatography (reverse phase chromatography, size exclusion chromatography, etc.), gel electrophoresis, and translation assays to assess the quality and purity of nucleic acid production. The quality of RNA preparations may also be determined using a Bioanalyzer chip-based electrophoresis system. In vitro efficacy may be analyzed, for example, by transfecting RNA transcripts into human cell lines (e.g., HeLA, PBMC, BJ fibroblasts, Hek 293). Protein expression of the polypeptide of interest may be quantified using methods such as enzyme-linked immunosorbent assay (ELISA), Western blot, or flow cytometry.

[0118] Various methods have been used to detect and / or quantify dsRNA using dsRNA-specific antibodies. These include ELISA, such as sandwich ELISA (Schonborn et al. (1991) Nucleic Acids Res 19:2993-3000), dot blot (for quantification, specificity testing) (Kariko et al. (2011) Nucleic Acids Res 39:e142), and immunoprecipitation / immunoblotting. In an exemplary embodiment of the present invention, contaminants can be recognized using ELISA. K1 / J2 or K2 / J2 assays can be used to determine the abundance of dsRNA contaminants in a sample. An exemplary ELISA is a sandwich ELISA, as follows: Blocking: Pre-coat microtiter plates with protein, for example, 0.4 μg / well protein A, overnight at 4° C. Free binding sites are saturated with bovine serum albumin (BSA) (e.g., 2%) in a buffer (e.g., PBS), then the plate is washed with a buffer (e.g., PBS) and stored at 4°C. dsRNA-specific J2 monoclonal antibody (IgG2a) is immobilized on the protein A layer by incubating hybridoma supernatant (e.g., 100 μl per well, overnight at 4°C). The plate is washed multiple times with a buffer, e.g., PBS plus Tween 20 (e.g., 0.5% (v / v) Tween 20), and the nucleic acid sample is added to the buffer (e.g., TE buffer, 37°C, 2 hours). After washing as above, bound nucleic acid (i.e., J2 antigen) is identified by sequential incubation with dsRNA-specific K2 IgM monoclonal antibody, followed by diluted hybridoma supernatant (e.g., 1:2) of alkaline phosphatase-conjugated secondary antibody (e.g., 1:5000 diluted goat anti-mouse IgM). Both incubation steps are carried out for approximately 1-2 hours at 37° C. Washing, substrate incubation and absorbance readings are performed according to art-recognized methods.

[0119] A similar assay using dot blots is described in Kariko et al., Nuc. Acids Res. 2011;39(21):e142. The assay is performed by blotting RNA (200 ng) onto a supercharged Nytran membrane, where it is dried and blocked with 5% nonfat dry milk in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4). The sample is then incubated with dsRNA-specific K1 or J2 monoclonal antibodies (IgG) for 1 hour. The membrane is washed with TBS-T and may be incubated, for example, with an HRP-conjugated anti-goat polyclonal antibody. The membrane is washed again and the signal is detected with TMB. The signal is developed by adding TMB. This assay is useful for detecting dsRNA duplexes that are more than 40 base pairs in length.

[0120] Cytokine assays may also be used to detect RNA contamination. Numerous cytokine assays are known in the art. This assay may test for the induction of any cytokine associated with impure IVT products. These cytokines include, for example, interleukins (IL), interference (IFN) alpha, beta, and gamma, and TNF. In one embodiment, an IFN-β cell-based assay may be used. The results have been shown to correlate with the presence of RNaseIII substrates detected by LC-MS. Other cell-based cytokine assays, such as IL or multiplex cytokine assays, may also be used.

[0121] In an exemplary BJF IFNβ and hEPO expression assay, BJ fibroblasts (ATCC) are seeded in cell culture plates. Approximately 24 hours after seeding, cells are transfected with mRNA using lipofectamine or other delivery agents. After transfection, supernatants are collected and IFNβ expression is measured using a human IFNβ ELISA kit, High Sensitivity, according to the manufacturer's instructions (PBL Assay Science). Briefly, human IFN-β is measured in cell supernatants by indirect enzyme-linked immunosorbent assay (ELISA). Pre-coated plates are incubated with cell supernatants and then washed to remove non-specifically bound material. IFN-β expression is analyzed by incubating wells with an anti-IFN-β antibody followed by a secondary antibody conjugated to horseradish peroxidase (HRP). Tetramethylbenzidine (TMB) is the HRP substrate used for detection. Human Epo levels are measured using an Epo Human ELISA Kit (Thermo Fisher).

[0122] In an exemplary Luminex assay, serum from mice is collected to assess cytokine levels using Luminex screening assay technology (R&D Systems). Briefly, analyte-specific antibodies are pre-coated onto color-coded beads. Beads, standards, and samples are pipetted into wells to allow the immobilized antibodies to bind to the analytes of interest. After washing away unbound material, a biotinylated antibody cocktail specific for the analyte of interest is added to each well. After washing to remove unbound biotinylated antibodies, streptavidin-phycoerythrin conjugate (streptavidin-PE), which binds to the biotinylated detection antibody, is added to each well. A final wash removes unbound streptavidin-PE, and the beads are resuspended in buffer and read using a Luminex analyzer (R&D Systems). The first laser is bead-specific and determines which analyte is being detected. The second laser determines the magnitude of the signal from the PE, which is directly proportional to the amount of analyte bound.

[0123] Surrogate Assay for Purity In an exemplary embodiment of the present invention, it may be desirable to determine purity by using a surrogate assay that is suitable for highly qualitative and / or quantitative detection of products and / or impurities. Thus, the present invention contemplates the determination of the purity of an RNA composition produced by a particular IVT method, e.g., IVT RNA, by determining the purity of a surrogate RNA (e.g., model RNA) produced by the same conditions. In this way, the purity may be determined indirectly through a highly qualitative and / or quantitative detection method in the surrogate system, which purity determination correlates to the purity of the IVT RNA produced in the production system. Additionally, reconstituted or surrogate type assays may be used to indirectly determine the amount and identity of contaminants in a given RNA preparation. In some cases, it may be difficult to detect low levels of contaminants or contaminants with similar structural characteristics as the RNA transcript. Reconstituted systems may be used to test biological activities such as immune stimulatory activity, e.g., cytokine activity associated with the contaminant, by adding a putative contaminant missing from the RNA preparation of the present invention, compared to biological activity from the RNA composition produced by traditional IVT methods. Reconstitution of a putative contaminant with a pure RNA preparation of the invention can demonstrate the absence of the contaminant in the pure RNA preparation.

[0124] In addition, model RNA (surrogate mRNA) may be used. Model RNA is generated from a DNA template encoding the model RNA under the same IVT conditions used to generate the IVT RNA. Model RNA or surrogate mRNA as used herein refers to a non-coding RNA transcript consisting of only the 5'UTR, 3'UTR, and polyA tail. Short model RNA may also be used. Short model RNA is a shorter version of the model RNA (consisting of only the 5'UTR and a shorter polyA tail (A20)). The amount of model RNA indicates the amount of reverse complement transcript or cytokine-induced species in the composition, and therefore the amount of reverse complement transcript or cytokine-induced species in the composition is determined by LC-MS or other analytical methods. The amount and nature of the contaminant detected in the assay correlates with and predicts the amount and nature of the contaminant that would be obtained using the same IVT reaction conditions to generate full-length mRNA.

[0125] The RNA preparations of the present invention are high quality preparations. In some embodiments, the RNA preparations obtained directly from the IVT process may be used directly as research reagents or diagnostic or therapeutic reagents without further purification. In some embodiments, the RNA preparations may be subjected to one or more purification steps. For example, the RNA preparations may be purified from truncated RNA, DNA template, and residual enzymes using oligo dT chromatography. An exemplary oligo dT chromatography assay involves loading 20mer polythymidine sepharose (3 ml) onto a 5 mL SPE column on a solid phase extraction vacuum manifold. The RNA transcripts are applied to the column, followed by washing and elution. The oligo dT purified RNA transcripts are diafiltered into water and concentrated to 1.22 mg / mL using a 100 kDa MWCO Amicon spin filter (EMD Millipore). The RNA may be recovered and its concentration determined using Bioanalyzer gel electrophoresis.

[0126] Analysis of the RNA preparation to determine sample purity and quality may be performed before or after capping. Alternatively, analysis may be performed before or after polyA capture-based affinity purification. In another embodiment, analysis may be performed before or after optional additional purification steps, such as anion exchange chromatography.

[0127] Mass spectrometry encompasses a wide range of techniques for identifying and characterizing compounds in a mixture. Different types of mass spectrometry-based approaches may be used to analyze a sample to determine its composition. Mass spectrometry involves converting the sample being analyzed into a number of ions by an ionization process. When placed in a force field, each resulting ion moves through the force field along a trajectory such that its acceleration is inversely proportional to its mass-to-charge ratio. A mass spectrum of a molecule is thus generated, which displays a plot of the relative abundance of precursor ions versus their mass-to-charge ratio. When a subsequent stage of mass spectrometry, such as tandem mass spectrometry, is used to further analyze the sample by subjecting the precursor ions to higher energies, each precursor ion may undergo dissociation into fragments called product ions. The resulting fragments may be used to provide information about the nature and structure of their precursor molecules.

[0128] MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time of Flight) mass spectrometry provides spectroscopic measurement of the mass of low volatility, low ionization analytes or analytes that are easily fragmented by embedding them in a matrix of light absorbing material and measuring the weight of the molecule as it becomes ionized and goes into flight by volatilization. A combination of electric and magnetic fields is applied to the sample to move the ionized material according to the molecule's individual mass and charge. U.S. Patent No. 6,043,031 issued to Koster et al. describes an exemplary method for identifying single base mutations in DNA using MALDI-TOF and other mass spectrometry methods.

[0129] HPLC (High Performance Liquid Chromatography) is used for analytical separation of biopolymers based on their properties. HPLC may be used to separate nucleic acid sequences based on size charge and base composition. Nucleic acid sequences that differ from another nucleic acid by one base pair can be separated using HPLC. Thus, nucleic acid samples that are identical except for a single nucleotide can be differentially separated using HPLC to identify the presence or absence of a particular nucleic acid fragment. Preferably, the HPLC is HPLC-UV.

[0130] In some embodiments, the RNA may be purified without using a dsRNase step. For example, RNase III may not be used. The composition may be produced by a process that does not use a reverse phase chromatography purification step. In one embodiment, the RNA composition may be produced without using high performance liquid chromatography (HPLC) purification. Thus, the composition does not contain residual organic reagents or contaminants associated with traditional purification processes.

[0131] In some cases, the methods of the present invention are used to determine the purity of an RNA sample. The term "pure" as used herein refers to a material that has only a target nucleic acid activator such that the presence of unrelated nucleic acids is reduced or eliminated, i.e., impurities or contaminants, including RNA fragments, are reduced or eliminated. For example, a purified RNA sample contains one or more target or test nucleic acids, but is preferably substantially free of other nucleic acids detectable by the described methods. As used herein, the term "substantially free" is used operationally in the context of analytical testing of a material. Preferably, a purified material is substantially free of one or more impurities or contaminants, including reverse complement transcripts and / or cytokine-induced RNA contaminants as described herein, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% pure; more preferably at least 98% pure, and even more preferably at least 99% pure. In some embodiments, a pure RNA sample consists of 100% target or test RNA and is free of other RNAs.

[0132] In some embodiments, RNA is separated using capillary electrophoresis (CE). An electric field is applied to the sample so that it flows through an electrolyte solution, such as an aqueous buffer, through a capillary tube into a vial of interest. The analytes migrate differentially based on their electrophoretic mobility and are detected at the outlet end of the capillary. The output data is recorded and then displayed as an electropherogram. This may be used in conjunction with mass spectrometry to determine the identity of sample components. The capillary electrophoresis system is fully automated in the FRAGMENT ANALYZER™, which can detect differences of as little as three base pairs.

[0133] In some embodiments, RNA may be quantified and purified using a Fragment Analyzer (FA), which automates capillary electrophoresis and HPLC.

[0134] In some embodiments, the RNA molecule is an mRNA molecule. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded peptide polypeptide of interest. mRNA is transcribed from a DNA sequence by an RNA polymerase enzyme and interacts with ribosomes to synthesize the genetic information encoded by the DNA. In general, mRNA is classified into two subclasses: pre-mRNA and mature mRNA. Precursor mRNA (pre-mRNA) is mRNA that has been transcribed by RNA polymerase but has not undergone any post-transcriptional processing (e.g., 5' capping, splicing, editing, and polyadenylation). Mature mRNA has been modified through post-transcriptional processing (e.g., spliced ​​and polyadenylated to remove introns) and can interact with ribosomes to carry out protein synthesis. mRNA may be isolated from tissues or cells by a variety of methods. For example, total RNA extraction may be performed on cells or cell lysates, and the resulting extracted total RNA may be purified (eg, on a column containing oligo-dT beads) to obtain extracted mRNA.

[0135] Alternatively, mRNA may be synthesized in a cell-free environment, for example, by in vitro transcription (IVT). As used herein, "in vitro transcription template" refers to a deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some embodiments, an IVT template encodes a 5' untranslated region, contains an open reading frame, and encodes a 3' untranslated region and a polyA tail. The specific nucleotide sequence composition and length of the IVT template will depend on the mRNA of interest encoded by the template.

[0136] "5' untranslated region (UTR)" refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript that is translated by the ribosome) that does not code for a protein or peptide.

[0137] "3' untranslated region (UTR)" refers to the region of an mRNA that does not code for a protein or peptide and is immediately downstream (i.e., 3') of a stop codon (i.e., a codon in an mRNA transcript that signals the end of translation).

[0138] An "open reading frame" is a contiguous stretch of DNA that begins with a start codon, such as methionine (ATG), and ends with a stop codon, such as TAA, TAG, or TGA, that encodes a protein or peptide.

[0139] A "poly-A tail" is a region of an mRNA downstream, e.g., immediately downstream (i.e., 3'), from the 3'UTR that contains multiple consecutive adenosine monophosphates. A poly-A tail may contain between 10 and 300 adenosine monophosphates. For example, a poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, or 600 adenosine monophosphates. In some embodiments, a poly-A tail contains between 50 and 250 adenosine monophosphates. In relevant biological settings (e.g., within a cell, in vivo, etc.), the poly(A) tail functions to protect the mRNA from enzymatic degradation, for example in the cytoplasm, as well as assisting in transcription termination, transport of the mRNA from the nucleus, and translation.

[0140] Thus, in some embodiments, a polynucleotide comprises: (a) a first region of linked nucleotides encoding a polypeptide of interest; (b) a first terminal region located 5' to said first region, said first terminal region comprising a 5' untranslated region (UTR); (c) a second terminal region located 3' to said first region; and (d) a tailing region. The terms polynucleotide and nucleic acid are used interchangeably herein.

[0141] In some embodiments, the polynucleotide comprises about 1 to about 3,000, 10 to about 3,000, 20 to about 3,000, 30 to about 3,000, 40 to about 3,000, 50 to about 3,000, 100 to about 3,000, 200 to about 3,000 nucleotides (e.g., 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, and 2,000 to 3,000).

[0142] IVT RNAs can function as RNAs, but are distinct from wild-type RNAs in their functional and / or structural design features, which help overcome the existing problems of effective polypeptide generation using nucleic acid-based therapeutics. For example, IVT RNAs can be structurally or chemically modified. As used herein, a "structural" modification is a modification in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Since chemical bonds are necessarily broken and reformed to result in structural modifications, structural modifications are of a chemical nature and are therefore chemical modifications. However, structural modifications will result in different nucleotide sequences. For example, a polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG", where a dinucleotide "CC" is inserted, resulting in a structural modification to the polynucleotide.

[0143] The cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. This system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. The NTPs may be homemade, selected from a supplier, or synthesized as described herein. The NTPs may be selected from the NTPs described herein, including but not limited to natural and non-natural (modified) NTPs. The polymerase may be selected from, but not limited to, T7 RNA polymerase, T3 RNA polymerase, and mutant polymerases, including but not limited to polymerases capable of incorporating polynucleotides (e.g., modified nucleic acids).

[0144] chemically modified RNA Thus, in an exemplary embodiment, the polynucleotide of the present invention may contain at least one chemical modification. The polynucleotide may contain various substitutions and / or insertions from natural or naturally occurring polynucleotides. As used herein in reference to polynucleotides, the term "chemical modification" or, where appropriate, "chemically modified" refers to modifications of adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of the following positions, patterns, percentages or populations. In general, these terms are not intended herein to refer to ribonucleotide modifications in naturally occurring 5'-terminal RNA cap moieties.

[0145] The modification may be a variety of different modifications. In some embodiments, the region may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide introduced into a cell may exhibit reduced degradation within the cell compared to the unmodified polynucleotide.

[0146] Modifications of a polynucleotide include, but are not limited to, those listed in detail below. A polynucleotide may contain naturally occurring, non-naturally occurring modifications, or a polynucleotide may contain both naturally occurring and non-naturally occurring modifications.

[0147] The polynucleotides of the invention may include any useful modifications, such as to the sugar, nucleobase, or internucleotide linkage (e.g., to the linked phosphate / to the phosphodiester linkage / to the phosphodiester backbone). One or more atoms of the pyrimidine nucleobase may be replaced or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, a modification (e.g., one or more modifications) is present in each of the sugar and the internucleotide linkage. The modification according to the invention may be a modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Additional modifications are described herein.

[0148] Non-naturally occurring modified nucleotides may be introduced into a polynucleotide during or after synthesis of the chain to achieve a desired function or property. Modifications may be to the internucleotide group, the purine or pyrimidine base, or the sugar. Modifications may be introduced at the end of the chain or elsewhere in the chain (by chemical synthesis or polymerase enzyme). Any region of a polynucleotide may be chemically modified.

[0149] The present disclosure provides polynucleotides consisting of unmodified or modified nucleosides and nucleotides and combinations thereof. As described herein, a "nucleoside" is defined as a compound that includes a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleotides) as described herein. The polynucleotide may include a region(s) of linked nucleotides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotide will include a region of nucleotides. Any combination of bases / sugars or linkers may be incorporated into the polynucleotides of the present invention.

[0150] In some embodiments, the RNA of the invention comprises a combination of one or more of the aforementioned modified nucleobases (eg, a combination of two, three or four of the aforementioned modified nucleobases).

[0151] Nucleic acid modifications that are useful in the present invention include, but are not limited to, those in the table below.

[0152] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

[0153] In some embodiments, the modified nucleobase is pseudouridine (Ψ), N1-methylpseudouridine (m 1 Ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the RNA of the invention comprises a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0154] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m 1 Ψ), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5C), pseudouridine (Ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the mRNA of the invention comprises a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0155] In some embodiments, the RNA is a 5-methyl-cytidine (m 5 In some embodiments, the RNA comprises 1-methyl-pseudouridine (m 1 In some embodiments, the RNA comprises 1-methyl-pseudouridine (m 1 Ψ) and 5-methyl-cytidine (m 5 In some embodiments, the RNA comprises 2-thiouridine (sC). 2 In some embodiments, the RNA comprises 2-thiouridine and 5-methyl-cytidine (mU). 5 In some embodiments, the RNA comprises 5-methoxy-uridine (moC). 5 In some embodiments, the RNA comprises 5-methoxy-uridine (moU). 5 U) and 5-methyl-cytidine (m 5 In some embodiments, the RNA comprises 2'-O-methyluridine. In some embodiments, the RNA comprises 2'-O-methyluridine and 5-methyl-cytidine (mC). 5 In some embodiments, the RNA comprises N6-methyl-adenosine (mC). 6 In some embodiments, the RNA comprises N6-methyl-adenosine (mA). 6 A) and 5-methyl-cytidine (m 5 C) is included.

[0156] In certain embodiments, the RNA of the present invention is uniformly modified (i.e., completely modified, modified throughout the entire sequence) for a particular modification. For example, the RNA may be modified with 5-methyl-cytidine (m 5Alternatively, all cytosine residues in the RNA sequence may be uniformly modified with 5-methyl-cytidine (MC), in which case all cytosine residues in the RNA sequence are modified with 5-methyl-cytidine (MC). 5 C). Similarly, the RNA of the present invention may be uniformly modified by replacing any type of nucleotide residue present in the sequence with a modified residue as described above.

[0157] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases, nucleosides, and nucleotides having modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0158] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases, nucleosides, and nucleotides having modified uridine include 5-cyanouridine or 4'-thiouridine.

[0159] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases, nucleosides, and nucleotides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine.

[0160] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases, nucleosides, and nucleotides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0161] In one embodiment, the polynucleotides of the invention, such as IVT polynucleotides, may have uniform chemical modifications of all or any of the same nucleotide types, or a population of modifications resulting from a simple downward titration of the same starting modifications in all or any of the same nucleotide types, or a measured percentage of chemical modifications of any of the same nucleotide types but randomly incorporated, such as when all uridines are replaced with uridine analogs, e.g., pseudouridines. In another embodiment, the polynucleotides may have two, three, or four uniform chemical modifications of the same nucleotide type throughout the polynucleotide (such as all uridines and all cytosines being similarly modified). When the polynucleotides of the invention are chemically and / or structurally modified, the polynucleotides may be referred to as "modified polynucleotides."

[0162] Generally, the length of an IVT polynucleotide (e.g., IVT RNA) encoding a polypeptide of interest is greater than about 30 nucleotides (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, , 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or more, up to a maximum of 100,000 nucleotides.

[0163] In some embodiments, the IVT polynucleotide (e.g., IVT RNA) is from about 30 to about 100,000 nucleotides (e.g., 30-50, 30-100, 30-250, 30-500, 30-1,000, 30-1,500, 30-3,000, 30-5,000, 30-7,000, 30-10,000, 30-25,000, 30-50,000, 30-70,000, 100-250, 100-500, 100-1,000, 100-1,500, 00~3,000, 100~5,000, 100~7,000, 100~10,000, 100~25,000, 100~50,000, 100~70,000, 100~100,000, 500~1,000, 500~1,500, 500~2,000, 500~3,000, 500~5,000, 500~7,000, 500~10,000, 500~25,000, 500~50,000, 00~70,000, 500~100,000, 1,000~1,500, 1,000~2,000, 1,000~3,000, 1,000~5,000, 1,000~7,000, 1,000~10,000, 1,000~25,000, 1,000~50,000, 1,000~70,000, 1,000~100,000, 1,500~3,000, 1,500~5,000, 1,500~7 ,000, 1,500-10,000, 1,500-25,000, 1,500-50,000, 1,500-70,000, 1,500-100,000, 2,000-3,000, 2,000-5,000, 2,000-7,000, 2,000-10,000, 2,000-25,000, 2,000-50,000, 2,000-70,000, and 2,000-100,000).

[0164] In some embodiments, the nucleic acids described herein are chimeric polynucleotides. The chimeric polynucleotides or RNA constructs maintain a similar modular structure as the IVT polynucleotides, but the chimeric polynucleotides contain one or more structural and / or chemical modifications or alterations that confer useful properties to the polynucleotide. Thus, the chimeric polynucleotides that are modified RNA molecules of the present invention are referred to as "chimeric modified RNAs" or "chimeric RNAs". The chimeric polynucleotides have portions or regions that differ in size and / or chemical modification pattern, chemical modification position, chemical modification percentage or population, and combinations thereof.

[0165] Polypeptide of interest In some embodiments of the invention, the invention is one or more of the following: mRNA, modified mRNA, unmodified RNA, lncRNA, self-replicating RNA, circular RNA, CRISPR guide RNA, etc. In this embodiment, the RNA is an RNA that encodes a polypeptide, such as an mRNA or a self-replicating RNA.

[0166] In an exemplary aspect of the invention, the highly pure RNA composition is used to generate a polypeptide of interest, e.g., a therapeutic protein, a vaccine antigen, etc. In some embodiments, the nucleic acid is a therapeutic RNA. As used herein, the term "therapeutic mRNA" refers to an mRNA that encodes a therapeutic protein. Therapeutic proteins mediate various effects in a host cell or subject to treat a disease or ameliorate the signs and symptoms of a disease. For example, therapeutic proteins may replace a protein that is missing or abnormal, enhance the function of an endogenous protein, provide a new function to a cell (e.g., inhibit or activate an endogenous cellular activity), or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNAs may be useful in the treatment of the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders.

[0167] Thus, the polynucleotides of the present invention may be used as therapeutic or preventive agents. They are provided for use in the medical field. For example, RNA described herein may be administered to a subject, where the polynucleotide is translated in vivo to generate a therapeutic peptide. Compositions, methods, kits, and reagents are provided for the diagnosis, treatment, or prevention of diseases or conditions in humans and other mammals. The active therapeutic agents of the present invention include polynucleotides, cells containing polynucleotides, or polypeptides translated from polynucleotides.

[0168] The polynucleotide can be induced to translate in a cell, tissue or organism. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue or organism is contacted with an effective amount of a composition containing a polynucleotide that contains an RNA polynucleotide.

[0169] An "effective amount" of a polynucleotide is provided based, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., size and degree of modified nucleotides), as well as other components and other determinants of the polynucleotide. In general, an effective amount of a polynucleotide provides for induced or enhanced peptide production in a cell, preferably more efficiently than a composition containing a corresponding unmodified polynucleotide encoding the same peptide. Increased peptide production can be demonstrated by increased cell transfection, increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., as indicated by increased duration of protein translation from a modified polynucleotide), or altered peptide production in the host cell.

[0170] The RNA of the invention may be designed to encode a polypeptide of interest selected from any of several target categories including, but not limited to, a biologic, an antibody, a vaccine, a therapeutic protein or peptide, a cell penetrating peptide, a secreted protein, a plasma membrane protein, a cytoplasmic or cytoskeletal protein, an intracellular membrane associated protein, a nuclear protein, a protein associated with a human disease, a targeting moiety, or a protein encoded by the human genome that has not been identified for a therapeutic indication but that nevertheless has utility in the fields of research and discovery. A "therapeutic protein" refers to a protein that has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect when administered to a cell.

[0171] The RNA disclosed herein may code for one or more biologics. As used herein, a "biologic" is a polypeptide-based molecule that can be produced by the methods provided herein and used to treat, cure, alleviate, prevent, or diagnose serious or life-threatening diseases or conditions. Biologics according to the present invention include, but are not limited to, allergenic extracts (e.g., for allergy injections and testing), blood components, gene therapy products, human tissue or cell products used for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytic drugs, and immunomodulatory drugs, among others.

[0172] According to the present invention, one or more biologics currently on the market or in development may be encoded by the RNA of the present invention. Without wishing to be bound by theory, it is believed that the incorporation of coding polynucleotides of known biologics into the RNA of the present invention provides improved therapeutic efficacy, at least in part, due to the specificity, purity, and / or selectivity of the construct design.

[0173] The RNA disclosed herein may encode one or more antibodies or fragments thereof. The term "antibody" includes monoclonal antibodies (including full-length antibodies with immunoglobulin Fc regions), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site.

[0174] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include, but are not limited to, "primatized" antibodies that contain variable domain antigen-binding sequences from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0175] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; nanobodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0176] Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, may be encoded by the RNA of the present invention, including heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. Polynucleotide sequences encoding the subclasses gamma and mu are also included. Thus, any subclass of antibody may be encoded in part or in whole, and includes the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In accordance with the present invention, one or more antibodies or fragments currently commercially available or in development may be encoded by the RNA of the present invention.

[0177] Antibodies encoded by the RNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas including, but not limited to, hematology, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory system, sensory system and anti-infective.

[0178] In one embodiment, the RNA disclosed herein may encode a monoclonal antibody and / or variants thereof. Antibody variants also include, but are not limited to, substitution variants, conservative amino acid substitutions, insertion variants, deletion variants, and / or covalent derivatives. In one embodiment, the RNA disclosed herein may encode an immunoglobulin Fc region. In another embodiment, the RNA may encode a variant immunoglobulin Fc region.

[0179] The mRNA disclosed herein can encode one or more vaccine antigens. As used herein, a "vaccine antigen" is a biological preparation that improves immunity against a particular disease or infectious agent. According to the present invention, one or more vaccine antigens currently on the market or under development can be encoded by the RNA of the present invention.

[0180] Vaccine antigens encoded in the RNA of the present invention may be utilized to treat conditions or diseases in many therapeutic areas, including but not limited to cancer, allergies, and infectious diseases. In some embodiments, the cancer vaccine may be a personalized cancer vaccine in the form of individual RNAs encoding concatemers or peptide epitopes, or a combination thereof.

[0181] The RNA of the invention can be designed to encode one or more antimicrobial peptides (AMPs) or antiviral peptides (AVPs). AMPs and AVPs have been isolated and described from a wide range of animals, including, but not limited to, microbes, invertebrates, plants, amphibians, birds, fish, and mammals. The antimicrobial polypeptides described herein can block cell fusion and / or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, the antimicrobial polypeptides can comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of a viral envelope protein, e.g., the transmembrane subunit of HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of HIV-1 gp120 or gp41 are described, for example, in Kuiken et al., (2008) “HIV Sequence Compendium,” Los Alamos National Laboratory.

[0182] In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein sequence.

[0183] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of the capsid binding protein. In some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding sequence of the capsid binding protein.

[0184] The antimicrobial polypeptides described herein can block protease dimerization and inhibit cleavage of viral proproteins (e.g., HIV Gag-pol processing) into functional proteins, thereby preventing the release of one or more enveloped viruses (e.g., HIV, HCV). In some embodiments, the antimicrobial polypeptides can have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence.

[0185] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a protease binding protein, hi some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of the protease binding protein.

[0186] A non-limiting list of infectious diseases that the RNA vaccine antigens or antimicrobial peptides may treat are as follows: human immunodeficiency virus (HIV), HIV causing mycobacterial infections, AIDS-associated cachexia, AIDS-associated cytomegalovirus infection, HIV-associated nephropathy, lipodystrophy, AIDS-associated cryptococcal meningitis, AIDS-associated neutropenia, Pneumocystis jiroveci (Pneumocystis carinii infections, AIDS-associated toxoplasmosis, Hepatitis A, B, C, D or E, Herpes, Shingles (Chickenpox), Rubella (Rubella Virus), Yellow Fever, Dengue Fever etc. (Flaviviruses), Influenza (Influenza Virus), Hemorrhagic Infections (Marburg or Ebola Virus), Bacterial Infections such as Legionnaires' Disease (Legionella), Gastric Ulcers (Helicobacter), Cholera (Vibrio), E. coli infections, Staphylococcus aureus infections, Salmonella infections or Streptococcus aureus infections, Tetanus (Clostridium tetani), Protozoal Infections (Malaria, Sleeping Sickness, Leishmaniasis, Toxic Substances), xoplasmosis, i.e. infections caused by Plasmodium, Trypanosoma, Leishmania and Toxoplasma), diphtheria, leprosy, measles, whooping cough, rabies, tetanus, tuberculosis, typhoid, chickenpox, diarrheal infections, e.g. amebiasis, Clostridium difficile associated diarrhea (CDAD), cryptosporidiosis, giardiasis, cyclosporiasis and rotavirus gastroenteritis, encephalitis such as Japanese encephalitis, Western equine encephalitis and tick-borne encephalitis (TBE), fungal skin diseases, e.g. candidiasis, onychomycosis, tinea capitis / tinea gracilis, tinea corporis (Tinea corporis), corporis / body ringworm, Jock itch, Sporotrichosis and Tinea pedis / Athlete's foot, Meningitis, e.g. Haemophilus influenzae type b (Hib), Meningitis, viral, meningococcal and pneumococcal infections, Neglected tropical diseases, e.g. Argentine hemorrhagic fever, Leishmaniasis, Nematode / worm infections, Ross River virus infection and West Nile virus (WNV) disease, Non-HIVSTDs such as Trichomoniasis, Human Papilloma Virus (HPV) infections, Sexually Transmitted Chlamydia Disease, Chancroid and Syphilis, Non-specific bacterial infections such as Cellulitis, Lyme Disease, MRSA infections, Pseudomonas, Staphylococcus aureus infections, Button Fever, Leptospirosis, Rheumatic Fever, Botulism, Rickettsiosis and Mastoiditis, Parasitic infections such as Cysticercosis, Echinococcosis, Trematode / Fluke Infections infection), Trichinellosis, Babesiosis, Cattle fly larvae, Diphyllobothriasis and Trypanosomiasis, Respiratory infections such as Adenovirus infection, Aspergillosis infection, Avian (H5N1) influenza, Influenza, RSV infection, Severe Acute Respiratory Syndrome (SARS), Sinusitis, Legionnaires' disease, Coccidioidomycosis and Swine (H1N1) influenza, Sepsis such as bacteremia, Sepsis / septic shock, Sepsis in premature infants, Urinary tract infections such as Vaginal infections (bacterial), Vaginal infections (fungal) and Gonococcal infections, Viral skin diseases such as B19 parvovirus infection, Warts, Genital herpes, Orofacial herpes, Shingles, Inner ear infections, Fetal cytomegalovirus syndrome, Foodborne infections such as Brucellosis (Brucella spp.), Clostridium perfringens (Epsilon toxin), E. coli O157:H7 (Escherichia coli), Salmonellosis (Salmonella spp.), Shinglellosis (Shingella), Vibriosis and Listeriosis, Bioterrorism and Potentially Endemic Diseases such as Ebola Hemorrhagic Fever, Lassa Fever, Marburg Hemorrhagic Fever, Plague, Nipah Virus Disease, Hantavirus, Smallpox, Glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydia psittaci), Q Fever (Coxiella burnetii), Tularemia (Fancisella tularensis), Rubella, Mumps and Polio.

[0187] The RNA disclosed herein may encode one or more confirmed or "in-test" therapeutic proteins or peptides. According to the present invention, one or more therapeutic proteins or peptides currently on the market or in development may be encoded by the RNA of the present invention. The therapeutic proteins and peptides encoded by the RNA of the present invention may be utilized to treat conditions or diseases in many therapeutic areas, including, but not limited to, hematology, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, genetics, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory system, sensory system, and anti-infective.

[0188] The RNA disclosed herein may code for one or more cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide" or CPP refers to a polypeptide that can facilitate intracellular uptake of a molecule. The cell-penetrating polypeptide of the present invention may include one or more detectable labels. The polypeptide may be partially labeled or completely labeled throughout. The RNA may completely, partially, or not at all encode a detectable label. The cell-penetrating peptide may also include a signal sequence. As used herein, "signal sequence" refers to a sequence of amino acid residues attached to the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-penetrating polypeptide.

[0189] In one embodiment, the RNA may also code for a fusion protein. The fusion protein may be made by operably linking a charged protein to a therapeutic protein. As used herein, "operably linked" refers to a therapeutic protein and a charged protein that are linked in a manner that allows for the expression of the complex when introduced into a cell. As used herein, "charged protein" refers to a protein that carries a positive, negative or overall neutral charge. Preferably, the therapeutic protein may be covalently linked to the charged protein in the formation of a fusion protein. The ratio of surface charge to total or surface amino acids may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.

[0190] The cell-penetrating polypeptide encoded by the RNA can form a complex after translation, which can include a charged protein linked, e.g., covalently attached, to the cell-penetrating polypeptide.

[0191] In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, "protein-binding partner" includes, but is not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be secreted from a cell into which the RNA may be introduced. The cell-penetrating polypeptide may also penetrate the first cell.

[0192] In one embodiment, the RNA can code for a cell-permeable polypeptide that can include a protein-binding partner.The protein-binding partner can include, but is not limited to, an antibody, a supercharged antibody, or a functional fragment.The RNA can be introduced into a cell into which the cell-permeable polypeptide that includes the protein-binding partner is introduced.

[0193] Human and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane-bound compartment, or organelle, contains different proteins essential for the function of the organelle. Cells use "sorting signals," amino acid motifs located within proteins, to direct proteins to specific organelles. One type of sorting signal, called a signal sequence, signal peptide, or leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER).

[0194] Proteins targeted to the ER by signal sequences can be released into the extracellular space as secreted proteins. Similarly, proteins present on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of the "linker" that holds the protein to the membrane. Without wishing to be bound by theory, the molecules of the present invention can be used to exploit the above cellular transport. Thus, in some embodiments of the present invention, RNA is provided to express secreted proteins. In one embodiment, these may be used to manufacture large quantities of valuable human gene products.

[0195] In some embodiments of the invention, RNA is provided to express a plasma membrane protein.

[0196] In some embodiments of the invention, RNA is provided to express a cytoplasmic or cytoskeletal protein.

[0197] In some embodiments of the invention, RNA is provided to express an intracellular membrane-associated protein.

[0198] In some embodiments of the invention, RNA is provided to express nuclear proteins.

[0199] In some embodiments of the invention, RNA is provided to express a protein associated with a human disease.

[0200] The RNA may have the nucleotide sequence of a natural or naturally occurring RNA, or may have a nucleotide sequence that codes for a natural or naturally occurring peptide. Alternatively, the RNA may have a nucleotide sequence that has a certain percent identity to the nucleotide sequence of a natural or naturally occurring RNA, or the mRNA may have a nucleotide sequence that codes for a peptide that has a certain percent identity to the nucleotide sequence of a natural or naturally occurring peptide. The term "identity" as known in the art refers to the relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences with gap alignment (if any) that is addressed by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related peptides can be easily calculated by known methods.Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0201] Thus, in some embodiments, the peptide encoded by the RNA is a polypeptide variant that may have the same or similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) compared to the reference polypeptide. In general, a variant of a particular polynucleotide or polypeptide of the present invention has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to the particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Tools for such alignment include the BLAST suite of tools (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997) and "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402.). Other tools are described herein, particularly in the definition of "identity". Default parameters in the BLAST algorithm include, for example, expectation threshold 10, word size 28, match / mismatch score 1, -2, gap cost linear. Optional filters may be applied, as well as selection for species-specific repeats, such as Homo sapiens.

[0202] According to the present invention, a polynucleotide comprises an RNA for encoding one or more polypeptides of interest or fragments thereof. A polypeptide of interest includes, but is not limited to, a whole polypeptide, multiple polypeptides, or fragments of a polypeptide. As used herein, the term "polypeptide of interest" refers to any polypeptide selected to be encoded in a primary construct of the present invention. As used herein, "polypeptide" refers to a polymer (natural or non-natural) of amino acid residues most often linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the encoded polypeptide is smaller than about 50 amino acids, so the polypeptide is referred to as a peptide. When the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the above. A polypeptide may be a single molecule or a multi-molecular complex such as a dimer, trimer, or tetramer. They may also include single or multi-chain polypeptides, such as antibodies or insulin, and may be associated or linked. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acids.

[0203] The term "polypeptide variants" refers to molecules whose amino acid sequences differ from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least about 50% identity to the native or reference sequence, and preferably, they are at least about 80%, more preferably at least about 90% identical to the native or reference sequence.

[0204] In some embodiments, "mutant mimics" are provided. As used herein, the term "mutant mimics" includes one or more amino acids that will mimic an activation sequence. For example, glutamate can serve as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, mutant mimics may result in inactivation or may result in an inactivated product containing the mimic, for example, phenylalanine may act as an inactivating replacement for tyrosine; or alanine may act as an inactivating replacement for serine.

[0205] The present invention contemplates several types of polypeptide-based compositions, including variants and derivatives. These include substitutional, insertional, deletional and covalent variants and derivatives. The term "derivative" is used interchangeably with the term "variant," but generally refers to a molecule that is modified and / or altered in some way relative to a reference or starting molecule.

[0206] As such, RNAs encoding polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications with respect to a reference sequence, particularly the polypeptide sequences disclosed herein, are included within the scope of the present invention. For example, sequence tags or amino acids such as one or more lysines may be added to the peptide sequences of the present invention (e.g., at the N-terminus or C-terminus). Sequence tags may be used for peptide purification or for localization. Lysines may be used to increase the solubility of the peptide or to allow biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein may be optionally deleted to provide a truncated sequence. Certain amino acids (e.g., C- or N-terminal residues) may instead be deleted depending on the use of the sequence, for example, expression of the sequence as part of a larger sequence that is soluble or bound to a solid.

[0207] A "substitutional variant" when referring to a polypeptide is one in which at least one amino acid residue in a native or starting sequence has been removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been replaced, or multiple, where two or more amino acids have been replaced in the same molecule.

[0208] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine. Furthermore, the substitution of a basic residue, such as lysine, arginine, or histidine, for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another, are further examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, methionine, for a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a non-polar residue.

[0209] An "insertion variant" when referring to a polypeptide is one that has one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native or starting sequence. "Directly adjacent to" an amino acid means bound to either the alpha-carboxy or alpha-amino functionality of that amino acid.

[0210] "Deletion variants" when referring to polypeptides are those in which one or more amino acids in a native or starting amino acid sequence have been removed. Typically, deletion variants will have one or more amino acids deleted in a particular region of the molecule.

[0211] "Covalent derivatives" when referring to polypeptides include modifications of native or starting proteins with organic protein or non-protein derivative derivatizing agents and / or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of proteins with organic derivatizing agents capable of reacting with selected side chains or terminal residues, or by exploiting post-translational modification mechanisms operative in selected recombinant host cells. The resulting covalent derivatives are useful in programs directed at identifying residues important for biological activity, immunoassays, or preparation of anti-protein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the skill of the art and are performed without undue experimentation.

[0212] Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the polypeptides produced according to the invention.

[0213] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, and methylation of the alpha amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).

[0214] The term "domain," as used herein with respect to polypeptides, refers to a motif in a polypeptide having one or more identifiable structural or functional features or characteristics (e.g., a binding capacity to serve as a site of protein-protein interaction).

[0215] As used herein with respect to polypeptides, the term "site" with respect to amino acid-based embodiments is used interchangeably with "amino acid residue" and "amino acid side chain." A site represents a position within a peptide or polypeptide that can be modified, manipulated, altered, derivatized, or diversified within a polypeptide-based molecule of the invention.

[0216] As used herein, the term "terminal" or "terminal" refers to the end of a peptide or polypeptide when referring to a polypeptide. Such ends are not limited to only the first or last site of a peptide or polypeptide, but may include additional amino acids in the terminal region. The polypeptide-based molecules of the invention may be characterized as having both an N-terminus (terminating with an amino acid having a free amino group (NH2)) and a C-terminus (terminating with an amino acid having a free carboxyl group (COOH)). The proteins of the invention are in some cases composed of multiple polypeptide chains held together by disulfide bonds or by non-covalent forces (multimers, oligomers). These types of proteins will have multiple N-terminuses and C-terminuses. Alternatively, the ends of the polypeptide may be modified to begin or end with non-polypeptide moieties, such as organic complexes, in some cases.

[0217] Once any features have been identified or defined as desired components of the polypeptide encoded by the RNA of the present invention, any of the manipulations and / or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that the manipulation of features may result in the same outcome as a modification to the molecules of the present invention. For example, manipulations including deleting domains will result in alteration of the length of the molecule, as will modifying the nucleic acid to encode less than full-length molecules.

[0218] Modification and manipulation may be accomplished by methods known in the art, such as, but not limited to, site-directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays, such as those described herein, or any other suitable screening assay known in the art.

[0219] Formulations / pharmaceutical compositions The present invention provides polynucleotides and pharmaceutical compositions thereof, optionally in combination with one or more pharma- ceutically acceptable excipients. Pharmaceutical compositions may optionally contain one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. Pharmaceutical compositions of the present invention may be sterile and / or pyrogen-free. General considerations in the manufacture of formulations and / or medicaments may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety.

[0220] In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to a polynucleotide, e.g., a polynucleotide encoding an mRNA, that is delivered as described herein.

[0221] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the steps of bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients and then, as necessary and / or desirable, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

[0222] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as on the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100%, such as from 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0223] The polynucleotides of the invention may be formulated with one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g., from a depot formulation); (4) alter biodistribution (e.g., target specific tissues or cell types); (5) increase translation of the encoded protein in vivo; and / or (6) alter the release profile of the encoded protein in vivo. In addition to conventional excipients such as any solvent, dispersion medium, diluent, or other liquid vehicle, dispersing or suspending aid, surfactant, isotonicity agent, thickening or emulsifying agent, preservative, excipient, excipients of the invention include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, and combinations thereof.

[0224] In some embodiments, the nucleic acid molecule of the present invention may be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.In one embodiment, the pharmaceutical composition of the nucleic acid molecule comprises lipid nanoparticles (LNPs).In some embodiments, the lipid nanoparticles are MC3-based lipid nanoparticles.

[0225] In one embodiment, the polynucleotide may be formulated into a lipid-polycation complex. The formation of the lipid-polycation complex may be achieved by methods known in the art. As a non-limiting example, the polycation may include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In another embodiment, the polynucleotide may be formulated into a lipid-polycation complex, which may further include non-cationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0226] Liposome formulations may be influenced by biophysical parameters, including but not limited to cationic lipid component selection, cationic lipid saturation, PEGylation nature, total component ratio, and size.In one example by Semple et al. (Semple et al. Nature Biotech. 2010 28:172-176: incorporated herein by reference in its entirety), liposome formulations were composed of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, varying the composition of cationic lipids can more effectively deliver siRNA to various antigen-presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; incorporated herein by reference in its entirety). In some embodiments, the liposome formulation may comprise about 35% to about 45% cationic lipid, about 40% to about 50% cationic lipid, about 50% to about 60% cationic lipid, and / or about 55% to about 65% cationic lipid. In some embodiments, the lipid to RNA ratio in the liposome may be about 5:1 to about 20:1, about 10:1 to about 25:1, about 15:1 to about 30:1, and / or at least 30:1.

[0227] In some embodiments, the percentage of PEG in a lipid nanoparticle (LNP) formulation may be increased or decreased, and / or the carbon chain length of the PEG lipid may be changed from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the LNP formulation. As a non-limiting example, the LNP formulation may include a lipid molar ratio of about 0.5% to about 3.0%, about 1.0% to about 3.5%, about 1.5% to about 4.0%, about 2.0% to about 4.5%, about 2.5% to about 5.0%, and / or about 3.0% to about 6.0% PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In another embodiment, the PEG-c-DOMG may be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol) and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

[0228] In one embodiment, the polynucleotide is formulated into a nanoparticle that may include at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In another aspect, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipid may be a lipid described in U.S. Patent Publication No. 20130150625, the entirety of which is incorporated herein by reference, and / or may be a lipid produced by a method therein. As non-limiting examples, cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 in US20130150625); or any pharma- ceutically acceptable salt or stereoisomer thereof.

[0229] Lipid nanoparticle formulations typically include lipids, particularly ionizable cationic lipids such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), as well as neutral lipids, sterols, and molecules that may reduce particle aggregation, such as PEG or PEG-modified lipids.

[0230] In one embodiment, the lipid nanoparticle formulation consists essentially of: (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; and 0.5-15% PEG-lipid.

[0231] In one embodiment, the formulation comprises from about 25% to about 75% on a molar basis, e.g., from about 35 to about 65%, about 45 to about 65%, about 60%, about 57.5%, about 50%, or about 40%, of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0232] In one embodiment, the formulation comprises about 0.5% to about 15% neutral lipid on a molar basis, for example, about 3 to about 12%, about 5 to about 10%, or about 15%, about 10%, or about 7.5% on a molar basis. Exemplary neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In one embodiment, the formulation comprises about 5% to about 50% sterol on a molar basis (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis). An exemplary sterol is cholesterol. In one embodiment, the formulation comprises about 0.5% to 20% PEG or PEG-modified lipid on a molar basis (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis). In one embodiment, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2,000, e.g., about 1,500 Da, about 1,000 Da, or about 500 Da. Exemplary PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA.

[0233] In one embodiment, the formulation of the invention comprises, on a molar basis, 25-75% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% neutral lipid, 5-50% sterol, and 0.5-20% PEG or PEG-modified lipid.

[0234] In one embodiment, the formulation of the invention comprises, on a molar basis, 35-65% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% neutral lipid, 15-45% sterol, and 0.5-10% PEG or PEG-modified lipid.

[0235] In one embodiment, the formulation of the invention comprises, on a molar basis, 45-65% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and Di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% neutral lipid, 25-40% sterol, and 0.5-10% PEG or PEG-modified lipid.

[0236] In one embodiment, a formulation of the invention comprises, on a molar basis, about 60% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% neutral lipid, about 31% sterol, and about 1.5% PEG or PEG-modified lipid.

[0237] In one embodiment, a formulation of the invention comprises, on a molar basis, about 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 38.5% sterol, and about 1.5% PEG or PEG-modified lipid.

[0238] In one embodiment, a formulation of the invention comprises, on a molar basis, about 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 35% sterol, about 4.5% or about 5% PEG or PEG-modified lipid, and about 0.5% target lipid.

[0239] In one embodiment, a formulation of the invention comprises, on a molar basis, about 40% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% neutral lipid, about 40% sterol, and about 5% PEG or PEG-modified lipid.

[0240] In one embodiment, a formulation of the invention comprises about 57.2% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1% neutral lipid, about 34.3% sterol, and about 1.4% PEG or PEG-modified lipid on a molar basis.

[0241] In one embodiment, a formulation of the invention comprises, on a molar basis, about 57.5% cationic lipid selected from PEG lipids, PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005) (the entire contents of which are incorporated herein by reference)), about 7.5% neutral lipid, about 31.5% sterol, and about 3.5% PEG or PEG-modified lipid.

[0242] In a preferred embodiment, the lipid nanoparticle formulation consists essentially of a lipid mixture having a molar ratio of about 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid; more preferably, a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

[0243] In certain embodiments, the lipid molar ratio is about 50 / 10 / 38.5 / 1.5 (mol % cationic lipid / neutral lipid, e.g., DSPC / cholesterol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1134.3 / 1.4 (mol % cationic lipid / neutral lipid, e.g., DPPC / cholesterol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol % cationic lipid / neutral lipid, e.g., DSPC / cholesterol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (mol % cationic lipid / neutral lipid, e.g., DSPC / cholesterol / PEG-modified lipid, e.g., , PEG-DSG), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid, e.g. DSPC / cholesterol / PEG-modified lipid, e.g. PEG-DMG), 40 / 10 / 40 / 10 (mol % cationic lipid / neutral lipid, e.g. DSPC / cholesterol / PEG-modified lipid, e.g. PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol % cationic lipid / neutral lipid, e.g. DSPC / cholesterol / PEG-modified lipid, e.g. PEG-DMG or PEG-cDMA) or 52 / 13 / 30 / 5 (mol % cationic lipid / neutral lipid, e.g. DSPC / cholesterol / PEG-modified lipid, e.g. PEG-DMG or PEG-cDMA).

[0244] Exemplary lipid nanoparticle compositions and methods of making the same are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51:8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578, the contents of each of which are incorporated by reference in their entirety.

[0245] In one embodiment, the lipid nanoparticle formulations described herein may include cationic lipids, PEG lipids, and structured lipids, and may optionally include non-cationic lipids. As a non-limiting example, the lipid nanoparticles may include about 40-60% cationic lipids, about 5-15% non-cationic lipids, about 1-2% PEG lipids, and about 30-50% structured lipids. As another non-limiting example, the lipid nanoparticles may include about 50% cationic lipids, about 10% non-cationic lipids, about 1.5% PEG lipids, and about 38.5% structured lipids. As yet another non-limiting example, the lipid nanoparticles may include about 55% cationic lipids, about 10% non-cationic lipids, about 2.5% PEG lipids, and about 32.5% structured lipids. In one embodiment, the cationic lipids may be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0246] In one embodiment, the lipid nanoparticle formulation described herein may be a four-component lipid nanoparticle. The lipid nanoparticle may comprise a cationic lipid, a non-cationic lipid, a PEG lipid, and a structured lipid. As a non-limiting example, the lipid nanoparticle may comprise about 40-60% cationic lipid, about 5-15% non-cationic lipid, about 1-2% PEG lipid, and about 30-50% structured lipid. As another non-limiting example, the lipid nanoparticle may comprise about 50% cationic lipid, about 10% non-cationic lipid, about 1.5% PEG lipid, and about 38.5% structured lipid. As yet another non-limiting example, the lipid nanoparticle may comprise about 55% cationic lipid, about 10% non-cationic lipid, about 2.5% PEG lipid, and about 32.5% structured lipid. In one embodiment, the cationic lipid may be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0247] In one embodiment, the lipid nanoparticle formulations described herein may include cationic lipids, non-cationic lipids, PEG lipids, and structured lipids. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-KC2-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DOMG, and about 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-MC3-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DOMG, and about 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-MC3-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DMG, and about 38.5% structured lipid cholesterol. As yet another non-limiting example, the lipid nanoparticles comprise about 55% cationic lipid L319, about 10% non-cationic lipid DSPC, about 2.5% PEG lipid PEG-DMG, and about 32.5% structural lipid cholesterol.

[0248] In one embodiment, the polynucleotide of the present invention has a diameter of about 10 to about 100 nm, for example, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm.

[0249] In one embodiment, the lipid nanoparticles may have a diameter of about 10-500 nm. In one embodiment, the lipid nanoparticles may have a diameter of more than 100 nm, more than 150 nm, more than 200 nm, more than 250 nm, more than 300 nm, more than 350 nm, more than 400 nm, more than 450 nm, more than 500 nm, more than 550 nm, more than 600 nm, more than 650 nm, more than 700 nm, more than 750 nm, more than 800 nm, more than 850 nm, more than 900 nm, more than 950 nm, or more than 1000 nm. In some embodiments, the cationic lipid nanoparticles have an average diameter of 50-150 nm. In some embodiments, the cationic lipid nanoparticles have an average diameter of 80-100 nm.

[0250] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. For example, the composition may contain 0.1%-99% (w / w) active ingredient. By way of example, the composition may contain 0.1%-100%, e.g., 0.5-50%, 1-30%, 5-80%, at least 80% (w / w) active ingredient.

[0251] In one embodiment, the composition contains a polynucleotide as described herein formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC and PEG2000-DMG, buffer trisodium citrate, sucrose, and water for injection. As a non-limiting example, the composition comprises: 2.0 mg / mL drug substance, 21.8 mg / mL MC3, 10.1 mg / mL cholesterol, 5.4 mg / mL DSPC, 2.7 mg / mL PEG2000-DMG, 5.16 mg / mL trisodium citrate, 71 mg / mL sucrose, and about 1.0 mL water for injection.

[0252] The polynucleotides of the present invention may be administered by any route that produces a therapeutically effective result. The present invention provides a method that includes administering a polynucleotide according to the present invention to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its method of administration, its method of activity, and the like. The compositions according to the present invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present invention may be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or suitable imaging dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the particular composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the particular compound employed; the duration of treatment; drugs used in combination or simultaneously with the particular compound used; and similar factors well known in the medical arts.

[0253] In certain embodiments, compositions according to the invention may be administered one or more times per day at a dosage level sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg of subject body weight per kg per day to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). When multiple administrations are employed, split dosing schedules, such as those described herein, may be used.

[0254] The polynucleotide pharmaceutical compositions described herein may be formulated into dosage forms described herein, such as intranasally, intratracheally, or for injection (e.g., intravenously, intraocularly, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0255] The present invention provides pharmaceutical compositions comprising polynucleotides (eg, RNA molecules) and polynucleotide compositions and / or complexes, optionally in combination with one or more pharma- ceutically acceptable excipients.

[0256] The present invention provides polynucleotides (e.g., RNA molecules) and related pharmaceutical compositions and complexes, optionally in combination with one or more pharma- ceutical acceptable excipients. Pharmaceutical compositions may optionally include one or more additional active agents, such as therapeutic and / or prophylactic active agents. Pharmaceutical compositions of the present invention may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical products may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety.

[0257] In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to the polynucleotide (e.g., RNA molecule) to be delivered as described herein.

[0258] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals are well understood, and a skilled veterinary pharmacologist may design and / or perform such modifications, if any, using no more than routine experimentation. Subjects to which the pharmaceutical compositions are intended to be administered include, but are not limited to, humans and / or other primates; mammals, such as commercially relevant mammals, e.g., cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, such as commercially relevant birds, e.g., poultry, chickens, ducks, geese, and / or turkeys.

[0259] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include the steps of bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients and then, as necessary and / or desired, dividing, shaping and / or packaging the product into desired single or multiple dosage units.

[0260] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as on the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100%, such as from 0.5 to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) active ingredient.

[0261] The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and may be practiced or carried out in various ways. Moreover, the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," "having," "containing," "including," and variations thereof herein means the inclusion of the items listed thereafter and equivalents thereof, as well as additional items. EXAMPLES

[0262] Example 1. Preparation of polynucleotides In accordance with the present invention, production of polynucleotides and / or portions or regions thereof may be accomplished utilizing the methods taught in WO2014 / 152027, filed March 15, 2013, entitled "Manufacturing Methods for Production of RNA Transcripts" (Attorney Docket No. M500), the contents of which are incorporated herein by reference in their entireties. Purification methods may include those taught in International Patent Applications WO2014 / 152030 and WO2014 / 152031, each of which is incorporated herein by reference in its entirety.

[0263] Methods for detecting and characterizing polynucleotides may be performed as taught in WO2014 / 144039, which is incorporated herein by reference in its entirety.

[0264] Characterization of the polynucleotides of the present disclosure may be accomplished using a procedure selected from the group consisting of polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, and detection of RNA impurities, where characterization includes determining the RNA transcript sequence, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript. Such methods are taught, for example, in WO2014 / 144711 and WO2014 / 144767, the contents of each of which are incorporated herein by reference in their entirety.

[0265] Example 2. Chimeric Polynucleotide Synthesis Preface In accordance with the present invention, two regions or portions of a polynucleotide may be joined or linked using triphosphate chemistry.

[0266] According to this method, an initial region or portion of up to 100 nucleotides is chemically synthesized with a 5' monophosphate and a terminal 3' desOH or blocked OH. If the region is longer than 80 nucleotides, it can be synthesized as a double strand for ligation.

[0267] If the initial region or portion is synthesized as a non-positionally modified region or portion using in vitro transcription (IVT), this may be followed by conversion of the 5' monophosphate followed by capping of the 3' end.

[0268] The monophosphate protecting group may be selected from any protecting group known in the art.

[0269] The second region or portion of the polynucleotide may be synthesized using either chemical synthesis or IVT techniques. IVT techniques may include an RNA polymerase that can utilize a primer with a modified cap. Alternatively, a cap of up to 130 nucleotides may be chemically synthesized and attached to the IVT region or a portion thereof.

[0270] Regarding the ligation method, note that ligation with DNA T4 ligase, followed by treatment with DNAse should avoid easy ligation.

[0271] It is not necessary for the entire polynucleotide to be produced with a phosphate-sugar backbone. If one of the regions or portions encodes a polypeptide, it is preferred that such region or portion comprises a phosphate-sugar backbone.

[0272] Ligation is then carried out using any known click chemistry, ortho-click chemistry, solulink, or other bioconjugate chemistry known to those of skill in the art.

[0273] Synthetic Route Chimeric polynucleotides are generated using a series of starting segments, including: (a) A capped and protected 5' segment (SEG.1) consisting of a normal 3'OH. (b) a 5' triphosphate segment (SEG.2) that may include a coding region for a polypeptide and that contains a normal 3' OH group; (c) a 5' monophosphate segment (SEG.3) at the 3' end (e.g., tail) of the polynucleotide that contains cordycepin or does not contain a 3' OH.

[0274] After synthesis (chemical or IVT), segment 3 (SEG.3) is treated with cordycepin and then pyrophosphatase to generate the 5' monophosphate.

[0275] Segment 2 (SEG.2) is then ligated to SEG.3 using RNA ligase. The ligated polynucleotide is then purified and treated with pyrophosphatase to cleave the diphosphate. The treated SEG.2-SEG.3 construct is then purified and SEG.1 is ligated to the 5' end. Further purification steps of the chimeric polynucleotide may be performed.

[0276] Where the polynucleotide encodes a polypeptide, the linked or connected segments may be represented as 5'UTR (SEG.1), open reading frame or ORF (SEG.2) and 3'UTR plus polyA (SEG.3).

[0277] The yield of each step may be about 90 to 95%.

[0278] Example 3: PCR for preparation of DNA templates The PCR procedure for preparation of cDNA is carried out using 2x KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, MA). The system contains 12.5 μl 2x KAPA ReadyMix; 0.75 μl forward primer (10 uM); 0.75 μl reverse primer (10 uM); template cDNA-100 ng; and dH2O diluted to 25.0 μl. The reaction conditions are 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, then 58°C for 15 seconds, then 72°C for 45 seconds, then 72°C for 5 minutes, then 4°C.

[0279] Clean up the reaction using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, CA) according to the manufacturer's instructions (up to 5 μg). Larger reactions will require clean up using a larger volume of product. After clean up, the cDNA is quantified using NANODROP™ and analyzed by agarose gel electrophoresis to ensure that the cDNA is the expected size. The cDNA is then subjected to sequencing analysis before proceeding to the in vitro transcription reaction.

[0280] Example 4. IVT and IFN-β analysis of short model RNA-1 IVT reactions can be "spiked" with specific nucleotides to minimize cytokine contamination and improve yields. In some processes, the ratio of GTP to other NTPs is increased, resulting in higher total NTP loading and a higher NTP:Mg ratio, as shown in Table 1. 2+ The molar ratio was increased. These specific nucleotides are called short model RNAs. The purpose of the short model RNAs was to reproduce the same cytokine signal / equimolar effect (prior art) as observed in full-length RNA (prior art) with the method of the present invention. This construct was small enough to detect full-length impurities by LC / MS (which is not currently feasible with full-length RNA).

[0281] [Table 1]

[0282] To determine whether short model transcripts prepared using two different IVT processes mimic the in vitro (BJ fibroblast) IFNβ response observed for the full-length transcript, an independent experimental process (i.e., crude IVT, ultrafiltration, or dT purification) study was set up. A surrogate RNA, short model RNA-1, produced under the same IVT conditions and from the same DNA template duplex as the target RNA was generated. The cytokine response mimicked that of full-length RNA in the IFN-β assay in BJ fibroblast cells (Figure 1). The short model transcripts prepared using an equimolar IVT process have a higher IFNβ response than material prepared using the alpha IVT process. After ultrafiltration and dT purification, the cytokine response was preserved.

[0283] Furthermore, impurity profiling of short model RNA-1 (all uridines modified to pseudouridine and all cytidines modified by 5'O-methyl) showed the presence of reverse complements (dsRNA) in the equimolar process but not in the alpha reaction. Nine different species were identified: the peaks overlapping between the two represent abortive species, while the peaks seen only in the equimolar preparation are reverse complement species. Screening was performed on species with varying amounts of uridine and cytidine modifications, and reverse complement and abortive species were found in all three (Figure 2). The abortive transcripts are sense and the reverse complements are antisense transcripts. IFN-β levels also varied between the three (Figure 2). At the top, G0 = standard A, G, C, U; in the middle, G5 = standard A, G, C and 1-methyl pseudo-U; at the bottom, G6 = standard A, G, C and 5-methoxyuridine. Short model transcripts prepared by two different processes in three different chemistries had similar impurity profiles by LCMS despite differences in the BJF assay and appear to be sensitive to the different chemistries.

[0284] LCMS analysis of model and intact transcripts prepared by IVT at 25°C for 6 hours. Short and full-length transcripts were prepared using equimolar and alpha processes at 25°C for 6 hours (rather than our regular IVT at 37°C for 2 hours). Low temperature IVT reactions (<30°C) generate higher amounts of reverse complement RNA than 37°C. Impurity profiles of samples were analyzed by LCMS. Samples prepared using both processes both contained abortive / truncated products. Transcripts prepared using equimolar process contained polyU species of various lengths. Alpha process mitigates reverse complement formation even at 25°C where the abundance was greater than standard conditions using equimolar process (Figure 3).

[0285] Complementary / antisense RNA detection Since T7 polymerizes from nascent transcribed RNA, the immune stimulatory impurities appear to be caused by RNA-templated RNA transcription. The generated complementary / antisense RNA (dsRNA) exhibits mixed bases, polyU components, and 5' triphosphate (5'ppp), which starts at any base. RNA-templated transcription was performed. Reverse-phase purified hEPO G5 (cold) was incubated at 4 mg / mL (consistent with the typical yield of in vitro transcription reactions) with all IVT components, except for the DNA template, which was determined by qPCR to be below the threshold required for in vitro transcription, to explore the RNA-templated transcription phenomenon. The material was analyzed by UPLC and the in vitro BJF IFNβ assay. UPLC analysis demonstrated that aberrant RNA transcripts, and most notably polyU species, were produced via RNA-templated transcription (in the absence of a DNA template). Reactions performed with RNA and all IVT components were IFNβ hot, whereas reactions performed without RNA were cold. (Figure 4).

[0286] Double-stranded RNA may be removed from reverse phase (RP) purified transcribed RNA. Capillary electrophoresis analysis of RNase III treated hEPO G5 material was performed. hEPO G5 was prepared using equimolar or alpha conditions and a portion was purified by RP. The samples were then treated with RNase III and analyzed by capillary electrophoresis. Material prepared by the alpha process contained less RNase III substrate than that prepared by the equimolar process. RP purification removes most of the RNase III substrate from the equimolar and alpha material. The alpha process and reverse phase purification appear to provide a synergistic reduction in RNase III substrates (e.g., dsRNA) (Figure 5A). Capillary electrophoresis analysis of RNase III treated hEPO G0 material was performed. hEPO G0 was prepared using the equimolar process and a portion was purified by RP. The samples were then treated with RNase III and analyzed by capillary electrophoresis (blue: treated; black: untreated). RP-purified material contained less RNase III substrate than non-RP-purified material (state of the art) (Figure 5B).

[0287] RNase III is a dsRNA-specific nuclease. RNA preparations are subjected to RNase III treatment for a period of time. RNA purity / impurity profiles are compared before and after RNase III treatment and measured by HPLC or capillary electrophoresis. The amount of full-length product degraded was proportional to the level of double-stranded RNA impurities present in the RNA preparation. The amount degraded as indicated by the change in apparent size / retention time was considered to be the amount of RNase III substrate. Samples that do not contain dsRNA should show nearly identical purity before and after RNase III treatment. Samples that contain significant amounts of dsRNA will show substantial cleavage product formation and depletion of full-length RNA present in the untreated feedstock as seen by HPLC or capillary electrophoresis. (For example, if 80% of the original mRNA remains after RNase III treatment, 20% was a substrate for RNase III).

[0288] IFNβ and hEPO expression analysis of samples from Figures 5A and 5B was performed. Treated and untreated samples were analyzed to determine how RNAse III treatment affects IFNβ response and hEPO expression in BJ fibroblasts (in vitro). hEPO A100 G5 equimolar material was found to be similarly expressed for untreated and RIII treated samples. Cytokine levels of the equitreated RP samples were decreased after treatment with RIII. hEPO A100 G5 alpha materials were all similarly expressed and had zero / low IFNβ response. TL material was not expressed. However, RIII treatment reduced CK levels for both + and -RP samples. The G0 hEPO A100 material saw the greatest effect from RNase III treatment. Both + and -RP purified samples saw increased expression and decreased IFNβ levels after RNase III treatment (Figure 6).

[0289] We performed capillary electrophoresis analysis of short transcripts transcribed using different processes and treated with RNase III. The question was whether the effect of RNase III treatment could be characterized using short model transcripts. Surrogate RNA constructs were transcribed using equimolar or alpha process, then treated with RNase III, and analyzed by capillary electrophoresis. The equimolar material appeared to contain mostly RNase III substrate, whereas the alpha process material contained no substrate, according to CE (capillary electrophoresis). Upon RNase III treatment, the model RNA peak shifts by 5-7 nucleotides on the fragment analyzer. Equimolar IVT formed an intense second peak 216 nucleotides shorter than the main peak, which was also observed for the ORF-containing mRNA (Figure 7). Thus, using model RNA and LC / MS, we could accurately characterize which components were cleaved and, by inference, which components remain. HPLC purity methods showed polyU upon isolation and concentration of the cleavage products (Figure 9). We performed RP analysis of RNA surrogate 2 EQ transcripts treated with RNase III (same construct as in the top panel of Figure 7...separate analysis). After treating the surrogate RNA transcripts with RNase III, several species were observed by RP analysis. Since this RP method was a tail-selective method, we hypothesize that these early eluting peaks are short oligos and / or tail variants (Figure 8A). We performed RP analysis of the RNA surrogate 2 alpha construct transcribed with alpha process transcripts treated with RNase III. We observed no emergence of additional impurity peaks and no obvious change to overall purity in the RP traces for the surrogate RNA alpha material treated with RIII. Thus, we may conclude that the alpha process does not create the same dsRNA species as the equimolar process (Figure 8B).

[0290] dsRNA abundance by RNase III and cytokine data from RP fractions RP fractionation of hEPO with and without RNase III treatment was performed. hEPO G5 mRNA transcribed via both the equimolar and alpha processes was purified by reversed-phase HPLC. Fractions were collected across an elution gradient. Fractions were treated with RNase III and subsequently analyzed by capillary electrophoresis comparing untreated RNA and RNase III treatment (overlapped). RNA fractions were also transfected into BJ fibroblasts and IFN-B induction was assessed before and after RNase III treatment (Figure 9).

[0291] Capillary electrophoresis analysis of RP fractions hEPO EQ+ / -RIII treated was performed. hEPO EQ was treated with RNase III, then RP purified, fractionated, and analyzed by CE (blue: RIII treated; black: untreated). The initial equimolar fractions containing RNA impurities (fractions 1-6) show significant amounts of dsRNA / RNase III substrate. dsRNA was enriched in the initial fractions. This was confirmed by high IFN-B levels. Equimolar fractions 7-9 showed lower levels of dsRNA / RNase III substrate by CE, which was also confirmed by reduced levels of IFN-B. RNase III treatment of each fraction reduced IFN-B induction to basal levels, again supporting that the IFN-B impurities consist of dsRNA (Figure 10A-C). In vitro IFNβ analysis of hEPO EQ G5, untreated or after RNase III treatment (FIG. 10D).

[0292] Capillary electrophoresis analysis of RP fractions hEPO alpha + / - RIII treated was performed. hEPO alpha was treated with RNase III, then RP purified, fractionated, and analyzed by CE (capillary electrophoresis) (blue: RIII treated; black: untreated) (Figure 11A-C). In vitro IFNβ analysis of hEPO alpha G5 untreated or after RNase III treatment (Figure 11D). All fractions show trace levels of dsRNA (RNase III substrate) by both capillary electrophoresis runs, as the overlay electropherograms are virtually identical. This was confirmed by basal levels of IFN-B in both treated and untreated fractions. Alpha-processed transcribed RNA was devoid of dsRNA, despite the presence of non-full-length / truncated RNA impurities in earlier fractions.

[0293] ELISA detection of dsRNA abundance The J2 / K2 dsRNA ELISA was developed to measure dsRNA abundance. Plates were coated with J2 monoclonal (IgG) antibody and then blocked. RNA of interest was then added at a given concentration and incubated for 1 hour. K2 monoclonal antibody was added (IgM) and HRP-conjugated anti-IgM goat polyclonal antibody was added. TMB was added to develop the signal. The assay detects duplexes greater than 40 base pairs in length. J2 can aid in the determination of RIII endpoints. Knockdown of dsRNA was observed with RP or RNase III treatment (Figure 12). The J2 assay suggests that there was significantly more dsRNA in the equimolar material than with alpha. The RP process removes dsRNA material from the EQ sample to the same extent as the RIII treatment. The alpha-reacted mRNA had less dsRNA in the feedstock compared to the equimolar IVT product. It was also observed that knockdown in the dT-purified RNase III material was greater than with TrisRP. The assay also showed that dsRNA was removed by RNase III treatment (Figure 13). dsRNA levels detected by J2 vary based on construct / process / chemistry, but RNase III treatment appears to reduce most dsRNA levels to baseline. LCMS analysis after nuclease P1 treatment showed that additional NTPs were present in the FFB mRNA, as non-G primed reverse complements were more abundant in the equimolar group compared to the α-reacted group (Table 2).

[0294] [Table 2]

[0295] Relaxation of RNA-templated transcription during low-temperature IVT IFNβ responses to hEPO constructs prepared using different processes and chemistries were performed. As shown in the IVT characterization studies, the alpha response was less sensitive to cold-induced cytokine spikes (Figure 14). Standard equimolar IVT when performed at 25°C produces enhanced IFN-B-induced impurity abundance. Total nucleotide analysis was performed on hEPO constructs prepared using different processes. When performing IVT at both 37°C and 25°C, both alpha process conditions (GDP and GTP) mitigate exogenous IFN-B-induced impurity formation. Nucleotide distribution is consistent across the control region in the nuclease P1 study (grey box), with the same hEPO plasmid showing consistent cleavage (Figure 15). When the equimolar IVT process is performed at 25°C, there is a higher amount of uridine / 1-methyl pseudo-U than at 37°C, likely due to poly-U evolution, and the alpha process (GDP and GTP) shows a consistent nucleotide distribution even at 25°C, further supporting mitigation of impurity formation. The difference in bar height is likely due to differences in concentration. In standard IVT at 25°C, the U content increased with time. The molar corrected nucleotide compositions are shown in Tables 3 and 4. G and U are at theoretical values, while A was over-represented and C was under-represented. The deviations between temperature conditions with the alpha reaction were small, with the largest deviation observed for U in standard IVT.

[0296] [Table 3]

[0297] [Table 4]

[0298] "Forced" RNA-templated transcription Impurity analysis was performed by LCMS of RNA-based IVT in various chemistries using G5 eGFP EQ. IVTs were set up (in various chemistries) using 4 mg / mL eGFP G5 etc. RNA and no DNA template. PolyU species are generated from RNA-templated transcription. RNA-templated transcription occurs independently of chemistry. Reverse-phase purified eGFP G5 (cold) was incubated at 4 mg / mL (consistent with typical yields of in vitro transcription reactions) with all IVT components except the DNA template, which was determined by qPCR to be below the threshold required for in vitro transcription, to explore the RNA-templated transcription phenomenon. The material was analyzed by LC / MS (Figure 16A (equimolar process), 16B (alpha process)).

[0299] IFNβ analysis was performed on the RNA-templated IVT products. Material was from Figure 16. Following RNA-based IVT, samples were analyzed in vitro to determine if there was a correlation between LCMS analysis (polyU species) and IFNβ response. Alpha (A100) RNA-based IVT produces material with a lower CK response than equimolar material (G0 or G5); again suggesting that the alpha process mitigates the formation of RNA-templated transcripts. G6 was all cold; (essentially cold despite impurity generation) (Figure 17).

[0300] To determine whether dsRNA may be capped, a study was performed to determine whether dsRNA may be capped by vaccinia. Forward and reverse complementary oligos, both with a 5' triphosphate, were annealed and then subjected to the capping process using vaccinia to determine whether dsRNA may be capped. The pppF oligo (containing 5' pppG) can be capped to cap1 using vaccinia. The pppRC oligo (containing 5' pppU) cannot be capped using vaccinia. The dsRNA with pppF+pppRC cannot be capped using vaccinia (Figure 18).

[0301] Dephosphorylation of dsRNA using CIP was performed to confirm whether calf intestinal phosphatase dephosphorylates dsRNA with different 5' / 3' overhangs. Various F and RC oligos were annealed and then treated with CIP and analyzed by LCMS for dephosphorylation efficiency. dsRNA with 5' overhangs can be completely dephosphorylated. Fully double-stranded dsRNA can be partially dephosphorylated. dsRNA with 3' overhangs cannot be completely dephosphorylated. This explains why phosphatases were not 100% effective in reducing CK response (Figure 19).

[0302] CE (capillary electrophoresis) purity of the administered mRNA in the aforementioned in vivo study was performed as shown by electropherograms (Figure 20). Structure may play a role as the 5'UTR may affect the efficacy of the alpha reaction (Figure 20).

[0303] In vivo studies RIII treatment showed clear cytokine knockdown in mice using both unmodified and fully modified species (where all uridines were 1-methylpseudouridines), which can be used as an independent method to knockdown cytokines to near basal levels. Female mice (n=5 per group) were injected once with 0.5 mg / kg of the selected test material.

[0304] IFNβ analysis of G0 and G5 hEPO mRNA transcribed using different IVT processes and purification permutations was performed to determine what the IFNβ response was to material produced with different chemicals, processes, and treatments (in vitro; BJ fibroblasts). +RIII, +RP, and alpha processes have similar effects (reduction) on the in vitro IFNβ response of G5. G0 demonstrates that alpha+RIII was superior to iso+RIII (Figure 21).

[0305] In vivo (Balb-C mice) expression of hEPO prepared using G0 or G5 chemistry, equimolar amounts or the alpha process, and RNase III treatment by ELISA was performed to examine how expression is affected by hEPO prepared using different processes (chemistry, IVT process, RIII treatment) using the same materials as in Figures 12, 20, 21, 22. There is no significant difference in hEPO expression using RNA produced by the different processes (Figure 22).

[0306] A cytokine (luminex) panel for in vivo (Balb-C mice) G0 and G5 hEPO mRNA transcribed using different IVT processes and purification permutations with and without RNaseIII treatment was performed and determined for the same material as in Figures 12, 20, 21, 22. Cytokine analysis after treatment of Balb-C mice with 0.5 mpk formulated RNA. Untreated alpha material has a similar CK response as IP10 EQ material (G5). Overall, alpha process combined with RNase III treatment appears to confer less immunostimulatory activity than equimolar comparator (+RNase III), especially in G0. RIII treatment reduces the CK response (Figures 23A-23D).

[0307] B cell activation from spleens of Balb-C mice treated with hEPO prepared using G0 or G5 chemistry, equimolar or alpha process, and RNase III treatment was performed. Activated B cells (CD86+ CD69+) were analyzed for their response after treatment with hEPO prepared using the various processes. B cell activation roughly correlates with the cytokine (luminex) panel. RIII treatment reduces the CK response compared to untreated controls (Figure 24).

[0308] In vitro analysis of short 5' triphosphorylated oligos was performed to determine whether they stimulate IFNβ responses in vitro (BJF, IFNβ). ssRNA and dsRNA less than 12 nucleotides or base pairs do not stimulate IFNβ in BJF (Figure 25).

[0309] In vitro analysis of polyU and dsRNA standards was performed to determine what stimulated the IFNβ response in BJF, whether or not using the 5' triphosphorylated oligo standard. pppF+pppRC dsRNA of >20 bp was immunostimulatory. 5' triphosphorylated polyU was not immunostimulatory as were ssRNA or dsRNA of <25 nt or 25 bp (Figure 26).

[0310] In vitro analysis of dsRNA standards with 3' overhangs was performed to determine whether there was a 3' overhang length dependency on IFNβ response for the dsRNA standards. The longer the 3' overhang, the lower the IFNβ response (Figure 27).

[0311] In vitro analysis of dsRNA standards with 5' overhangs, full duplexes, and different lengths of 3' overhangs was performed to determine whether the length / identity of the overhangs depended on the IFNβ response. The ssRNA pppRC oligo is hot. dsRNA with 5' overhangs (and approximately 20 bp duplexes) is less immunostimulatory than the full duplex. dsRNA with 3' overhangs has equal or less immunostimulatory power than the full duplex. The longer the dsRNA duplex, the higher the CK response (Figure 28).

[0312] In vitro analysis of polyU species was performed to determine what polyU species were required to simulate an IFNβ response. ssRNA containing 5' triphosphate polyU standard is slightly immunostimulatory. As dsRNA with OH-F30 (30 bp duplex), there was an additive CK response. However, polyU standard is not immunostimulatory in the presence of full-length mRNA (A100) (Figure 29).

[0313] In vitro analysis of ssRNA oligonucleotide standards was performed to determine what the IFNβ response to the ssRNA standards was. (F or forward oligo represents the abortive or truncated species; R or RC or reverse complement oligo represents the reverse complement species generated by RNA-templated transcription). Forward oligos containing a 5' triphosphate do not stimulate IFNβ. Reverse complement oligos with a 5' triphosphate stimulate IFNβ at lengths greater than 25 nt (Figure 30).

[0314] To determine whether the 5' functional group (triphosphate and hydroxyl) affects the IFNβ response, in vitro analysis of dsRNA oligo standards with different 5' functional groups was performed. 5' triphosphate on the F oligo and 5' hydroxyl on the RC oligo simulates IFNβ at >20 bp. 5' triphosphate on the F oligo and 5' hydroxyl on the RC oligo simulates IFNβ at >25 bp. 5' hydroxyl on the F oligo and 5' triphosphate on the RC oligo stimulates IFNβ at >20 bp. Only one triphosphate was required to simulate IFNβ >20 bp (Figure 31).

[0315] IFNβ analysis of CIP-treated dsRNA oligo standards was performed. dsRNA oligo standards of various overhang lengths were treated with CIP and analyzed for IFNβ response. Samples that were observed to be dephosphorylated (i.e., 5' overhang) by LCMS (Figure 19) did not stimulate IFNβ as much as untreated samples. Samples that were not dephosphorylated by CIP (i.e., full duplex and 3' overhang) showed no change in IFNβ response + / - CIP (Figure 32).

[0316] A ssRNA impurity dose response (IFNβ in BJ fibroblasts) was performed to determine what the dose dependency of ssRNA impurity standards in vitro was (BJF, IFNβ). How much of either type of impurity was needed to stimulate a response? <20mer ssRNA (RC) was not hot. >30mer ssRNA (RC) stimulated IFNβ at approximately >1ng / uL (or >2.5ng / transfection). There was an apparent length dependency on the ssRNA CK response (RC). The longer the oligo, the higher the CK response (Figure 33).

[0317] A dsRNA impurity dose response (IFNβ in BJ fibroblasts) was performed to determine the dose dependency of dsRNA impurity standards in vitro (BJF, IFNβ). 20-30 bp dsRNA stimulates IFNβ at approximately >1 ng / uL (>2.5 ng / transfection). >30 bp dsRNA stimulates IFNβ at approximately 0.1 ng / uL (>0.25 ng / transfection). >1000x dilution of >30 bp dsRNA was required to silence the IFNβ response...indicating that only a few molecules of these impurities are sufficient to stimulate a response. There was an apparent length dependency on the dsRNA CK response...the longer the duplex, the higher the CK response (Figure 34).

[0318] IFNβ response to modified 5' nucleotides of the forward oligo standard was performed to determine whether the 5' nucleotide (triphosphorylation) affected the CK response. When 5'A / C / U was added to the F oligo, the CK response was induced. 5'non-G did not change the IFNβ response (still cold). This suggests that there was sequence dependency (since RC is hot) (Figure 35).

[0319] To determine whether the 5' nucleotide (triphosphorylation) affects the CK response, the IFNβ response to modified 5' nucleotides of the reverse complement oligo standard was performed. When a 5'G was added to the RC oligo, the CK response was induced. The 5'pppG on the RC oligo silences the IFNβ response. This suggests that the 5'G silences the CK response, regardless of sequence identity (Figure 36).

[0320] IFNβ responses of 5' hydroxyl functionalized dsRNA were performed to determine the extent to which the 5' functionality (ppp vs. OH) affects the cytokine response of dsRNA. From 5'ppp-F / 5'ppp-RC duplexes to 5'OH-F / 5'OH-RC were tested. There was an IFNβ response of >30 bp dsRNA with either 5'ppp or 5'OH. This suggests that dsRNA longer than 30 bp stimulated an IFNβ response in BJF, regardless of the 5' functionality (Figure 37).

[0321] Speculation RNA-templated transcription as an IVT by-product was significantly reduced and nearly eliminated in the alpha-reaction process. As a result of mitigating reverse complement formation, the desired impurities of both dsRNA (with 5' ppp) and non-pppG-initiated RNA (pppA, pppC, and pppU) are addressed.

[0322] RNA template transcription was enhanced at IVT reaction temperatures below 37 °C (e.g., 25 °C). Ramp times to achieve 37 °C heating while the IVT reaction is proceeding can result in higher impurity levels, especially as the time ramp from 25 °C to 37 °C increases. The Alpha Reaction process was more tolerant at 25 °C, as trace amounts of RNA template-transcribed species were detected at ambient temperatures.

[0323] RP was more effective with the alpha reaction than with the equimolar process. The amount of impurities was lower in the alpha reaction, resulting in improved separation. "Purity" could be elucidated more clearly, potentially increasing loading challenges and increasing productivity. A single RP cycle is sufficient to knock the unmodified species to baseline using the GDP-alpha reaction, whereas 2-3 successive RP cycles are required to obtain two fractions of unmodified species using equimolar IVT.

[0324] Example 5. RNA produced using the Alpha Process has <40% run-on transcripts. hEPO RNA was produced using either equimolar or alpha process. RNA was digested with Rnase T1 and tail fragments were analyzed by LCMS. The fragment at the tail of 3'mP indicates a run-on transcript. The alpha process produced a 3'OH (clean) calculated to be 32780 Da and a 5'OH / 3'mP (run-on) calculated to be 32860 Da. The equimolar process produced a 5'OH / 3'mP (run-on) of 32861 Da and much less 3'OH (clean) (Figure 38).

[0325] Example 6. Total digestion shows that RNA from equimolar process has higher abundance of non-GTP 5' nucleotides than RNA made by alpha process Short open reading frame RNAs were generated using four different conditions: 37°C for 2 hours (standard) vs. 25°C for 6 hours and equimolar vs. alpha. Each RNA was enzymatically digested to single nucleotides and then analyzed by LCMS for 5' nucleotide abundance (e.g., pppG or GTP, pppA or ATP, etc.). The 5'G is the first template nucleotide, meaning that if an impure RNA population is generated, the majority of the 5' nucleotide will be present as ATP, CTP, or UTP (e.g., equimolar process), and if a pure RNA population is generated, the majority of the 5' nucleotide will be GTP (e.g., alpha process).

[0326] Example 7. Short open reading frame RNAs generated by the alpha process generate fewer reverse complements Equimolar or alpha processes containing either 32P-GTP or 32P-CTP were used to measure G0 (wild type) and G5 (m 1 In the Ψ) chemistry, short open reading frame RNA was generated. 32P-GTP labeled abortive transcripts and 32P-CTP labeled reverse complement transcripts. There were no differences in the abortive or RC profiles between G0 and G5 chemistries. The equimolar and alpha processes have similar abortive profiles. The equimolar process generates more reverse complements than the alpha process.

[0327] Example 8. RNase T1 digestion is informative on run-on transcript populations RNase T1 is an endonuclease that specifically cleaves RNA after a guanosine nucleotide, leaving a 5' hydroxide and a 3' monophosphate. In constructs containing a templated guanosine nucleotide at the 3' end, RNase T1 can be used to discriminate the population of run-on transcripts that leave a 3' monophosphate compared to clean transcripts that contain a 3' hydroxide. hEPO RNA was produced using either the equimolar or alpha process, then digested with RNase T1 and analyzed by mass spectrometry. The 3' oligo fragments were analyzed and quantified for their 3' heterogeneity. RNA produced with the equimolar process contains approximately 70-80% run-on transcripts, whereas RNA produced with the alpha process contains 30-40% run-on transcripts.

[0328] Example 9. Total Digestion of RNA to Determine Sample Purity Short model RNA constructs (surrogate RNA1) were generated using equimolar or Alpha process with incubation at 37°C or 25°C. Each sample was purified with oligo dT resin to remove unreacted NTPs. Each sample was enzymatically digested to single nucleotides with S1 and benzonase nuclease. The abundance of 5' nucleotides that are triphosphorylated at the 5' end was analyzed by mass spectrometry. Extracted ion peaks were integrated and the % NTP abundance is summarized in Table 5. Pure RNA populations are indicated by high %GTP values ​​since 5'GTP is the first templated nucleotide. Similarly, impure RNA populations are indicated by relatively low amounts of GTP. Samples generated using the EQ process contain more than 5% non-GTP 5' nucleotides, while samples generated using the Alpha process have less than 5% non-GTP 5' nucleotides.

[0329] [Table 5]

[0330] Example 10. dsRNA ELISA shows the presence of dsRNA hEPO RNA constructs were produced using either the equimolar or alpha process and purified by either oligo-dT resin only (-RP) or reverse phase purification (+RP). An ELISA using a dsRNA-specific antibody is used to determine the relative differences in purity of the RNA produced using the different processes. RNA produced using the equimolar process contains significantly more dsRNA than RNA produced using the alpha process (Figure 39). Reverse phase purification improves the purity of the -RP RNA.

[0331] Example 11. Radioactive Sequencing Gel Analysis Determines Sample Purity Short model RNA constructs (surrogate RNA 1) were generated using equimolar or alpha processes. Each IVT reaction labels abortive transcripts 32 Labels P-GTP or reverse complement transcripts 32P-CTP. RNA samples were analyzed by sequencing gel. 32 Based on the P-GTP data, there is no difference in abortive transcript abundance between the two processes. 32 Based on the P-CTP data, the equimolar process generates more reverse complements than the alpha process.

[0332] Example 12. In vivo analysis of dsRNA doped into mRNA Reverse-phase purified hEPO mRNA was doped with 5%, 0.5% or 0.05% w / w of 60bp dsRNA corresponding to the first 60 nucleotides of the intact hEPO construct. The mRNA samples were formulated in MC3 and then administered IV at 0.5mpk to C57BL / 6 and Balb-c mice. After 24 hours, spleens were harvested and homogenized to generate single cell suspensions. Splenocytes were stained for B cell markers and then analyzed by flow cytometry. Activated B cell populations were analyzed based on their expression of CD86 and CD69 markers. The 60bp dsRNA has the following sequence:

[0333] [ka]

[0334] The group given hEPO mRNA produced by the alpha process had lower B cell activation than the group given hEPO with doped dsRNA. Serum was also collected 6 hours later and analyzed by cytokine luminex panel. The expression trends for IP-10, IFN-gamma, and IFN-alpha markers from the luminex panel were consistent with the trends from the B cell activation analysis, indicating that the dsRNA in the doped samples induced the type I interferon pathway, which led to B cell activation.

[0335] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the scope of the claims appended hereto.

[0336] All references disclosed herein, including patent documents, are incorporated by reference in their entirety.

Claims

1. A composition comprising an in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition being substantially free of reverse-transcribed complement products.

2. The composition of claim 1 , wherein greater than about 80% of the RNA mass comprises single-stranded full-length transcripts.

3. The composition of claim 2, wherein greater than about 90% of the RNA mass comprises single-stranded full-length transcripts.

4. The composition of claim 2, wherein greater than about 95% of the RNA mass comprises single-stranded full-length transcripts.

5. The composition of claim 2, wherein greater than about 96% of the RNA mass comprises single-stranded full-length transcripts.

6. The composition of claim 2, wherein greater than about 97% of the RNA mass comprises single-stranded full-length transcripts.

7. The composition of claim 2, wherein greater than about 98% of the RNA mass comprises single-stranded full-length transcripts.

8. The composition of claim 2 , wherein greater than about 99% of the RNA mass comprises single-stranded full-length transcripts.

9. The composition of claim 2, wherein greater than about 99.5% of the RNA mass comprises single-stranded full-length transcripts.

10. The composition of claim 2 , wherein 100% of the mass of the single-stranded RNA comprises a single-stranded full-length transcript.

11. The composition of any one of claims 2 to 10, wherein the composition is substantially free of RNAse III insensitive fragments.

12. The composition of any one of claims 2 to 10, wherein the composition comprises a population of single-stranded partial RNA transcripts in a sense orientation, and more than 80% of the population of single-stranded partial RNA transcripts in a sense orientation have a nucleotide length of 100 nucleotides or less.

13. The composition of claim 12, wherein more than 90% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

14. The composition of claim 12, wherein more than 95% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

15. The composition of claim 12, wherein more than 98% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

16. The composition of claim 12, wherein more than 99% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

17. The composition of claim 12, wherein 100% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

18. The composition of claim 1 , wherein less than about 0.5% of the mass of the RNA in the composition is a reverse complement transcript.

19. The composition of claim 1 , wherein less than about 0.25% of the mass of the RNA in the composition is a reverse complement transcript.

20. The composition of claim 1 , wherein less than about 0.1% of the mass of the RNA in the composition is a reverse complement transcript.

21. The composition of claim 1 , wherein less than about 0.05% of the mass of RNA in the composition is a reverse complement transcript.

22. The composition of claim 1 , wherein less than about 0.01% of the mass of the RNA in the composition is a reverse complement transcript.

23. The composition of claim 1 , wherein less than about 0.005% of the mass of the RNA in the composition is a reverse complement transcript.

24. The composition of claim 1 , wherein less than about 0.001% of the mass of the RNA in the composition is a reverse complement transcript.

25. The composition of claim 1 , wherein less than about 5% of the mass of the RNA in the composition is a reverse complement transcript.

26. The composition of any one of claims 1 to 25, wherein the mass of the RNA in the composition is determined by LC or MS.

27. A composition comprising in vitro transcribed (IVT) RNA and a pharma- ceutically acceptable excipient, the composition being substantially free of cytokine-induced RNA contaminants.

28. 28. The composition of claim 27, wherein greater than about 80% of the RNA mass comprises single-stranded full-length transcripts.

29. 30. The composition of claim 28, wherein greater than about 90% of the RNA mass comprises single-stranded full-length transcripts.

30. 30. The composition of claim 28, wherein greater than about 95% of the RNA mass comprises single-stranded full-length transcripts.

31. 30. The composition of claim 28, wherein greater than about 96% of the RNA mass comprises single-stranded full-length transcripts.

32. 30. The composition of claim 28, wherein greater than about 97% of the RNA mass comprises single-stranded full-length transcripts.

33. 30. The composition of claim 28, wherein greater than about 98% of the RNA mass comprises single-stranded full-length transcripts.

34. 30. The composition of claim 28, wherein greater than about 99% of the RNA mass comprises single-stranded full-length transcripts.

35. 30. The composition of claim 28, wherein greater than about 99.5% of the RNA mass comprises single-stranded full-length transcripts.

36. 30. The composition of claim 28, wherein 100% of the mass of the single-stranded RNA comprises a single-stranded full-length transcript.

37. The composition of any one of claims 28 to 36, wherein the composition is substantially free of RNAse III insensitive fragments.

38. The composition of any one of claims 28 to 37, wherein the composition comprises a population of single-stranded partial RNA transcripts in a sense orientation, and more than 80% of the population of single-stranded partial RNA transcripts in a sense orientation have a nucleotide length of 100 nucleotides or less.

39. 39. The composition of claim 38, wherein more than 90% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

40. 39. The composition of claim 38, wherein more than 95% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

41. 39. The composition of claim 38, wherein more than 98% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

42. 39. The composition of claim 38, wherein greater than 99% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

43. 39. The composition of claim 38, wherein 100% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

44. 28. The composition of claim 27, wherein less than about 0.5% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

45. 28. The composition of claim 27, wherein less than about 0.25% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

46. 28. The composition of claim 27, wherein less than about 0.1% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

47. 28. The composition of claim 27, wherein less than about 0.05% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

48. 28. The composition of claim 27, wherein less than about 0.01% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

49. 28. The composition of claim 27, wherein less than about 0.005% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

50. 28. The composition of claim 27, wherein less than about 0.001% of the mass of the RNA in the composition is a cytokine-induced RNA contaminant.

51. The composition of any one of claims 12 to 17, wherein the mass of the RNA in the composition is determined by LC or MS.

52. 2. The composition of claim 1, wherein the reverse complement transcript is a dsRNA that includes a strand that includes a sequence that is the reverse complement of at least a portion of the IVT RNA or is a dsRNA that includes a strand that includes a polyU-containing sequence.

53. 53. The composition of claim 52, wherein the strand containing a sequence that is the reverse complement of the IVT RNA or the strand containing the polyU sequence starts with a 5' triphosphate (5'-PPP).

54. 2. The composition of claim 1, wherein the reverse complement transcript comprises a reverse complement of the 5' end of the IVT RNA and / or a reverse complement of the 3' end of the RNA that encodes a polypeptide of interest.

55. 2. The composition of claim 1, wherein the IVT RNA is an RNA encoding a polypeptide of interest, and the reverse complement of the RNA encoding the polypeptide of interest comprises a sequence complementary to all or a portion of the open reading frame of the RNA encoding the polypeptide of interest.

56. 54. The composition of claim 53, wherein the reverse complement of the 5' end of the IVT RNA comprises a sequence complementary to all or a portion of a 5' UTR of an RNA encoding the polypeptide of interest.

57. 54. The composition of claim 53, wherein the reverse complement of the 3' end of the IVT RNA comprises a sequence complementary to all or part of the poly A tail of an RNA encoding the polypeptide of interest.

58. 28. The composition of claim 27, wherein the cytokine-induced RNA contaminant is a dsRNA that includes a strand that includes an inverted sequence that is the reverse complement of the IVT RNA, or is a dsRNA that includes a strand that includes a polyU sequence.

59. 59. The composition of claim 58, wherein the strand containing the sequence that is the reverse complement of the IVT RNA or the strand containing the polyU sequence starts with a 5' triphosphate (5'-PPP).

60. 59. The composition of claim 58, wherein the cytokine-induced RNA contaminant comprises a reverse complement of the 5' end of the IVT RNA and / or a reverse complement of the 3' end of the IVT RNA.

61. 61. The composition of claim 60, wherein the reverse complement of the 5' end of the IVT RNA comprises a sequence complementary to all or a portion of the 5' UTR of the IVT RNA.

62. 61. The composition of claim 60, wherein the reverse complement of the 3' end of the RNA encoding the polypeptide of interest comprises a sequence complementary to all or part of the polyA tail of the IVT RNA.

63. 60. The composition of claim 52 or 58, wherein the dsRNA comprises a strand of 10-250 nucleotides in length.

64. 60. The composition of claim 52 or 58, wherein the dsRNA is double-stranded and about 20 to about 50 bp in length.

65. 61. The composition of claim 54 or 60, wherein the reverse complement comprises a sequence complementary to the first 10-15 nucleotides of the 5'UTR.

66. 3. The composition of claim 1 or 2, wherein less than about 0.5% of the mass of the RNA in the composition is dsRNA greater than 40 base pairs in size.

67. The composition of any one of claims 1 to 66, wherein the amount of reverse complement transcript or cytokine-induced RNA contaminant is determined by high performance liquid chromatography (e.g., reverse phase chromatography, size exclusion chromatography), Bioanalyzer chip-based electrophoresis system, ELISA, flow cytometry, reconstitution or surrogate assay.

68. 68. The composition of any one of claims 1 to 67, wherein the mass of the RNA is determined by mass spectrometry, such as MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time of Flight).

69. The composition of any one of claims 1 to 68, wherein the composition comprises an abortive transcript.

70. The RNA is (a) forming a reaction mixture comprising a DNA template, NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and a magnesium-containing buffer; (b) incubating the reaction mixture under conditions such that the RNA is transcribed, wherein 1) the concentration of at least one of GTP, CTP, ATP, and UTP is at least two-fold higher than the concentration of any one or more of ATP, CTP, or UTP; or 2) the reaction further comprises a nucleotide diphosphate (NDP) or a nucleotide analog, the concentration of the NDP or nucleotide analog being at least two times higher than the concentration of any one or more of ATP, CTP or UTP; 70. The composition of any one of claims 1 to 69, produced by a process comprising:

71. 71. The composition of claim 70, wherein the ratio of the concentration of at least one of GTP, CTP, ATP, and UTP to the concentration of any one or more of ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, or at least 10:

1.

72. 71. The composition of claim 70, wherein the reactants further comprise a nucleotide diphosphate (NDP) or a nucleotide analog, and the ratio of the concentration of the NDP or nucleotide analog to the concentration of any one or more of ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, or at least 10:

1.

73. 71. The composition of claim 70, wherein the ratio of the concentration of GTP to the concentrations of ATP, CTP and UTP is 2:1, 4:1 and 4:1, respectively.

74. 71. The composition of claim 70, wherein the ratio of the concentration of GTP to the concentrations of ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.

75. 71. The composition of claim 70, wherein the reaction mixture comprises GTP and GDP, and the ratio of the concentration of GTP + GDP to the concentration of any one of ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 or at least 6:

1.

76. 76. The composition of claim 75, wherein the ratio of the concentration of GTP + GDP to the concentrations of ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.

77. The RNA is (a) forming a reaction mixture comprising a DNA template, NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP), and a buffer containing magnesium; (b) incubating the reaction mixture under conditions such that the RNA is transcribed, wherein the effective concentration of phosphate in the reaction is at least 150 mM phosphate, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, at least 200 mM, at least 210 mM, or at least 220 mM; The composition of any one of claims 1 to 76, produced by a process comprising:

78. 78. The composition of claim 77, wherein the effective concentration of phosphate in the reaction is 180 mM.

79. 78. The composition of claim 77, wherein the effective concentration of phosphate in the reaction is 195 mM.

80. 78. The composition of claim 77, wherein the effective concentration of phosphate in the reaction is 225 mM.

81. The RNA is (a) forming a reaction mixture comprising a DNA template, NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP) and optionally guanosine diphosphate (GDP), and a magnesium-containing buffer; (b) incubating the reaction mixture under conditions such that the RNA is transcribed; wherein the magnesium-containing buffer contains Mg2+, and the molar ratio of the concentration of ATP+CTP+UTP+GTP and optionally GDP to the concentration of Mg2+ is at least 1.0, at least 1.25, at least 1.5, at least 1.75, at least 1.85 or more; The composition according to any one of claims 1 to 80.

82. 82. The composition of claim 81, wherein the molar ratio of the concentration of ATP + CTP + UTP + GTP and optionally GDP to the concentration of Mg2+ is 1.

5.

83. 82. The composition of claim 81, wherein the molar ratio of the concentration of Mg2+ to the concentration of ATP + CTP + UTP + GTP and optionally GDP to the concentration of Mg2+ is 1.

88.

84. 84. The composition of any one of claims 1 to 83, produced by a process that does not include a dsRNase (e.g., RNase III) treatment step.

85. 84. The composition of any one of claims 1 to 83, produced by a process that does not include a reversed phase (RP) chromatographic purification step.

86. 84. The composition of any one of claims 1 to 83, produced by a process that does not include a high performance liquid chromatography (HPLC) purification step.

87. 84. The composition of any one of claims 1 to 83, produced by a process further comprising RP purification and / or RNase III treatment.

88. The composition of any one of claims 1 to 83, wherein the RNA is a modified mRNA.

89. 84. The composition of any one of claims 1 to 83, wherein the RNA comprises UTP, and the UTP is modified UTP.

90. 90. The composition of claim 89, wherein the RNA is 100% modified.

91. 90. The composition of claim 89, wherein only a portion of the RNA is modified.

92. The amount of reverse complement transcript or cytokine-induced species is: (a) generating a composition comprising the model RNA from a DNA template encoding the model RNA under the same IVT conditions used to produce the IVT RNA; and (b) determining the amount of a reverse complement transcript or a cytokine-induced species in the composition comprising the model RNA by LC-MS; wherein the amount of reverse complement transcript or cytokine-induced species by LC-MS in the composition comprising the model RNA indicates the amount of reverse complement transcript or cytokine-induced species in the composition comprising the IVT RNA; The composition of any one of claims 1 to 83, which is indirectly determined by a process.

93. An in vitro transcribed (IVT) RNA composition, wherein the RNA is not subjected to RNaseIII treatment and / or RP purification.

94. A composition comprising in vitro transcribed (IVT) single-stranded RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, wherein greater than 98% of the RNA is single-stranded, and wherein the single-stranded RNA comprises transcripts of different lengths, and wherein greater than about 80% of the single-stranded RNA comprises a single-stranded full-length transcript.

95. 95. The composition of claim 94, wherein greater than about 90% of the single-stranded RNA comprises a single-stranded full-length transcript.

96. 95. The composition of claim 94, wherein greater than about 95% of the single-stranded RNA comprises a single-stranded full-length transcript.

97. 95. The composition of claim 94, wherein greater than about 96% of the single-stranded RNA comprises a single-stranded full-length transcript.

98. 95. The composition of claim 94, wherein greater than about 97% of the single-stranded RNA comprises a single-stranded full-length transcript.

99. 95. The composition of claim 94, wherein greater than about 98% of the single-stranded RNA comprises a single-stranded full-length transcript.

100. 95. The composition of claim 94, wherein greater than about 99% of the single-stranded RNA comprises a single-stranded full-length transcript.

101. 95. The composition of claim 94, wherein greater than about 99.5% of the single-stranded RNA comprises a single-stranded full-length transcript.

102. 102. The composition of any one of claims 94 to 101, wherein the composition is substantially free of RNAse III insensitive fragments.

103. The composition of any one of claims 94 to 101, wherein the single-stranded RNA comprises a population of sense-orientated single-stranded partial RNA transcripts, and more than 80% of the population of sense-orientated single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

104. The composition of claim 103, wherein more than 90% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

105. The composition of claim 103, wherein more than 95% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

106. The composition of claim 103, wherein more than 98% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

107. The composition of claim 103, wherein greater than 99% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

108. The composition of claim 103, wherein 100% of the population of sense-oriented single-stranded partial RNA transcripts have a nucleotide length of 100 nucleotides or less.

109. 95. The composition of claim 94, wherein the single-stranded RNA comprising transcripts of different lengths comprises a full-length transcript and an abortive transcript.

110. 95. The composition of claim 94, wherein 80-98% of the population of sense-orientated single-stranded partial RNA transcripts comprises abortive transcripts.

111. The composition of claim 94, wherein 95-98% of the population of sense-orientated single-stranded partial RNA transcripts comprises abortive transcripts.

112. A unit of use composition comprising an in vitro transcribed (IVT) single-stranded RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, the use composition being free of residual organic solvents or alkylammonium ions.

113. A composition comprising an in vitro transcribed (IVT) single-stranded RNA encoding a polypeptide of interest and a pharma- ceutically acceptable excipient, said composition being non-immunogenic, and said single-stranded RNA comprising transcripts of different lengths.

114. The composition of claim 113, wherein the single-stranded RNA comprising transcripts of different lengths comprises a full-length transcript and an abortive transcript.

115. 1. A method for preparing RNA, comprising: (a) forming a reaction mixture comprising a DNA template, NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP) or optionally guanosine diphosphate (GDP) or analogs of each nucleotide, and a magnesium-containing buffer; (b) incubating the reaction mixture under conditions such that the RNA is transcribed; Where: 1) the concentration of at least one of GTP, CTP, ATP, and UTP is at least two-fold greater than the concentration of any one or more of ATP, CTP, or UTP; or 2) The method, wherein the reaction further comprises a nucleotide diphosphate (NDP) or a nucleotide analog, and the concentration of the NDP or nucleotide analog is at least two times greater than the concentration of any one or more of ATP, CTP, or UTP.

116. 116. The method of claim 115, wherein the ratio of the concentration of GTP to the concentration of any one of ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1, or at least 6:1 to produce RNA.

117. 116. The method of claim 115, wherein the ratio of the concentration of GTP to the concentrations of ATP, CTP and UTP is 2:1, 4:1 and 4:1, respectively.

118. 116. The method of claim 115, wherein the ratio of the concentration of GTP to the concentrations of ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.

119. The method of claim 115, wherein the reaction mixture comprises GTP and GDP, and the ratio of the concentration of GTP + GDP to the concentration of any one of ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 or at least 6:

1.

120. 120. The method of claim 119, wherein the ratio of the concentration of GTP + GDP to the concentrations of ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.

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