Sequential precipitation for mRNA purification

Abstract

Aspects of the disclosure relate to methods of purifying mRNA by precipitating the mRNA, washing the precipitate to remove impurities and salts, and resuspending the washed mRNA to produce an mRNA composition. The disclosure describes reagents and methods useful for precipitating, washing, and resuspending mRNA, as well as compositions produced by the methods described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 306,212, filed February 3, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Messenger RNA (mRNA) is a new alternative to traditional small molecule and protein therapeutics due to the potency and programmability of mRNA. The mRNA encoding a desired therapeutic protein can be administered to a subject for in vivo expression of the protein for therapeutic effects such as vaccination or replacement of the protein encoded by a mutant gene. However, there is a need for improved methods of purifying the produced mRNA. Summary of the Invention

[0003] Provided herein is a method for purifying nucleic acids (e.g., mRNA) by precipitation, filtration, and resuspension. Nucleic acids such as mRNA are commonly purified by chromatography-based methods (e.g., oligo-dT chromatography and / or high performance liquid chromatography), but such methods are limited by the dynamic binding capacity of the stationary phase used in the chromatography. Exceeding the binding capacity of the column will cause excess mRNA to flow through the column, resulting in loss of mRNA and a manufacturing process with reduced efficiency. To address these challenges, mRNA is purified by precipitating dissolved mRNA to produce a composition containing solid mRNA, while impurities remain dissolved. The impurities are then removed by separating the liquid phase of the composition from the precipitated mRNA, such as by aspiration or filtration. One or more washing processes are then performed in which a washing solution is added to dilute the soluble impurities, whereby subsequent separation of this washing solution from the precipitated mRNA reduces the total abundance of soluble impurities associated with the mRNA. This washing process may be repeated multiple times to successively dilute and remove impurities until a desired purity is achieved or no detectable impurities remain. In addition, the wash solution may contain reagents useful for removing certain impurities from the mRNA composition, such as those used to produce the mRNA, but whose presence is undesirable in the final mRNA composition. For example, DNA templates and RNA polymerases are useful for producing mRNA by in vitro transcription, but are not useful for translating the mRNA in vivo, and therefore must be removed before downstream applications. Thus, DNase and protease may be added to digest the DNA template and proteins (e.g., RNA polymerase) into DNA and peptide fragments, respectively, which can then be separated from the precipitated mRNA by filtration. After removal of impurities, the mRNA may be resolubilized and resuspended to produce an mRNA composition that contains fewer or no impurities than the original mRNA composition, and is therefore useful for downstream applications (e.g., administration for in vivo translation of the encoded protein).These methods can be performed in a continuous manner, where the mRNA is continuously input into the process, manipulated through intermediate steps, and output without interruption. In contrast, batch-based methods that require the input to be loaded in separate batches must be interrupted to transfer materials between steps and remove the output. Thus, continuous methods that precipitate, filter, wash, and resuspend the mRNA allow for the removal of impurities in a faster, more streamlined manner than batch-based methods.

[0004] Thus, in some embodiments, described herein is a continuous method for removing one or more impurities from a composition comprising messenger ribonucleic acid (mRNA), the continuous method comprising: (a) precipitating mRNA from the composition to produce a precipitated mRNA composition; (b) removing one or more impurities from the precipitated mRNA composition; and then (c) resuspending the precipitated mRNA. In some embodiments, the precipitating comprises adding a high salt buffer and / or alcohol to the composition comprising the mRNA to generate a precipitated mRNA composition. In some embodiments, the removing one or more impurities comprises contacting the precipitated mRNA composition with a filter membrane. In some embodiments, the removing one or more impurities comprises contacting the precipitated mRNA composition with a wash solution. In some embodiments, the removing one or more impurities comprises contacting the precipitated mRNA composition with a second filter membrane. In some embodiments, the removing one or more impurities comprises contacting the precipitated mRNA composition with a second wash solution.

[0005] In some aspects, the continuous methods described herein include: (i) adding a high salt buffer to the mRNA composition to produce a precipitated mRNA composition; (ii) contacting the precipitated mRNA composition with a filter membrane to remove one or more impurities from the precipitated mRNA composition; (iii)(a) contacting the precipitated mRNA composition with a wash solution; (iii)(b) contacting the precipitated mRNA composition with a second filter membrane to remove one or more impurities from the precipitated mRNA composition; (iv) contacting the precipitated mRNA composition with a resuspension solution to resuspend the mRNA.

[0006] In some embodiments, the high salt buffer comprises a salt selected from the group consisting of lithium chloride, lithium acetate, lithium sulfate, sodium chloride, sodium acetate, sodium sulfate, ammonium chloride, ammonium acetate, and ammonium sulfate. In some embodiments, the high salt buffer comprises ammonium sulfate. In some embodiments, the high salt buffer has a salt concentration of about 0.1 M to about 5.0 M. In some embodiments, the high salt buffer has a conductivity of about 5 mS / cm or greater, optionally the high salt buffer has a conductivity of about 5 mS / cm to about 300 mS / cm. In some embodiments, the precipitation step further comprises adding an alcohol to the mRNA composition. In some embodiments, the alcohol is ethanol or isopropanol. In some embodiments, the filter membrane is a tangential flow filtration (TFF) membrane. In some embodiments, the filter membrane is a hollow fiber membrane. In some embodiments, the filter membrane has a molecular weight cutoff of about 20 kDa to about 150 kDa.

[0007] In some embodiments, the wash solution comprises a detergent, a salt, DNase, and / or RNase III.

[0008] In some embodiments, the wash solution comprises a surfactant. In some embodiments, the wash solution comprises a detergent. In some embodiments, the detergent is Triton X-100 detergent.

[0009] In some embodiments, the cleaning solution comprises a salt. In some embodiments, the salt comprises a monovalent or divalent cation. In some embodiments, the monovalent or divalent cation is selected from the group consisting of lithium, sodium, ammonium, calcium, and magnesium. In some embodiments, the cleaning solution comprises an anion selected from the group consisting of chloride, sulfate, and acetate. In some embodiments, the cleaning solution comprises sodium chloride, calcium chloride, and / or ammonium sulfate. In some embodiments, the salt concentration in the cleaning solution is about 50 mM to about 800 mM.

[0010] In some embodiments, the wash solution comprises a Tris buffer. In some embodiments, the wash solution comprises EDTA. In some embodiments, the resuspension solution comprises an RNase inhibitor.

[0011] In some embodiments, the wash solution comprises DNase. In some embodiments, the DNase is DNase I. In some embodiments, the wash solution comprises RNase III. In some embodiments, the wash solution comprises a protease. In some embodiments, the protease is proteinase K.

[0012] In some embodiments, the second filter membrane is a tangential flow filtration (TFF) membrane. In some embodiments, the second filter membrane is a hollow fiber membrane. In some embodiments, the second filter membrane has a molecular weight cutoff of about 20 kDa to about 150 kDa.

[0013] In some embodiments, the method further comprises repeating steps (iii)(a) and (iii)(b). In some embodiments, steps (iii)(a) and (iii)(b) are each performed 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more times prior to step (iv). In some embodiments, one or more repetitions of step (iii)(a) use a different wash solution than the first repetition of step (iii)(a). In some embodiments, the method comprises contacting the precipitated mRNA composition with a salt, a detergent, a protease, a DNase, and an RNase III prior to resuspending the mRNA.

[0014] In some embodiments, the resuspension solution has a salt concentration of 20 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less. In some embodiments, the resuspension solution comprises Tris. In some embodiments, the resuspension solution comprises EDTA. In some embodiments, the resuspension solution comprises an RNase inhibitor. In some embodiments, the resuspension solution has a pH of about 5.0 to about 7.5. The resuspension solution has a conductivity of 10 mS / cm or less, 8 mS / cm or less, 6 mS / cm or less, 5 mS / cm or less, 4 mS / cm or less, 3 mS / cm or less, 2.5 mS / cm or less, 2.0 mS / cm or less, 1.5 mS / cm or less, 1.0 mS / cm or less, or 0.5 mS / cm or less.

[0015] In some aspects, the disclosure relates to a purified mRNA composition produced by any of the methods described herein, comprising resuspended mRNA, the resuspended mRNA comprising an open reading frame encoding a vaccine antigen or a therapeutic protein. In some embodiments, the purified mRNA composition has a salt concentration of 20 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less. In some embodiments, 1% or less, 0.8% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less of the mRNA molecules in the purified mRNA composition are double-stranded RNA (dsRNA) molecules. In some embodiments, the concentration of double-stranded RNA (dsRNA) in the isolated mRNA composition is 1% (w / w) or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, or 0.008% or less, 0.006% or less, 0.004% or less, 0.002% or less, or 0.001% or less. In some embodiments, the purified mRNA composition has a protein concentration of 1% (w / w) or less, 0.8% or less, 0.6% or less, 0.4% or less, or 0.2% or less. In some embodiments, the purified mRNA composition has a DNA concentration of 1% (% w / w) or less, 0.8% or less, 0.6% or less, 0.4% or less, or 0.2% or less. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the mRNA in the purified mRNA composition comprises a poly(A) tail. [Brief description of the drawings]

[0016] [Figure 1]1 shows a diagram outlining an exemplary process for sequential mRNA purification by precipitation and filtration: mRNA and a high salt buffer are mixed to precipitate the mRNA, the precipitated mRNA is filtered to remove soluble impurities, one or more wash solutions are added and filtered to remove any remaining impurities, and a resuspension solution is mixed with the precipitated mRNA to resuspend the precipitated mRNA, thereby producing a purified mRNA composition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] An embodiment of the present disclosure relates to a method of purifying an mRNA composition by (i) precipitating mRNA, (ii) removing the liquid phase of the composition containing soluble impurities from the precipitated mRNA, (iii)(a) adding a wash solution to the precipitated mRNA, (iii)(b) removing the wash solution from the precipitated mRNA, and (iv) adding a resuspension solution to the precipitated mRNA to resuspend the mRNA. Although mRNA is useful for multiple applications, including expression of a desired protein in vivo, the mRNA production process may introduce impurities that must be removed before the mRNA can be used in such applications. Purifying mRNA by the methods described herein has multiple advantages, such as not being limited by the binding capacity of the column stationary phase used in conventional chromatographic approaches and eliminating the need for chromatographic reagents. Additionally, multiple different wash solutions may be applied sequentially to the precipitated mRNA composition to remove specific impurities from the mRNA composition, such as impurities known to be introduced by the manufacturing process (e.g., in vitro transcriptase and DNA template) or impurities detected by analysis of the mRNA composition (e.g., dsRNA). Finally, the integration of precipitation, filtration, washing, and resuspension steps into a continuous method allows impurities to be removed from an mRNA composition by automated means, eliminating the need for separate input loading, intermediate transfer, and output discharge steps.

[0018] Precipitation Some embodiments relate to methods of precipitating mRNA in an mRNA composition to produce a precipitated mRNA composition comprising precipitated mRNA and soluble impurities. After the mRNA is precipitated, other components of the mRNA composition (e.g., proteins, DNA, free nucleotide triphosphates, short oligonucleotides) can be removed, for example, by filtering the solution and washing the precipitated mRNA. After washing and filtering the precipitated mRNA to remove impurities, the RNA can be resolubilized and dissolved in a solvent to produce a purified mRNA composition. The sugar-phosphate backbone of nucleic acids such as mRNA contains negatively charged phosphate ions, making individual mRNA molecules hydrophilic and dissolving them in water. Adding a salt containing a positive ion, such as sodium ion, ammonium ion, or lithium ion, neutralizes the negative charge of these phosphate groups, making the mRNA molecules less hydrophilic and reducing the tendency of individual mRNA molecules to repel one another. Without being bound by theory, it is believed that this reduced solubility of mRNA, coupled with the addition of salts that increase the concentration of other solutes in the mRNA composition, can facilitate precipitation of the mRNA while other components of the solution remain dissolved.

[0019] In some embodiments, the methods described herein relate to precipitating mRNA in an mRNA composition by adding a high salt buffer to the composition. As explained in the previous section, the positive ions of the salt can balance the negative charges on the phosphate moieties on the sugar:phosphate backbone of the nucleic acid, reducing the electrostatic repulsion between the mRNA molecules and precipitating the mRNA in the composition. The high salt buffer for precipitating the mRNA in the composition can contain any salt, or salts, that release positive ions when dissolved in water. Non-limiting examples of salts suitable for precipitating mRNA include lithium chloride, sodium chloride, sodium acetate, ammonium acetate, calcium chloride, magnesium chloride, and ammonium sulfate. In some embodiments, the high salt buffer comprises a salt that includes a monovalent cation. A monovalent cation is a cation that contains one more proton than electrons, and thus has a 1+ charge. Non-limiting examples of monovalent cations include lithium ions, sodium ions, ammonium ions, and potassium ions. In some embodiments, the high salt buffer comprises a salt that includes a divalent cation. A divalent cation is a cation that contains two more protons than electrons, and thus has a 2+ charge. Non-limiting examples of divalent cations include magnesium ions and calcium ions. In some embodiments, the high salt buffer solution comprises a cation selected from the group consisting of lithium ions, sodium ions, potassium ions, ammonium ions, magnesium ions, and calcium ions, and an anion selected from the group consisting of sulfate, acetate, and chloride ions. In some embodiments, the high salt buffer comprises a salt selected from the group consisting of lithium chloride, sodium chloride, sodium acetate, ammonium acetate, calcium chloride, magnesium chloride, and ammonium sulfate. In some embodiments, the high salt buffer comprises lithium chloride. In some embodiments, the high salt buffer comprises sodium chloride. In some embodiments, the high salt buffer comprises sodium acetate. In some embodiments, the high salt buffer comprises ammonium acetate. In some embodiments, the high salt buffer comprises calcium chloride. In some embodiments, the high salt buffer comprises magnesium chloride.In some embodiments, the high salt buffer comprises ammonium sulfate.

[0020] The high salt buffer used to precipitate the mRNA can contain any salt concentration sufficient to reduce the dielectric constant of the mRNA composition. In some embodiments, the high salt buffer has a salt concentration of about 0.1 M to about 10 M. In some embodiments, the high salt buffer has a salt concentration of 0.1 M to 0.2 M, 0.2 M to 0.4 M, 0.4 M to 0.6 M, 0.6 M to 0.8 M, 0.8 M to 1.0 M, 1.0 M to 1.25 M, 1.25 M to 1.5 M, 1.5 M to 1.75 M, 1.75 M to 2.0 M, 2.0 M to 2.25 M, 2.25 M to 2.5 M, 2. ... In some embodiments, the high salt buffer has a salt concentration of 0.1 M to 1.0 M. In some embodiments, the high salt buffer has a salt concentration of 1.0 M to 2.0 M. In some embodiments, the high salt buffer has a salt concentration of 2.0 M to 3.0 M. In some embodiments, the high salt buffer has a salt concentration of 3.0 M to 4.0 M. In some embodiments, the high salt buffer has a salt concentration of 4.0 M to 5.0 M. In some embodiments, the high salt buffer has a salt concentration of about 1.0 M. In some embodiments, the high salt buffer has a salt concentration of about 1.5 M. In some embodiments, the high salt buffer has a salt concentration of about 2.0 M. In some embodiments, the high salt buffer has a salt concentration of about 2.5 M. In some embodiments, the high salt buffer has a salt concentration of about 3.0 M. In some embodiments, the high salt buffer has a salt concentration of about 3.5 M. In some embodiments, the high salt buffer has a salt concentration of about 4.0 M. In some embodiments, the high salt buffer has a salt concentration of about 4.5 M. In some embodiments, the high salt buffer has a salt concentration of about 5.0 M. In some embodiments, the salt concentration of the high salt buffer refers to the concentration of a single salt. In other embodiments, the salt concentration of the high salt buffer refers to the individual concentrations of two or more different salts. In other embodiments, the salt concentration of the high salt buffer refers to the concentration of salt cations dissolved in the high salt buffer.

[0021] In some embodiments, a high salt buffer is added to the mRNA composition to increase the salt concentration of the mRNA composition to a particular level or above a particular threshold. The salt concentration of the composition affects its dielectric constant, which in turn affects nucleic acid solubility, so precipitation can be achieved in some embodiments by increasing the salt concentration of the composition to a particular level or above a particular threshold. In some embodiments, the salt concentration of the mRNA composition after adding the high salt buffer is 0.1M to 0.2M, 0.2M to 0.4M, 0.4M to 0.6M, 0.6M to 0.8M, 0.8M to 1.0M, 1.0M to 1.25M, 1.25M to 1.5M, 1.5M to 1.75M, 1.75M to 2.0M, 2.0M to 2.25M, 2.2 ... In some embodiments, the salt concentration after addition of the high salt buffer is 0.1 M to 1.0 M. In some embodiments, the salt concentration after addition of the high salt buffer is 1.0 M to 2.0 M. In some embodiments, the salt concentration after addition of the high salt buffer is 2.0 M to 3.0 M. In some embodiments, the salt concentration after addition of the high salt buffer is 3.0 M to 4.0 M. In some embodiments, the salt concentration after addition of the high salt buffer is 3.0 M to 4.0 M. In some embodiments, the salt concentration after addition of the high salt buffer is 4.0M to 5.0M. In some embodiments, the salt concentration after addition of the high salt buffer is about 1.0M. In some embodiments, the salt concentration after addition of the high salt buffer is about 1.5M. In some embodiments, the salt concentration after addition of the high salt buffer is about 2.0M. In some embodiments, the salt concentration after addition of the high salt buffer is about 2.5M. In some embodiments, the salt concentration after addition of the high salt buffer is about 3.0M. In some embodiments, the salt concentration after addition of the high salt buffer is about 3.5M. In some embodiments, the salt concentration after addition of the high salt buffer is about 4.0M. In some embodiments, the salt concentration after addition of the high salt buffer is about 4.5M. In some embodiments, the salt concentration after addition of the high salt buffer is about 5.0M.The salt concentration after addition of a high salt buffer may refer to the concentration of a single salt, the total concentration obtained by adding the individual concentrations of multiple salts present in the mRNA composition, or the concentration of salt cations dissolved in the mRNA composition.

[0022] In some embodiments, the high salt buffer comprises a sugar. The presence of a sugar in a high salt buffer for precipitating mRNA increases the total solute concentration of the buffer, which reduces the solubility of the mRNA, thereby promoting mRNA nucleation, aggregation, and precipitation. Furthermore, the large size of sugar molecules allows them to act as volume excluding agents, reducing the available space through which dissolved mRNA can diffuse into the solution, further promoting mRNA nucleation, aggregation, and precipitation. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is selected from the group consisting of glucose, fructose, mannose, and galactose. In some embodiments, the sugar is a disaccharide. In some embodiments, the disaccharide is trehalose, lactose, or sucrose. In some embodiments, the sugar is a polysaccharide. In some embodiments, the polysaccharide is glycogen. In some embodiments, the high salt buffer has a sugar concentration of about 0.1 M to about 10 M. In some embodiments, the high salt buffer is 0.1M to 0.2M, 0.2M to 0.4M, 0.4M to 0.6M, 0.6M to 0.8M, 0.8M to 1.0M, 1.0M to 1.25M, 1.25M to 1.5M, 1.5M to 1.75M, 1.75M to 2.0M, 2.0M to 2.25M, 2.25M to 2.5M, 2.5M to 3.0M, 3.0M to 3.5M, 3.5 ... The high salt buffer has a sugar concentration of 0.1 M to 1.0 M. In some embodiments, the high salt buffer has a sugar concentration of 0.1 M to 1.0 M.

[0023] In some embodiments, the high salt buffer has a higher conductivity than the mRNA composition to which it is added, thus increasing the conductivity of the mRNA composition after addition. In some embodiments, the high salt buffer is added to increase the conductivity of the solution to a certain level or beyond a certain threshold. The conductivity of the solution serves as a measure of its salt concentration, which is negatively correlated with mRNA solubility. Thus, the conductivity of the high salt buffer and / or the mRNA composition after addition of the high salt buffer measures the extent to which the mRNA has increased. In some embodiments, the high salt buffer has a conductivity of at least 5 mS / cm, at least 6 mS / cm, at least 7 mS / cm, at least 8 mS / cm, at least 9 mS / cm, at least 10 mS / cm, at least 12 mS / cm, or at least 15 mS / cm. In some embodiments, the high salt buffer has a conductivity of 5-10 mS / cm, 5-15 mS / cm, 5-7 mS / cm, 6-9 mS / cm, 8-10 mS / cm, 9-12 mS / cm, or 10-15 mS / cm. In some embodiments, the high salt buffer has a conductivity of 5-300 mS / cm, 5-250 mS / cm, 5-200 mS / cm, 5-150 mS / cm, 5-100 mS / cm, 5-75 mS / cm, or 5-50 mS / cm. In some embodiments, after the high salt buffer is added, the mRNA composition has a conductivity of at least 5 mS / cm, at least 6 mS / cm, at least 7 mS / cm, at least 8 mS / cm, at least 9 mS / cm, at least 10 mS / cm, at least 12 mS / cm, or at least 15 mS / cm. In some embodiments, after the high salt buffer is added, the mRNA composition has a conductivity of 5-10 mS / cm, 5-15 mS / cm, 5-7 mS / cm, 6-9 mS / cm, 8-10 mS / cm, 9-12 mS / cm, or 10-15 mS / cm. In some embodiments, after the high salt buffer is added, the mRNA composition has a conductivity of 5-300 mS / cm, 5-250 mS / cm, 5-200 mS / cm, 5-150 mS / cm, 5-100 mS / cm, 5-75 mS / cm, or 5-50 mS / cm.

[0024] In some embodiments, the precipitating step includes adding alcohol to the mRNA composition. Alcohol is less polar than water, so the solubility of nucleic acids, such as mRNA, in alcohol is lower in alcohol than in water. Thus, the alcohol in the alcohol-based solution acts as a volume excluding agent, reducing the available space into which the mRNA can diffuse, thereby promoting nucleation, aggregation, and precipitation of the mRNA. In some embodiments, the alcohol-containing solution includes ethanol. In some embodiments, the alcohol-containing solution includes isopropanol. In some embodiments, the solution added to the mRNA during the precipitation step includes at least 60% (% w / v), at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 100% alcohol. In some embodiments, the alcohol-containing solution includes about 70% alcohol. In some embodiments, the alcohol-containing solution includes about 70% ethanol. In some embodiments, the alcohol-containing solution includes about 70% isopropanol.

[0025] One advantage of the methods described herein is that precipitation can be used to convert most or all of the mRNA in a composition into a solid form prior to purification, so precipitation-based methods are not limited by the binding capacity of a column. Thus, precipitation can be performed to precipitate a specific amount or percentage of mRNA in a composition in some embodiments. Methods for determining the percentage of mRNA precipitated by a process are routine in the art. For example, the amount of dissolved mRNA present in a sample can be measured before and after precipitation, and the measured amounts can be compared to calculate the percentage of mRNA precipitated. In some embodiments, at least 80% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 85% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 90% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 95% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 96% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 97% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 98% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, at least 99% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition. In some embodiments, up to 100% of the mRNA in the mRNA composition is precipitated before the liquid is removed from the composition.

[0026] filtration In some embodiments, after the mRNA is precipitated, the composition containing the precipitated mRNA is filtered to separate the solution containing impurities.

[0027] Non-limiting examples of impurities that may be removed by filtration include salts (e.g., salts of the mRNA production process (e.g., in vitro transcription) and / or salts used to precipitate the mRNA), proteins (e.g., in vitro transcriptase, DNase, protease, RNase III), and DNA from the precipitated mRNA. In some embodiments, the filtering step includes adding the precipitated mRNA and the supernatant to a hollow fiber filter. A hollow fiber filter is a membrane that includes a network of fibers and pores formed by the networked fibers. In contrast to tangential flow, where a liquid or composition flows parallel to the TFF membrane, the supernatant and precipitated mRNA are applied to the hollow fiber filter such that the direction of liquid flow is through the filter (axial flow). The liquid flow may occur due to gravity or may be assisted by the use of negative pressure below the filter to facilitate filtration. In some embodiments, a vacuum is applied to facilitate the flow of liquid through the filter. Liquids, dissolved solutes, and suspended particles smaller than the pores of the filter pass through the pores, while solid components larger than the pores are retained on the filter. Thus, the precipitated mRNA is retained on the filter while the supernatant passes through the filter. After the supernatant passes through the filter, a wash solution may be added to the precipitated mRNA to further remove any residual protein or DNA from the mRNA. In some embodiments, the hollow fiber filter has a pore size of 10 μm or less, 9 μm or less, 8 μm or less, 7 μm or less, 6 μm or less, 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, 1 μm or less, 0.9 μm or less, 0.8 μm or less, 0.7 μm or less, 0.6 μm or less, 0.5 μm or less, 0.4 μm or less, 0.3 μm or less, or 0.2 μm or less. In some embodiments, the hollow fiber filter has a pore size of 30 μm or less. In some embodiments, the hollow fiber filter has a pore size of 20 μm or less. In some embodiments, the hollow fiber filter has a pore size of 10 μm or less. In some embodiments, the hollow fiber filter has a pore size of 5 μm or less. In some embodiments, the hollow fiber filters have a pore size of 2 μm or less.In some embodiments, the hollow fiber filter has a pore size of 1 μm or less, hi some embodiments, the hollow fiber filter has a pore size of 0.5 μm or less.

[0028] In some embodiments, filtering the mRNA is accomplished by tangential flow filtration (TFF), which includes contacting the precipitated mRNA in the mRNA composition with a TFF membrane. In TFF, the mRNA composition flows over a filtration membrane (TFF membrane) that contains holes, and the holes of the membrane are oriented perpendicular to the direction of flow. Components of the mRNA composition flow through the holes, if possible, while components that do not pass through the holes are retained in the mRNA composition. Thus, TFF removes smaller impurities, such as peptide fragments, DNA fragments, amino acids, and nucleotides, from the mRNA composition, while larger molecules, such as full-length RNA transcripts, are retained in the mRNA composition. In addition, RNA polymerase can generate double-stranded RNA transcripts during IVT, including RNA:RNA hybrids of full-length RNA transcripts and another RNA with a complementary sequence. The second RNA hybridized to the full-length RNA transcript can be another full-length RNA or a smaller RNA that hybridizes to only a portion of the full-length transcript. Similar to the DNA fragments generated by DNase digestion of the DNA template, these small RNAs may also be removed during TFF, such that fewer dsRNA molecules are present in the filtered RNA composition.

[0029] The size of the pores of the TFF membrane affects which components are filtered (removed) from the mRNA composition and which components are retained in the mRNA composition. In general, TFF membranes are characterized in terms of molecular weight cutoff, where components smaller than the molecular weight cutoff are removed from the mRNA composition during TFF, while components larger than the molecular weight cutoff are retained in the mRNA composition. In some embodiments, the tangential flow filtration comprises using a TFF membrane with a molecular weight cutoff of 500 kDa or less, 200 kDa or less, 150 kDa or less, 100 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDa or less, or less. In some embodiments, the tangential flow filtration comprises using a TFF membrane with a molecular weight cutoff of 500 kDa or less. In some embodiments, the tangential flow filtration comprises using a TFF membrane with a molecular weight cutoff of 400 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 300 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 200 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 100 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 50 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 40 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 30 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 20 kDa or less. In some embodiments, the tangential flow filtration includes using a TFF membrane with a molecular weight cutoff of 20 kDa to 200 kDa. In some embodiments, the tangential flow filtration involves using a TFF membrane with a molecular weight cutoff of between 20 kDa and 150 kDa.

[0030] Removing liquid from an mRNA composition (e.g., by filtration) reduces the volume of the mRNA composition. If most or all of the mRNA in the composition is precipitated and not dissolved in the aqueous phase, little or no mRNA is removed by filtration. Thus, the loss of volume by filtration exceeds the loss of mRNA. As a result, the concentration of mRNA increases after filtration of the precipitated mRNA composition. Thus, in some embodiments, the concentration of mRNA in the precipitated mRNA composition increases by filtration. In some embodiments, the concentration of mRNA in the precipitated mRNA composition is increased by about 2-10,000, 2-5,000, 2-2,500, 2-1,000, 2-500, 2-100, 10-10,000, 10-5,000, 10-2,500, 10-1,000, 10-500, 10-100, 100-100,000, 100-50,000, 100-25,000, 100-10,000, 100-5,000, 100-2,500, 100-1,500, 100-1,000, 100-500, or 100-200 compared to the pre-filtration concentration of the mRNA composition.

[0031] Washing Some embodiments relate to a method of washing the precipitated mRNA of an mRNA composition to remove one or more impurities from the precipitated mRNA. A washing solution refers to a solution in which the solubility of the mRNA is minimal. Generally, the washing solution contains salts and / or alcohols that can precipitate the RNA. After the mRNA is precipitated, aspirating the supernatant removes many dissolved impurities from the mRNA composition. However, the precipitated mRNA contains many bound salt cations, and any liquid remaining in the mRNA composition still contains residual impurities. Adding a washing solution dilutes these impurities, thereby reducing the total abundance of impurities in the mRNA composition after the washing solution is removed. If the process of (a) adding a washing solution to the precipitated mRNA to dilute the impurities, and (b) removing the washing solution from the precipitated mRNA is repeated, the abundance of impurities in the precipitated mRNA composition is reduced during successive repetitions until the abundance of the impurities is minimized. Removing impurities by repeated steps of washing the precipitated mRNA and removing the washing solution has the added advantage that different washing solutions can be used in successive repetitions, which may improve the efficiency of impurity removal. For example, the first washing solution added to the precipitated mRNA may be one in which the salt used to precipitate the mRNA is soluble, reducing the salt concentration of the mRNA composition after precipitation. The precipitated mRNA may then be washed with a second washing solution containing an enzyme (e.g., DNase, RNase III, or protease) to digest impurities that may be present in the mRNA composition. This washing solution may contain one or more other components that promote enzyme activity (e.g., enzyme cofactors such as magnesium ions) to enhance the degradation of impurities targeted by the enzyme. The next washing solution may then contain components useful for removing proteins (e.g., enzymes or host cell proteins) from the composition.

[0032] In some embodiments, the wash solution comprises alcohol. Alcohols are generally less polar than water, so the solubility of nucleic acids such as mRNA in alcohol is lower than in water. However, the relative reduction in salt solubility is less than the reduction in mRNA solubility, so an alcohol-based wash solution can be used to dilute other impurities (e.g., salts, proteins, and / or DNA), allowing for their removal when the liquid wash solution is separated from the precipitated mRNA by aspiration or filtration. In some embodiments, the wash solution comprises ethanol. In some embodiments, the wash solution comprises isopropanol. In some embodiments, the wash solution comprises at least 60% (% w / v), at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or up to 100% alcohol. In some embodiments, the wash solution comprises about 70% alcohol. In some embodiments, the wash solution comprises about 70% ethanol. In some embodiments, the wash solution comprises about 70% isopropanol.

[0033] In some embodiments, the wash solution comprises a surfactant. Surfactant, as used herein, refers to a compound that reduces the surface tension between two liquids. In general, a surfactant promotes mixing between a first liquid and a second liquid that has a low ability to interact with the first liquid in the absence of the surfactant. For example, a surfactant promotes the interaction between an inorganic solvent, such as water, and an organic solvent, such as a hydrocarbon or acetonitrile. If an organic solvent is present in the mRNA composition, for example, if an organic solvent is used in an upstream process to separate the mRNA from other components of the mRNA composition, the use of a surfactant is useful to promote the interaction of the organic solvent with the wash solution, allowing aspiration or filtration of the wash solution to remove the organic solvent from the precipitated mRNA. In some embodiments, the wash solution comprises a detergent. Detergent, as used herein, refers to a surfactant or combination of surfactants in an aqueous solution. The dilution of the surfactant in the detergent promotes the interaction between the surfactant molecules and the water molecules, allowing the water molecules of the aqueous solution (e.g., the wash solution) to indirectly bind to impurities in the mRNA composition, so that removal of the aqueous phase by filtration also removes the surfactant-bound impurities. In some embodiments, the detergent is a Triton detergent, such as Triton X-100 detergent.

[0034] In some embodiments of the methods described herein, the wash solution comprises a salt. In addition to their use in precipitating mRNA, the use of high-salt wash solutions is useful for removing proteins from mRNA compositions. Salt ions dissolved in water associate with oppositely charged moieties of proteins, and these associated salt ions attract water molecules, forming bridges between the water molecules and amino acids on the protein surface. By promoting indirect binding of water molecules with amino acids, the salt ions then increase the solubility of the protein in water. Thus, removal of the wash solution in which the protein is highly soluble can separate protein impurities from the precipitated mRNA of the mRNA composition. Furthermore, salts present in the wash solution reduce the solubility of the mRNA, as discussed above. Therefore, the use of wash solutions containing one or more salts inhibits the dissolution of precipitated mRNA during washing and filtration of the wash solution, reducing the amount of mRNA lost during the washing steps.

[0035] A wash solution for washing the precipitated mRNA and / or removing proteins from the composition may contain any salt or salts that release positive and negative ions when dissolved in water. The wash solution may contain the same salt as used to precipitate the mRNA or a different salt. Non-limiting examples of salts suitable for washing the mRNA include lithium chloride, sodium chloride, sodium acetate, ammonium acetate, calcium chloride, magnesium chloride, and ammonium sulfate. In some embodiments, the wash solution comprises a salt comprising a monovalent cation. In some embodiments, the wash solution comprises a salt comprising a divalent cation. In some embodiments, the wash solution comprises a cation selected from the group consisting of lithium ions, sodium ions, potassium ions, ammonium ions, magnesium ions, and calcium ions, and anions selected from the group consisting of sulfate, acetate, and chloride ions. In some embodiments, the wash solution comprises a salt selected from the group consisting of lithium chloride, sodium chloride, sodium acetate, ammonium acetate, calcium chloride, magnesium chloride, and ammonium sulfate. In some embodiments, the wash solution comprises lithium chloride. In some embodiments, the wash solution comprises sodium chloride. In some embodiments, the wash solution comprises sodium acetate. In some embodiments, the cleaning solution comprises ammonium acetate. In some embodiments, the cleaning solution comprises calcium chloride. In some embodiments, the cleaning solution comprises magnesium chloride. In some embodiments, the cleaning solution comprises ammonium sulfate.

[0036] Wash solutions used to wash precipitated mRNA and / or remove proteins from the mRNA composition may contain any salt concentration sufficient to promote solubility of proteins in water and / or inhibit dissolution of precipitated mRNA. In some embodiments, the wash solution has a salt concentration of about 0.1 M to about 1.0 M. In some embodiments, the wash solution has a salt concentration of about 50 mM to about 800 mM. In some embodiments, the wash solution has a salt concentration of about 100 mM to about 600 mM. In some embodiments, the wash solution has a salt concentration of about 200 mM to about 500 mM. In some embodiments, the wash solution is 0.1M to 0.2M, 0.2M to 0.3M, 0.3M to 0.4M, 0.4M to 0.5M, 0.5M to 0.6M, 0.6M to 0.7M, 0.7M to 0.8M, 0.8M to 0.9M, 0.9M to 1.0M, 1.0M to 1.25M, 1.25M to 1.5M, 1.5M to 1.75M, 1.75M to 2.0M, 2.0M to 2.5M, 3.0M to 3.5M, 4.0M to 4.5M, 5.0M to 5.0M, 6.0M to 7.0M, 7.0M to 8.0M, 8.0M to 9.0M, 9.0M to 10.0M, 10.0M to 11.0M, 12.0M to 13.0M, 14.0M to 15.0M, 15.0M to 16.0M, 16.0M to 17.0M, 17.0M to 18.0M, 18.0M to 20.0M, 19.0M to 21.0M, 22.0M to 23.0M, 23.0M to 25.0M, 24.0M to 26.0M, 25.0M to 28.0M, 26.0M to 29.0M, 27.0M to 28.0M, 28.0M to 29.0M, 29.0M to 30.0M, 30.0M to 31.0M, 31.0M to The wash solution has a salt concentration of 0.25M, 2.25M to 2.5M, 2.5M to 2.75M, 2.75M to 3.0M, 3.0M to 3.5M, 3.5M to 4.0M, 4.5M to 5.0M, 5.0M to 6.0M, 6.0M to 6.5M, 6.5M to 7.0M, 7.0M to 7.5M, 7.5M to 8.0M, 8.5M to 9.0M, 9.0M to 9.5M, or 9.5M to 10M. In some embodiments, the wash solution has a salt concentration of 100 mM to 200 mM. In some embodiments, the wash solution has a salt concentration of 200 mM to 300 mM. In some embodiments, the wash solution has a salt concentration of about 300 mM to about 400 mM. In some embodiments, the washing solution has a salt concentration of 400 mM to 500 mM. In some embodiments, the washing solution has a salt concentration of 500 mM to 600 mM. In some embodiments, the washing solution has a salt concentration of 600 mM to 700 mM. In some embodiments, the washing solution has a salt concentration of 700 mM to 800 mM. In some embodiments, the washing solution has a salt concentration of 800 mM to 900 mM. In some embodiments, the washing solution has a salt concentration of 900 mM to 1.0 M. In some embodiments, the washing solution has a salt concentration of about 100 mM. In some embodiments, the washing solution has a salt concentration of about 150 mM.In some embodiments, the wash solution has a salt concentration of about 200 mM. In some embodiments, the wash solution has a salt concentration of about 250 mM. In some embodiments, the wash solution has a salt concentration of about 300 mM. In some embodiments, the wash solution has a salt concentration of about 400 mM. In some embodiments, the wash solution has a salt concentration of about 500 mM. In some embodiments, the salt concentration of the wash solution refers to the concentration of a single salt. In other embodiments, the salt concentration of the wash solution refers to the individual concentrations of two or more different salts. In other embodiments, the salt concentration of the wash solution refers to the concentration of salt cations dissolved in the high salt buffer.

[0037] In some embodiments, the wash solution comprises a protease. A protease is an enzyme that catalyzes the breakdown of a protein into smaller protein fragments, such as smaller polypeptides, oligopeptides, or individual amino acids. Generally, a protease catalyzes the hydrolysis of a peptide bond that connects amino acids in a polypeptide chain, and the hydrolysis of the peptide bond results in the release of two polypeptides, two amino acids, or an amino acid and a polypeptide, depending on the cleavage site. An exopeptidase is a protease that catalyzes a peptide bond between a terminal amino acid and an adjacent amino acid in a polypeptide, resulting in the release of the terminal amino acid from the remaining polypeptide. An endopeptidase is a protease that catalyzes a peptide bond between internal amino acids in a polypeptide, releasing two separate polypeptides or oligopeptides. Endopeptidases may differ in specificity and may cleave more readily before or after certain amino acid residues. For example, trypsin cleaves readily after arginine or lysine unless the arginine or lysine is followed by proline. Cleavage "after" the first amino acid refers to cleavage of the peptide bond connecting the first amino acid to the next amino acid in a protein, which is described by an amino acid sequence that lists the amino acids from the N-terminus to the C-terminus. The peptide fragments generated by protease-mediated cleavage of longer proteins are smaller than full-length mRNA and are more easily removed by filtration.

[0038] In some embodiments, the protease introduced into the mRNA composition is selected from the group consisting of proteinase K, Lys-C, trypsin, TPCK-treated trypsin, chymotrypsin, alpha-lytic protease, and endoproteinase AspN. In some embodiments, the protease is a serine protease, such as proteinase K. Proteinase K is an endopeptidase with broad specificity that cleaves peptide bonds adjacent to the carboxyl groups of aliphatic and aromatic amino acids. Exemplary amino acids that can serve as substrates for cleavage by proteinase K include alanine, glycine, isoleucine, leucine, proline, valine, tryptophan, tyrosine, and phenylalanine. An example of a DNA sequence encoding proteinase K is represented by accession number X14688. An example of an amino acid sequence of proteinase K is represented by accession number P06873. In some embodiments, the protease is proteinase K. In some embodiments, the protease is Lys-C. In some embodiments, the protease is TPCK-treated trypsin. In some embodiments, the protease is chymotrypsin. In some embodiments, the protease is alpha-lytic protease. In some embodiments, the protease is AspN.

[0039] In some embodiments, after the protease is introduced into the mRNA composition, the concentration of the protease in the mRNA composition is about 0.1 units / mL to about 100 units / mL. With respect to a protein, a "unit" ("U") refers to the amount of protein that can perform a specific function in a given amount of time. For example, one unit of proteinase K is defined as the amount of enzyme required to liberate Folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine in 1 minute at 37° C. in a total reaction volume of 250 μL. See, e.g., Anson. J Gen Physiol. 1938. 22(1):79-89. In some embodiments, the concentration of the protease in the mRNA composition is about 0.2 to about 50 units / mL, about 0.3 to about 25 units / mL, about 0.4 to about 10 units / mL, about 0.5 to about 5 units / mL, about 0.5 to about 3 units / mL, about 0.5 to about 2 units / mL, or about 0.5 to about 1 unit / mL. In some embodiments, the concentration of the protease in the mRNA composition is about 0.1 to about 2 units / mL. In some embodiments, the concentration of the protease in the mRNA composition is about 1 to about 10 units / mL. In some embodiments, the concentration of the protease in the mRNA composition is about 10 to about 100 units / mL.

[0040] In some embodiments, the amount of protease in the mRNA composition during the protease digestion step relative to the amount of RNA polymerase in the mRNA composition is at least 1:1,000,000 (1 unit protease:1,000,000 μmol RNA polymerase). In some embodiments, the protease concentration:RNA polymerase concentration in the mRNA composition is about 1:10 to about 1:100, about 1:100 to about 1:1,000, about 1:1,000 to about 1:10,000, about 1:10,000 to about 1:100,000, or about 1:100,000 to about 1:1,000,000. In some embodiments, the protease concentration:RNA polymerase concentration in the mRNA composition is about 1:1,000 to about 1:50,000. In some embodiments, the amount of protease in the mRNA composition during the protease digestion step relative to the amount of other proteins in the mRNA composition is at least 1:1,000,000 (1 unit protease:1,000,000 μmol of other proteins). In some embodiments, the protease concentration:protein concentration in the mRNA composition is about 1:10 to about 1:100, about 1:100 to about 1:1,000, about 1:1,000 to about 1:10,000, about 1:10,000 to about 1:100,000, or about 1:100,000 to about 1:1,000,000. In some embodiments, the protease concentration:protein concentration in the mRNA composition is about 1:1,000 to about 1:50,000.

[0041] In some embodiments, the mRNA composition is incubated after the addition of the protease for a sufficient time to allow protein digestion. In some embodiments, the protease digestion step is performed at about 37° C. In some embodiments, the protease digestion step is performed at a temperature of 70° C. or less, 60° C. or less, 50° C. or less, or 40° C. or less. In some embodiments, the protease digestion step is performed for about 10 minutes to about 6 hours. In some embodiments, the protease digestion step is performed for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, the protease digestion step is performed for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 5 hours. In some embodiments, the protease digestion step is performed for about 15 minutes. In some embodiments, the protease digestion step is performed for about 30 minutes. In some embodiments, the protease digestion step is performed for about 45 minutes. In some embodiments, the protease digestion step is carried out for about 60 minutes.

[0042] In some embodiments, the IVT mRNA composition includes one or more cations. In some embodiments, one or more cations are added to the IVT mRNA composition during or after IVT. The presence and concentration of cations can affect the activity of proteases. For example, some proteases are more active in the presence of divalent cations, such as magnesium (Mg2+) ions. Therefore, adding magnesium or other cations to the mRNA composition can improve the efficiency of protease digestion, thereby allowing more residual protein to be removed from the IVT mRNA composition to produce a purer RNA composition. In some embodiments, the cation present in or added to the mRNA composition is magnesium ion. In some embodiments, the cation present in or added to the mRNA composition is calcium ion. In some embodiments, the concentration of cations in the mRNA composition during protease digestion is about 10 mM to about 100 mM. In some embodiments, the concentration of the cation is about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 20 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 50 mM to about 60 mM, about 60 mM to about 70 mM, about 70 mM to about 80 mM, about 80 mM to about 90 mM, or about 90 to about 100 mM. In some embodiments, the concentration of the cation is about 10 mM. In some embodiments, the concentration of the cation is about 20 mM. In some embodiments, the concentration of the cation is about 30 mM. In some embodiments, the concentration of magnesium ions in the mRNA composition during protease digestion is about 10 mM to about 100 mM. In some embodiments, the magnesium ion concentration is about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 20 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 50 mM to about 60 mM, about 60 mM to about 70 mM, about 70 mM to about 80 mM, about 80 mM to about 90 mM, or about 90 to about 100 mM. In some embodiments, the magnesium ion concentration is about 10 mM.In some embodiments, the concentration of magnesium ions is about 20 mM. In some embodiments, the concentration of magnesium ions is about 30 mM.

[0043] In some embodiments, the wash solution comprises DNase. DNase is an enzyme that catalyzes the cleavage of phosphodiester bonds between nucleotides in DNA molecules, resulting in the digestion of DNA into multiple smaller DNA fragments. Thus, the addition of DNase allows the digestion of these DNA templates into DNA fragments that are more easily removed by DNase digestion. After DNase digestion, the RNA transcripts are larger than the other components of the mRNA composition, and therefore can be separated using size-based filtration methods, such as the TFF and hollow fiber-based filtration methods described herein. In some embodiments, the DNase is DNase I.

[0044] In some embodiments, the DNase is incubated for a period of time sufficient to cleave one or more DNAs of the mRNA composition. A "period of time" sufficient to achieve a result refers to the length of time that, if allowed to pass, allows the result to be achieved. A period of time sufficient to cleave one or more DNAs in an mRNA composition, or to cleave a specific percentage of DNA, can be determined by incubating the DNase in a composition containing the DNA, sampling the composition after multiple periods of time, and determining the extent of DNA cleavage at each sampling period. If a given result has been achieved after a given period of time, the period is said to be sufficient to achieve that result. A period of time sufficient to cleave a specific percentage of DNA refers to a period of time after which at least that percentage of DNA originally present at the start of incubation has been cleaved by the DNase. Thus, after a period of time sufficient to cleave 80% of the DNA in the mRNA composition, at least 80% of the DNA originally present will have been cleaved by the DNase. In some embodiments, the DNase digestion step is performed at about 37°C. In some embodiments, the DNase digestion step is performed at a temperature of 70°C or less, 60°C or less, 50°C or less, or 40°C or less. In some embodiments, the DNase digestion step is performed for about 10 minutes to about 6 hours. In some embodiments, the DNase digestion step is performed for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, the DNase digestion step is performed for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 5 hours. In some embodiments, the DNase digestion step is performed for about 15 minutes. In some embodiments, the DNase digestion step is performed for about 30 minutes. In some embodiments, the DNase digestion step is performed for about 45 minutes. In some embodiments, the DNase digestion step is performed for about 60 minutes.

[0045] In some embodiments, the wash solution comprises RNase III. Ribonuclease III (RNase III) is an endoribonuclease that binds and cleaves double-stranded RNA (dsRNA), and is therefore useful for digesting dsRNA produced during IVT to reduce the deleterious effects of dsRNA in downstream applications. Double-stranded RNA transcripts, in which at least a portion of the RNA transcript hybridizes to another RNA molecule, elicit an innate immune response when introduced into a cell, causing degradation of both strands of the dsRNA. Reducing the abundance of dsRNA molecules allows for the generation of less immunogenic, and therefore more stable, RNA compositions. RNase III enzymes catalyze the cleavage of phosphodiester bonds between nucleotides in dsRNA. In cells, RNase III enzymes typically generate shorter dsRNA fragments, 18-25 base pairs in length, with each RNA strand having two nucleotides at the 3' end that are not joined by complementary nucleotides on the opposite RNA strand. Such shorter dsRNA fragments play a role in RNA-mediated silencing of gene expression and are known as siRNAs. These smaller dsRNA fragments generated by RNase III digestion are smaller than the full-length single-stranded mRNA transcripts that code for proteins, and therefore are more easily separated from the desired mRNA.

[0046] RNase III is expressed in many organisms and is highly conserved (e.g., Mian et al., Nucleic Acids Res., 1997, 25, 3187-95). RNase III species cloned to date contain the RNase III signature sequence and vary in size from 25 to 50 kDa. Multiple functions have been ascribed to RNase III. In both Escherichia coli and Saccharomyces cerevisiae, RNase III is involved in the processing of pre-ribosomal RNA (pre-rRNA) (e.g., Elela et al., Cell, 1996, 85, 115-24). RNase III is also involved in processing small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) in S. cerevisiae (e.g., Chanfreau et al., Genes Dev. 1996, 11, 2741-51; Qu et al., Mol. Cell. Biol. 1996, 19, 1144-58). In E. coli, RNase III is involved in the degradation of several mRNA species (e.g., Court et al., Control of messenger RNA stability, 1993, Academic Press, Inc, pp. 71-116). In some embodiments, the RNase III is Escherichia coli, Thermotoga maritima, or Aquifex aeolicus RNase III.

[0047] In some embodiments, the RNase III is incubated for a period of time sufficient to cleave one or more dsRNAs in the mRNA composition. In some embodiments, the RNase III digestion step is performed at about 37° C. In some embodiments, the RNase III digestion step is performed at a temperature of 70° C. or less, 60° C. or less, 50° C. or less, or 40° C. or less. In some embodiments, the RNase III digestion step is performed for about 10 minutes to about 6 hours. In some embodiments, the RNase III digestion step is performed for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, the RNase III digestion step is performed for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 5 hours. In some embodiments, the RNase III digestion step is performed for about 15 minutes. In some embodiments, the RNase III digestion step is performed for about 30 minutes. In some embodiments, the RNase III digestion step is performed for about 45 minutes. In some embodiments, the RNase III digestion step is carried out for about 60 minutes.

[0048] In some embodiments, the wash solution containing RNase III also contains one or more ions that promote the activity of RNase III. The presence and concentration of different cations can affect the activity of RNase III. For example, some RNase III enzymes are more active in the presence of divalent cations, such as magnesium (Mg2+). The cations present during RNase III digestion also affect the specificity of the enzyme. For example, certain RNAse III enzymes are more specific in the presence of magnesium ions compared to equal concentrations of other cations, such as manganese (Mn2+) ions. In some embodiments, the cation present or added in the mRNA composition or composition is magnesium ion. In some embodiments, the concentration of magnesium ion in the mRNA composition or composition during RNase III digestion is about 10 mM to about 100 mM. In some embodiments, the concentration of magnesium ions is about 1 mM to about 5 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 20 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 50 mM to about 60 mM, about 60 mM to about 70 mM, about 70 mM to about 80 mM, about 80 mM to about 90 mM, or about 90 mM to about 100 mM. In some embodiments, the concentration of magnesium ions is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM. In some embodiments, the concentration of magnesium ions is about 10 mM. In some embodiments, the concentration of magnesium ions is about 20 mM. In some embodiments, the concentration of magnesium ions is about 30 mM.

[0049] In some embodiments, the washing solution comprises a buffer. The presence of a buffer in the solution reduces the magnitude of the change in pH when an acid or base is added to the solution compared to when the same acid or base is added to a similar composition that does not contain a buffer. Non-limiting examples of buffers for use herein include ethylenediaminetetraacetic acid (EDTA), succinic acid, citric acid, aspartic acid, glutamic acid, maleic acid, cacodylic acid, 2-(N-morpholino)-ethanesulfonic acid (MES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-2-ethanesulfonic acid (PIPES), 2-(N-morpholino)-2-hydroxyethyl ester (HYDROGEN) ... hydroxy-propanesulfonic acid (MOPSO), N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)-propanesulfonic acid (MOPS), N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid (TAPSO), 3-(N,N-bis [2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid) (HEPPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), N-[tris(hydroxymethyl)-methyl]glycine (tricine), N,N-bis(2-hydroxyethyl)glycine (bicine), [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), tris(hydroxymethyl)aminomethane (tris), and bis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (bis-tris). Other buffer compositions, buffer concentrations, and additional components of solutions for use herein will be apparent to those of skill in the art. In some embodiments, the wash solution comprises a phosphate buffer. In some embodiments, the wash solution comprises a Tris buffer. In some embodiments, the wash solution comprises an acetate buffer.In some embodiments, the wash solution comprises a histidine buffer. In some embodiments, the wash solution comprises a citrate buffer. Citrate solutions are generally less conductive than other salt solutions, such as lithium chloride or sodium acetate solutions. Furthermore, the presence of citrate in a composition comprising RNA can reduce the frequency of base hydrolysis, thereby maintaining the stability of full-length RNA transcripts in the precipitated RNA during washing. In some embodiments, the concentration of the buffer is about 2-10 mM.

[0050] In some embodiments, the washing solution comprises an RNase inhibitor. The RNase inhibitor limits the activity of RNase. The above-mentioned RNase III is useful for cleaving undesired dsRNA, while other RNases cleave single-stranded RNA, such as RNase T1, which cleaves at guanosine nucleotides, and RNase A, which cleaves at uridine and cytidine residues.Therefore, the use of RNase inhibitors in the washing solution is useful for inhibiting the cleavage of the mRNA in the mRNA composition by contaminating RNases.

[0051] In some embodiments, the wash solution includes EDTA. EDTA refers to ethylenediaminetetraacetic acid, which adsorbs divalent cations (e.g., Mg2+ and Ca2+ ions). Adsorption of divalent cations by EDTA sequesters the cations and reduces the concentration of those cations in solution. This reduced availability of divalent cations reduces the activity of enzymes that require divalent cations for enzymatic activity, such as RNase. In some embodiments, the concentration of EDTA in the wash solution is about 50 mM to about 1 M. In some embodiments, the concentration of EDTA in the wash solution is about 100 mM to about 500 mM.

[0052] In some embodiments, the washing and / or filtration steps are repeated. For example, a first washing solution is added to the precipitated mRNA composition and removed by filtration, followed by a second washing solution being added and removed by filtration. Each of the steps of (i) adding a washing solution and (ii) removing the washing solution added in (i) by filtration is referred to as one "iteration" of the step. Each iteration after the first is referred to as a "repeat" of the step. Thus, a method in which steps (i) and (ii) are repeated once includes two iterations of each step - a first iteration of step (i), a first iteration of step (ii), a second iteration of step (i), and a second iteration of step (ii). The washing solution added in each iteration of step (i) can be the same washing solution or a different washing solution. In some embodiments, the washing solution added in one iteration of step (i) contains the same components (e.g., salts) as the washing solution added in the previous iteration of step (i), but at a different concentration of one or more components. In some embodiments, the same wash solution added in one iteration of step (i) contains the same components at the same or similar concentrations as the wash solution added in a previous iteration of step (i). In some embodiments, the wash and filtration steps of (i) and (ii) are repeated such that the precipitated mRNA has been contacted with salts, detergents, proteases, DNases, and RNase III before the resuspension step. Adding each of these components to the precipitated mRNA composition allows for the removal of protein, organic solvent, protein, DNA, and dsRNA impurities, respectively. Adding these components during separate wash steps further allows each wash step to target a specific impurity, thus avoiding undesirable interference between components useful for removing different impurities. For example, the presence of detergents useful for removing organic solvents or other organic compounds may interfere with the activity of enzymes such as DNase and RNase III. In addition, the wash solution added in a later iteration may contain components useful for removing residual components of the wash solution added in a previous iteration.For example, after adding a protein (e.g., protease, DNase, RNase III) to digest impurities into smaller, more easily filtered fragments, a wash solution containing salts useful for solubilizing and removing proteins can be added in subsequent iterations to allow removal of existing protein impurities as well as proteins present in the previous wash solution.

[0053] In some embodiments, the washing and filtration steps are performed, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times, respectively, before the resuspension step. In some embodiments, the washing and filtration steps are performed 2-5, 5-10, 10-15, 15-20, 20-25, or 25-30 times, respectively, before the resuspension step. In some embodiments, the washing and filtration steps are performed until the concentration of one or more impurities falls below a certain amount. In some embodiments, the washing and filtration steps are performed a sufficient number of times to reduce the amount of impurities in the mRNA composition to undetectable levels. In some embodiments, the washing and filtration steps are repeated until the composition contains no detectable protein. In some embodiments, the washing and filtering steps are repeated until the composition does not contain detectable DNA. In some embodiments, the washing and filtering steps are repeated until the composition does not contain dsRNA. In some embodiments, the washing and filtering steps are repeated until the composition does not contain detectable nucleotide triphosphates.

[0054] Resuspension Some embodiments relate to a method of dissolving the precipitated mRNA by resuspending it in a solvent with low or no impurities to produce a purified mRNA composition. Resuspension and dissolution of the precipitated mRNA typically occurs after the salts and / or alcohols used to precipitate the mRNA and other impurities have been removed by one or more washing steps. Resuspending the precipitated mRNA in a solution in which the mRNA is soluble, such as an aqueous buffer with a low salt concentration, results in the precipitated mRNA dissolving in the resuspension solution. Thus, adding a resuspension solution to the precipitated mRNA to dissolve the mRNA is also referred to as "resolubilization".

[0055] After the salt ions are removed from the precipitated RNA, the conductivity of the liquid, such as the resuspension solution, added to the precipitated RNA is lower. Thus, the conductivity of the solution containing the resuspended mRNA serves as a measure of the solubility of the mRNA in the solution. In some embodiments, the resuspension solution has a conductivity of 10 mS / cm or less, 8 mS / cm or less, 6 mS / cm or less, 5 mS / cm or less, 4 mS / cm or less, 3 mS / cm or less, 2.5 mS / cm or less, 2.0 mS / cm or less, 1.5 mS / cm or less, 1.0 mS / cm or less, or 0.5 mS / cm or less after addition to the precipitated RNA. In some embodiments, the resuspension solution has a conductivity of 10 mS / cm or less after addition to the precipitated RNA. At low conductivity, the precipitated RNA can be redissolved by adding the resuspension solution. In general, the resuspension solution may contain low salt concentration or no salt. In the absence of dissolved salt ions, the phosphates of the precipitated RNA interact more easily with water molecules, allowing the RNA to dissolve in another solution. In some embodiments, the resuspension solution does not contain detectable salts. In some embodiments, the resuspension solution has a salt concentration of 20 mM or less. In some embodiments, the resuspension solution has a salt concentration of 15 mM or less. In some embodiments, the resuspension solution has a salt concentration of 10 mM or less. In some embodiments, the resuspension solution has a salt concentration of 5 mM or less.

[0056] In some embodiments, the resuspension solution comprises a Tris buffer. Tris refers to tris(hydroxymethyl)aminomethane, an organic compound commonly used in buffers for resuspension and storage of nucleic acids such as DNA and RNA. The buffering activity of Tris is useful for reducing the magnitude of pH change when the composition is contacted with acid or base, and preventing undesirable effects of hydrogen or hydroxide ions on the mRNA of the composition, such as base hydrolysis. Thus, the presence of Tris inhibits the degradation of full-length mRNA into RNA fragments. Furthermore, in some embodiments, the resuspension solution can have a desired pH, or a pH within a desired range, such that the resuspended mRNA composition has a suitable pH for the desired downstream application (e.g., formulation in a delivery vehicle or expression in vivo). In some embodiments, the resuspension solution has a pH of about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, about 6.0 to about 7.0, about 7.0 to about 8.0, or about 6.5 to about 7.5. In some embodiments, the resuspension solution has a pH of about 5.0 to about 8.0. In some embodiments, the resuspension solution has a pH of about 5.0 to about 7.5. In some embodiments, the resuspension solution has a pH of about 5.0 to about 7.0. In some embodiments, the resuspension solution has a pH of about 5.0 to about 6.5. In some embodiments, the resuspension solution has a pH of about 5.0 to about 6.0. In some embodiments, the resuspension solution has a pH of about 5.0 to about 5.2. In some embodiments, the resuspension solution has a pH of about 5.2 to about 5.4. In some embodiments, the resuspension solution has a pH of about 5.4 to about 5.6. In some embodiments, the resuspension solution has a pH of about 5.6 to about 5.8. In some embodiments, the resuspension solution has a pH of about 5.8 to about 6.0. In some embodiments, the resuspension solution has a pH of about 6.0 to about 6.2. In some embodiments, the resuspension solution has a pH of about 6.2 to about 6.4. In some embodiments, the resuspension solution has a pH of about 6.4 to about 6.6. In some embodiments, the resuspension solution has a pH of about 6.6 to about 6.8.In some embodiments, the resuspension solution has a pH of about 6.8 to about 7.0. In some embodiments, the resuspension solution has a pH of about 7.0 to about 7.2. In some embodiments, the resuspension solution has a pH of about 7.2 to about 7.4. In some embodiments, the resuspension solution has a pH of about 7.4 to about 7.6. In some embodiments, the resuspension solution has a pH of about 7.6 to about 7.8. In some embodiments, the resuspension solution has a pH of about 7.8 to about 8.0.

[0057] In some embodiments, the resuspension solution includes an RNase inhibitor. In some embodiments, the resuspension solution includes EDTA. In some embodiments, the concentration of EDTA in the resuspension solution is about 50 mM to about 1 M. In some embodiments, the concentration of EDTA in the resuspension solution is about 100 mM to about 500 mM.

[0058] nucleic acid Also provided are compositions comprising nucleic acids and methods for producing nucleic acids. As used herein, the term "nucleic acid" includes a plurality of nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base, either a substituted pyrimidine (e.g., cytosine (C), thymine (T), or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). The term nucleic acid includes polyribonucleotides as well as polydeoxyribonucleotides. The term nucleic acid also includes polynucleosides (i.e., polynucleotides minus phosphate) and any other organic base containing polymers. Non-limiting examples of nucleic acids include chromosomes, genomic loci, genes or gene segments encoding polynucleotides or polypeptides, coding sequences, non-coding sequences of genes (e.g., introns, 5'-UTR, or 3'-UTR), pri-mRNA, pre-mRNA, cDNA, mRNA, etc. Nucleic acids (e.g., mRNA) may include substitutions and / or modifications. In some embodiments, the substitution and / or modification is one or more of the bases and / or sugars. For example, in some embodiments, the nucleic acid (e.g., mRNA) comprises a nucleotide with an organic group, such as a methyl group, attached to the nucleobase at the N6 position. Thus, in some embodiments, the mRNA comprises one or more N6-methyladenosine nucleotides. The phosphate, sugar, or nucleobase of the nucleotide can also be replaced with another phosphate, sugar, or nucleobase. For example, the uridine base can be replaced with a pseudouridine base, and the uracil base is attached to the sugar by a carbon-carbon bond rather than a nitrogen-carbon bond. Thus, in some embodiments, the nucleic acid (e.g., mRNA) is heterogeneous in backbone composition, thereby containing any possible combination of polymer units linked together, such as peptide-nucleic acids (having an amino acid backbone with nucleobases).

[0059] The nucleic acid sequences provided herein include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or clonal DNA isolates, and chemically synthesized analogues or biologically synthesized analogues produced by heterologous systems.

[0060] An "engineered nucleic acid" is a nucleic acid that does not occur in nature. However, it should be understood that an engineered nucleic acid may not occur in nature as a whole, but may contain naturally occurring nucleotide sequences. In some embodiments, an engineered nucleic acid contains nucleotide sequences from different organisms (e.g., different species). For example, in some embodiments, an engineered nucleic acid contains bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences.

[0061] Engineered nucleic acids include recombinant and synthetic nucleic acids. A "recombinant nucleic acid" is a molecule constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or a combination thereof) and capable of replicating in a living cell in some embodiments. A "synthetic nucleic acid" is a molecule that has been amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically modified or otherwise modified, but are capable of base pairing with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules that result from replicating any of the foregoing. Nucleic acids can include naturally occurring and / or non-naturally occurring nucleotides, such as modified nucleotides.

[0062] In some embodiments, the nucleic acid is present in (or on) a vector. Examples of vectors include, but are not limited to, bacterial plasmids, phages, cosmids, phesmids, fosmids, bacterial artificial chromosomes, yeast artificial chromosomes, viruses, and retroviruses (e.g., vaccinia, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, Epstein-Barr virus, fowlpox virus, pseudorabies, baculovirus), and vectors derived therefrom. In some embodiments, the nucleic acid (e.g., DNA) used as input molecule for in vitro transcription (IVT) is present in a plasmid vector.

[0063] The term "isolated" when applied to a nucleic acid sequence indicates that the polynucleotide sequence has been removed from its natural genetic environment and is therefore free of other foreign or undesired coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators) and is in a form suitable for use in engineered protein production systems. Such isolated molecules are ones that are separated from their natural environment.

[0064] The terms 5' and 3' are used herein to describe properties of nucleic acid sequences related to either the location of genetic elements and / or the direction of events (5' to 3'), such as, for example, transcription by RNA polymerase or translation by ribosomes proceeding in a 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Conventionally, DNA sequences, gene maps, vector cards, and RNA sequences are drawn 5' to 3' from left to right, or the 5' to 3' direction is indicated by an arrow, the arrow head pointing in the 3' direction. Thus, 5' (upstream), if this convention is followed, indicates a genetic element located towards the left side, and 3' (downstream) indicates a genetic element located towards the right side.

[0065] Aspects of the present disclosure relate to populations of molecules. As used herein, a "population" of molecules (e.g., DNA molecules) generally refers to a preparation (e.g., a plasmid preparation) that contains multiple copies of a molecule of interest (e.g., DNA), such as a cell extract preparation that contains multiple expression vectors that code for a molecule of interest (e.g., DNA that codes for an RNA of interest). In some embodiments, the population is a homogenous population that contains a single RNA species. As used herein, an RNA species refers to RNA molecules that have a given nucleotide sequence. Two or more RNA molecules that have the same nucleotide sequence and backbone composition belong to the same RNA species, while two RNA molecules that have different nucleotide sequences and / or different backbone compositions belong to different RNA species. In some embodiments, the population is a heterogeneous population that contains two or more RNA species. In some embodiments, the heterogeneous population comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more RNA species.

[0066] Nucleic acids (e.g., mRNA) typically contain multiple nucleotides. A nucleotide contains a nitrogenous base, a pentose sugar (ribose or deoxyribose), and at least one phosphate group. Nucleotides include nucleoside monophosphates, nucleoside diphosphates, and nucleoside triphosphates. A nucleoside monophosphate (NMP) contains a nucleobase linked to a ribose and a single phosphate, a nucleoside diphosphate (NDP) contains a nucleobase linked to a ribose and two phosphates, and a nucleoside triphosphate (NTP) contains a nucleobase linked to a ribose and three phosphates. A nucleotide analog is a compound that has the general structure of a nucleotide or is structurally similar to a nucleotide. Nucleotide analogs include, for example, analogs of the nucleobase, sugar analogs, and / or phosphate group(s) of a nucleotide.

[0067] A nucleoside contains a nitrogenous base and a pentose sugar. Thus, the addition of a phosphate group to a nucleoside results in a nucleotide. A nucleoside analog is a compound that has the general structure of a nucleoside or is structurally similar to a nucleoside. Nucleoside analogs include, for example, nucleoside nucleobase analogs and / or sugar analogs.

[0068] The term "nucleotide" is understood to include naturally occurring, synthetic, and modified nucleotides, unless otherwise specified. Examples of naturally occurring nucleotides provided herein for use in, for example, the production of RNA in an IVT reaction include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and 5-methyluridine triphosphate (MTP). 5 In some embodiments, adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and / or uridine diphosphate (UDP) are used.

[0069] Examples of nucleotide analogs include antiviral nucleotide analogs, phosphate analogs (soluble or immobilized, hydrolyzable or non-hydrolyzable), dinucleotides, trinucleotides, tetranucleotides, such as cap analogs, or precursors / substrates for enzymatic capping (vaccinia or ligase), nucleotides labeled with functional groups to facilitate ligation / conjugation of the cap or 5' portion (IRES), 5'PO to facilitate ligation of the cap or 5' portion, 4 Examples of antiviral nucleotide / nucleoside analogs include, but are not limited to, nucleotides labeled with , or nucleotides labeled with functional groups / protecting groups that can be chemically or enzymatically cleaved. Examples of antiviral nucleotide / nucleoside analogs include, but are not limited to, ganciclovir, entecavir, telbivudine, vidarabine, and cidofovir.

[0070] Modified nucleotides may include modified nucleobases. For example, the RNA transcripts (e.g., mRNA transcripts) provided herein may include modified nucleobases selected from pseudouridine (ψ), 1-methylpseudouridine (m1ψ), 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 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-methyluridine, 5-methoxyuridine (mo5U), and 2'-O-methyluridine. In some embodiments, an RNA transcript (eg, an mRNA transcript) includes a combination of at least two (eg, 2, 3, 4 or more) of the foregoing modified nucleobases.

[0071] Some embodiments relate to mRNA produced by "in vitro transcription" or IVT. The IVT method provides a method for producing (synthesizing) an RNA transcript (e.g., an mRNA transcript) by contacting a DNA template (e.g., a first input DNA and a second input DNA) with an RNA polymerase (e.g., T7 RNA polymerase, a T7 RNA polymerase variant, etc.) under conditions that result in the production of an RNA transcript. IVT conditions typically require a purified DNA template containing a promoter, nucleoside triphosphates, a buffer system containing dithiothreitol (DTT) and magnesium ions, and an RNA polymerase. The exact conditions used for the transcription reaction depend on the amount of RNA required for a particular application. A typical IVT reaction is performed by incubating a DNA template with an RNA polymerase and nucleoside triphosphates containing GTP, ATP, CTP, UTP (or nucleotide analogs) in a transcription buffer. An RNA transcript with a 5'-terminal guanosine triphosphate is produced from this reaction.

[0072] In some embodiments, the methods described herein further comprise a step of separating (e.g., purifying) the in vitro transcript (e.g., mRNA) from other reaction components. In some embodiments, the separating comprises subjecting the mRNA composition to chromatography. In some embodiments, the method comprises reverse phase chromatography. In some embodiments, the method comprises reverse phase column chromatography. In some embodiments, the chromatography comprises size-based (e.g., length-based) chromatography. In some embodiments, the method comprises size exclusion chromatography. In some embodiments, the chromatography comprises oligo-dT chromatography.

[0073] The mRNA purified by the methods described herein and / or present in the mRNA compositions described herein may, in some embodiments, code for a vaccine antigen or a therapeutic protein. As used herein, a "vaccine antigen" is a biological preparation that improves immunity against a particular disease or infectious agent. Vaccine antigens encoded by the mRNA described herein 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.

[0074] The mRNAs described herein can be designed to encode one or more antimicrobial peptides (AMPs) or antiviral peptides (AVPs). AMPs and AVPs have been isolated and characterized from a wide range of animals, including, but not limited to, microorganisms, 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., HIV-1 gp120 or gp41 transmembrane subunit. 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.

[0075] 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.

[0076] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of a region, e.g., binding domain, of a 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.

[0077] The antimicrobial polypeptides described herein can block protease dimerization and inhibit cleavage of viral proproteins into functional proteins (e.g., HIV Gag-pol processing), 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.

[0078] In other embodiments, the antimicrobial polypeptide can comprise or consist of a synthetic peptide corresponding to a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of a region, e.g., binding domain, of a capsid binding protein. In some embodiments, the antimicrobial polypeptide can have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of a protease binding protein.

[0079] A non-limiting list of infectious diseases that may be treated with RNA vaccine antigens or antimicrobial peptides is given below: Human Immunodeficiency Virus (HIV), HIV causing mycobacterial infections, AIDS-associated Cacheixa, AIDS-associated cytomegalovirus infection, HIV-associated nephropathy, lipodystrophy, AIDS-associated cryptococcal meningitis, AIDS-associated neutropenia, Pneumocystis. carinii infection, AIDS-related toxoplasmosis, Hepatitis A, B, C, D, or E, Herpes, Shingles (Chickenpox), German measles (Rubella virus), Yellow fever, Dengue fever, etc. (Flaviviruses), Influenza (Influenza virus), Hemorrhagic infections (Marburg or Ebola viruses), Bacterial infectious diseases such as Legionnaires' disease (Legionella spp.), Gastric ulcers (Helicobacter spp.), Cholera (Vibrio spp.), E. coli infection, Staphylococcus aureus infection, Salmonella infection, or Streptococcus aureus infection, Tetanus (Clostridium tetani), protozoal infectious diseases (malaria, sleeping sickness, leishmaniasis, toxoplasmosis, i.e., infectious diseases caused by protozoa, trypanosomiasis, leishmania, and toxoplasmosis), diarrheal infections such as diphtheria, leprosy, measles, whooping cough, rabies, tetanus, tuberculosis, typhoid, chickenpox, and amebiasis, Clostridium difficile-associated diarrhea (CDAD), cryptosporidiosis, giardiasis, cyclosporosis, and rotavirus gastroenteritis, encephalitis such as Japanese encephalitis, Western equine encephalitis, and tick-borne encephalitis (TBE), fungal skin diseases such as candidiasis, onychomycosis, tinea capitis / scalp tinea, tinea corporis / body tinea, tinea cruris / vulva, sporotrichosis, and tinea pedis / athlete's foot, and meningitis such as Haemophilus type b. Neglected tropical diseases such as Argentine hemorrhagic fever, leishmaniasis, nematode / roundworm infections, Ross River virus infections, and West Nile virus (WNV) disease.STDs include trichomoniasis, human papillomavirus (HPV) infection, sexually transmitted chlamydia, chancroid, and syphilis; non-septic bacterial infections include cellulitis, Lyme disease, MRSA infection, pseudomonas infection, staphylococcal infection, boutonne fever, leptospirosis, rheumatic fever, botulism, rickettsiosis, and mastoiditis; parasitic infections include cysticercosis, echinococcosis, fluke / fasciola infection, trichinosis, babesiosis, hypodermatosis, diphyllobothriasis, and trypanosomiasis; and respiratory infections include adenovirus infection, aspergillosis, and avian (H5N1) influenza. swine flu, influenza, RSV infection, severe acute respiratory syndrome (SARS), sinusitis, legionnaires' disease, coccidioidomycosis, and swine (H1N1) influenza; sepsis such as bacteremia, sepsis / septic shock, and sepsis in premature infants; urinary tract infections such as vaginal infections (bacterial), vaginal infections (fungal), and glomerular infections; viral skin infections such as B19 parvovirus infection, warts, genital herpes, oral herpes, shingles, inner ear infections, and fetal cytomegalovirus syndrome; food-borne diseases such as brucellosis (Brucella species), Clostridium perfringens (epsilon toxin), E. Coli O157:H7 (Escherichia coli), Salmonellosis (Salmonellosis species), Shingelosis, Vibriosis, and Listeriosis; bioterrorism and potential epidemic diseases such as Ebola hemorrhagic fever, Lassa fever, Marburg hemorrhagic fever, plague, anthrax, Nipah virus disease, Hantavirus, smallpox, Burkholderia mallei, Burkholderia pseudomallei, Chlamydia psittaci, Q fever (Coxiella burnetii), tularemia (Fancisella tularensis), rubella, mumps, and polio.

[0080] The RNA disclosed herein may encode one or more validated or "testing" therapeutic proteins or peptides. In some embodiments, one or more therapeutic proteins or peptides currently on the market or in development may be encoded by the mRNA of the compositions and methods described herein. The therapeutic proteins or peptides encoded by the mRNA may be utilized to treat conditions or diseases in many therapeutic areas, such as, but not limited to, hematology, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, genetics, genitourinary, gastrointestinal, musculoskeletal, oncology, as well as immunology, respiratory, sensory, and anti-infective applications.

[0081] The mRNA of the compositions and methods described herein may encode one or more cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide" or CPP refers to a polypeptide that can facilitate cellular uptake of a molecule. A cell-penetrating polypeptide may contain one or more detectable labels. A polypeptide may be partially labeled or completely labeled throughout. An RNA may completely, partially, or not encode a detectable label at all. A cell-penetrating peptide may also include a signal sequence. As used herein, "signal sequence" refers to a sequence of amino acid residues that are attached at the amino terminus of a nascent protein during protein translation. A signal sequence may be used to signal the secretion of a cell-penetrating polypeptide.

[0082] In one embodiment, the RNA may also code for a fusion protein. A fusion protein may be created by operably linking a charged protein to a therapeutic protein. As used herein, "operably linked" refers to a therapeutic protein and a charged protein being connected in a manner that allows for expression of the complex when introduced into a cell. As used herein, "charged protein" refers to a protein that has 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 total surface charge or ratio to surface amino acids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0083] The cell-penetrating polypeptide encoded by the RNA may form a complex after translation, which may include a charged protein bound, e.g., covalently bound, to the cell-penetrating polypeptide.

[0084] In one embodiment, the cell-penetrating polypeptide may include a first domain and a second domain. The first domain may include a supercharged polypeptide. The second domain may include a protein binding partner. As used herein, "protein binding partner" includes, but is not limited to, an antibody and its functional fragments, a scaffold protein, or a peptide. The cell-penetrating polypeptide may further include an intracellular binding partner for the protein binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell into which the RNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating a first cell.

[0085] 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 that has the cell-permeable polypeptide that includes the protein-binding partner introduced therein.

[0086] Human and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane-bound compartment, or organelle, contains different proteins that are essential for the function of the organelle. Cells use "sorting signals," amino acid motifs located within proteins, to target 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).

[0087] Proteins that are targeted to the ER by a signal sequence can be released into the extracellular space as secretory 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, molecules can be used to take advantage of the above cellular transport. Thus, in some embodiments, mRNA expresses secretory proteins. In one embodiment, they can be used to manufacture large quantities of valuable human gene products.

[0088] In some embodiments, the mRNA can express a plasma membrane protein. In some embodiments, the mRNA can express a cytoplasmic protein or a cytoskeletal protein. In some embodiments, the mRNA can express an intracellular membrane-associated protein. In some embodiments, the mRNA can express a nuclear protein. In some embodiments, the mRNA can express a protein associated with a human disease.

[0089] Untranslated Regions An untranslated region (UTR) is a section of nucleic acid that is not translated, preceding the start codon (5'UTR) and following the stop codon (3'UTR). In some embodiments, a nucleic acid of the disclosure (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) that includes an open reading frame (ORF) encoding one or more proteins or peptides further includes one or more UTRs (e.g., a 5'UTR or a functional fragment thereof, a 3'UTR or a functional fragment thereof, or a combination thereof).

[0090] The UTR can be homologous or heterologous to the coding region in the nucleic acid. In some embodiments, the UTR is homologous to the ORF encoding one or more peptide epitopes. In some embodiments, the UTR is heterologous to the ORF encoding one or more peptide epitopes. In some embodiments, the nucleic acid comprises two or more 5'UTRs or functional fragments thereof, each of which has the same or different nucleotide sequence. In some embodiments, the nucleic acid comprises two or more 3'UTRs or functional fragments thereof, each of which has the same or different nucleotide sequence.

[0091] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof, is sequence optimized.

[0092] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, for example, 5-methoxyuracil.

[0093] UTRs can have features that provide a regulatory role, such as stability, localization, and / or increased or decreased translation efficiency. Nucleic acids containing UTRs can be administered to cells, tissues, or organisms, and one or more regulatory functions can be measured using conventional methods. In some embodiments, a functional fragment of a 5'UTR or 3'UTR comprises one or more regulatory functions of a full-length 5' or 3'UTR, respectively.

[0094] Naturally occurring 5'UTRs retain features that play a role in translation initiation. They contain signatures such as the Kozak sequence, which are commonly known to be involved in the process by which the ribosome initiates the translation of many genes. 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0095] Manipulation of features normally found in abundantly expressed genes of a particular target organ can enhance nucleic acid stability and protein production. For example, introduction of the 5'UTR of liver-expressed mRNAs such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII can enhance expression of the nucleic acid in hepatic cell lines or liver. Similarly, use of 5'UTRs from other tissue-specific mRNAs to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), endothelial cells (e.g., Tie-1, CD36), bone marrow cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (e.g., CD45, CD18), adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (e.g., SP-A / B / C / D).

[0096] In some embodiments, UTR is selected from a family of transcripts whose proteins share common functions, structures, characteristics, or properties.For example, the encoded polypeptide may belong to a family of proteins (i.e., share at least one function, structure, characteristic, localization, origin, or expression pattern) that are expressed in a particular cell, tissue, or at a certain time during development.The UTR from either gene or mRNA can be exchanged with other UTRs from the same or different protein families to create new nucleic acids.

[0097] In some embodiments, the 5'UTR and the 3'UTR may be heterologous. In some embodiments, the 5'UTR may be from a different species than the 3'UTR. In some embodiments, the 3'UTR may be from a different species than the 5'UTR.

[0098] International Patent Application No. PCT / US2014 / 021522 (Publication No. WO / 2014 / 164253) provides a list of exemplary UTRs that can be utilized as ORF flanking regions in the nucleic acids provided herein, and this publication is incorporated herein by reference for this purpose.

[0099] Additional exemplary UTRs that can be utilized in the nucleic acids provided herein include those from globins, such as α- or β-globin (e.g., Xenopus, mouse, rabbit, or human globin); strong Kozak translation initiation signals; CYBA (e.g., human cytochrome b-245α polypeptide); albumins (e.g., human albumin 7); HSD17B4 (hydroxysteroid (17-β) dehydrogenase); viruses (e.g., tobacco etch virus (TEV), Venezuelan equine encephalitis virus (VEEV), dengue virus, cytomegalovirus (CMV, e.g., CMV immediate early 1 (IE1)), hepatitis viruses (e.g., hepatitis B virus), sind bisvirus, or PAV barley yellow dwarf virus); heat shock proteins (e.g., hsp70); translation initiation factors (e.g., elF4G); glucose transporters (e.g., hGLUT1 (human glucose transporter 1)); actin (e.g., human α or β actin); GAPDH; tubulin; histones; citric acid cycle enzymes; topoisomerases (e.g., the 5´UTR of the TOP gene lacking a 5´TOP motif (oligopyrimidine tract)); ribosomal protein large 32 (L32); ribosomal proteins (e.g., human or mouse ribosomal proteins, such as rps9); ATP synthases (e.g., ATP5A1 or mitochondrial H +-β subunit of ATP synthase; growth hormone (e.g., bovine (bGH) or human (hGH)); elongation factors (e.g., elongation factor 1 alpha 1 (EEF1A1)); manganese superoxide dismutase (MnSOD); myocyte enhancer factor 2A (MEF2A); β-F1-ATPase, creatine kinase, myoglobin, granulocyte colony-stimulating factor (G-CSF); collagen (e.g., collagen type I alpha 2 (Col1A2), collagen type I alpha 1 (Col1A1), collagen type VI alpha 2 (Col6A 2), collagen type VI alpha 1 (Col6A1); ribophorin (e.g., ribophorin I (RPNI)); low density lipoprotein receptor-related protein (e.g., LRP1); cardiotrophin-like cytokine factor (e.g., Nnt1); calreticulin (Calr); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); and nucleobindin (e.g., Nucb1).

[0100] In some embodiments, the 5'UTR is selected from the group consisting of β-globin 5'UTR; a 5'UTR containing a strong Kozak translation initiation signal; cytochrome b-245 alpha polypeptide (CYBA) 5'UTR; hydroxysteroid (17-beta) dehydrogenase (HSD17B4) 5'UTR; tobacco etch virus (TEV) 5'UTR; Venezuelan equine encephalitis virus (TEEV) 5'UTR; the 5' proximal open reading frame of rubella virus (RV) RNA encoding a nonstructural protein; dengue virus (DEN) 5'UTR; heat shock protein 70 (Hsp70) 5'UTR; eIF4G 5'UTR; GLUT1 5'UTR; functional fragments thereof, and any combination thereof.

[0101] In some embodiments, the 3'UTR is selected from the group consisting of β-globin 3'UTR; CYBA 3'UTR; albumin 3'UTR; growth hormone (GH) 3'UTR; VEEV 3'UTR; Hepatitis B virus (HBV) 3'UTR; α-globin 3'UTR; DEN 3'UTR; PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; elongation factor 1 alpha 1 (EEF1A1) 3'UTR; manganese superoxide dismutase (MnSOD) 3'UTR; β subunit of mitochondrial H(+)-ATP synthase (β-mRNA) 3'UTR; GLUT1 3'UTR; MEF2A 3'UTR; β-F1-ATPase 3'UTR; functional fragments thereof, and combinations thereof.

[0102] Wild-type UTR from any gene or mRNA can be incorporated into the nucleic acid of the present disclosure.In some embodiments, UTR can be modified relative to wild-type or natural UTR, for example, by changing the orientation or position of UTR relative to ORF, or by including additional nucleotides, deleting nucleotides, exchanging or rearranging nucleotides, to produce variant UTR.In some embodiments, variants of 5' or 3' UTR can be utilized, for example, mutants of wild-type UTR, or variants in which one or more nucleotides are added or removed from the end of UTR.

[0103] In addition, one or more synthetic UTRs may be used in combination with one or more non-synthetic UTRs. See, for example, Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, and sequences available at www.addgene.org, the contents of each of which are incorporated herein by reference in their entirety. UTRs or portions thereof may be positioned in the same orientation as the transcript in which they are selected, or the orientation or position may be changed. Thus, 5' and / or 3' UTRs may be inverted, shortened, extended, or combined with one or more other 5' UTRs or 3' UTRs.

[0104] In some embodiments, the nucleic acid may comprise multiple UTRs, such as duplicated, triplet, or quadruple 5'UTRs or 3'UTRs. For example, a duplicated UTR comprises two copies of the same UTR in tandem or substantially in tandem. For example, a duplicated beta-globin 3'UTR can be used (e.g., US2010 / 0129877, the contents of which are incorporated herein by reference for this purpose).

[0105] The nucleic acids of the present disclosure can include a combination of features. For example, an ORF can be flanked by a 5'UTR that includes a strong Kozak translation initiation signal and / or a 3'UTR that includes an oligo(dT) sequence for templated addition of a polyA tail. The 5'UTR can include a first nucleic acid fragment and a second nucleic acid fragment from the same and / or a different UTR (see, e.g., US2010 / 0293625, which is incorporated herein by reference in its entirety for this purpose).

[0106] Other non-UTR sequences can be used as regions or subregions within the nucleic acids of the present disclosure. For example, introns or portions of intron sequences can be incorporated into the nucleic acids of the present disclosure. Incorporation of intron sequences can increase protein production and expression levels of the nucleic acids. In some embodiments, the nucleic acids of the present disclosure include an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the nucleic acid includes an IRES instead of a 5'UTR sequence. In some embodiments, the nucleic acid includes an IRES located between the 5'UTR and the open reading frame. In some embodiments, the nucleic acid includes an ORF encoding a viral capsid sequence. In some embodiments, the nucleic acid includes a synthetic 5'UTR in combination with a non-synthetic 3'UTR.

[0107] In some embodiments, the UTR may also include at least one translation enhancer nucleic acid, translation enhancer element, or translational enhancer element (collectively, "TEE", which refers to a nucleic acid sequence that increases the amount of polynucleotide or protein produced from a polynucleotide). As a non-limiting example, the TEE may include those described in US2009 / 0226470, which is incorporated by reference in its entirety for this purpose, and others known in the art. As a non-limiting example, the TEE may be located between the transcription promoter and the start codon. In some embodiments, the 5'UTR includes a TEE. In one aspect, the TEE is a conserved element within the UTR that can promote translational activity of the nucleic acid, such as, but not limited to, cap-dependent or cap-independent translation. In one non-limiting example, the TEE includes a TEE sequence in the 5' leader of a Gtx homeodomain protein. See, for example, Chappell et al., PNAS.2004.101:9590-9594, which is incorporated by reference in its entirety for this purpose.

[0108] Poly(A) Tail Some aspects relate to methods of producing RNA containing one or more poly-A tails. A "poly-A tail" is a region of an mRNA that is downstream, e.g., immediately downstream (i.e., 3'), from an open reading frame and / or a 3'UTR that contains multiple consecutive adenosine monophosphates. A poly-A tail can contain between 10 and 300 adenosine monophosphates. For example, a poly-A tail can 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, or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains between 50 and 250 adenosine monophosphates. In relevant biological environments (e.g., within a cell, in vivo, etc.), the poly(A) tail functions, for example, to protect the mRNA from enzymatic degradation in the cytoplasm and assists in transcription termination, transport of the mRNA from the nucleus, and translation.

[0109] As used herein, "polyA tail efficiency" refers to the amount (e.g., expressed as a percentage) of polyA-tailed mRNA produced by an IVT reaction using input DNA relative to the total number of mRNAs produced in the IVT reaction using input DNA. The polyA tail efficiency of an IVT reaction can vary depending on, for example, the RNA polymerase used, the amount or purity of input DNA used, etc. In some embodiments, the polyA tail efficiency of an IVT reaction is greater than 85%, 90%, 95%, or 99.9%. Methods are known to calculate polyA tail efficiency by determining the amount of polyA tail-containing mRNA relative to the total mRNA produced in an IVT reaction, for example, by column chromatography (e.g., oligo-dT chromatography).

[0110] In some embodiments, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% of the RNA in an RNA composition generated by the methods described herein comprises a poly-A tail. In some embodiments, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% of each RNA in an RNA composition generated by the methods described herein comprises a poly-A tail. The efficiency (e.g., percentage) of polyA-containing RNA in an RNA composition can be measured i) after the IVT reaction and before purification, or ii) after the RNA composition has been purified (e.g., by chromatography, such as oligo-dT chromatography).

[0111] The length of the unique polyA tail provides certain advantages to the nucleic acids provided herein. Generally, the length of the polyA tail, if present, is greater than 30 nucleotides in length. In another embodiment, the polyA tail is greater than 35 nucleotides in length (e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, or 3,000 nucleotides or more).

[0112] In some embodiments, the polyA tail is designed for the length of the entire nucleic acid or for the length of a particular region of the nucleic acid. This design can be based on the length of the coding region, the length of a particular feature or region, or the length of the final product expressed from the nucleic acid.

[0113] In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the nucleic acid or a feature thereof. The polyA tail can also be designated as a fraction of the nucleic acid to which it belongs. In this context, the polyA tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, the region of the construct, or the total length of the construct minus the polyA tail. Furthermore, conjugation of engineered binding sites and nucleic acid sites in polyA binding proteins can enhance expression.

[0114] mRNA composition Aspects of the present disclosure relate to compositions comprising mRNA purified by any of the methods described herein. In some embodiments, the mRNA compositions produced by the methods described herein are purer than mRNA compositions purified by alternative methods such as oligo-dT or high performance liquid chromatography. Whether a composition is purer than a composition produced by an alternative method can be determined by methods known in the art, including separating the purified composition into multiple equivalent samples, purifying each by a different method, and comparing the content of the resulting purified compositions. A first mRNA composition that contains a lower abundance or concentration of a given impurity than a second mRNA composition is said to be "more pure" than the second mRNA composition. If a first and a second purified mRNA composition are produced by purifying a similar sample of an initial mRNA composition by a first and a second method, and the first purified mRNA composition has a lower concentration of the impurity than the second purified mRNA composition, the first method is said to be more effective at removing the impurity than the second method.

[0115] In some embodiments, the purified mRNA composition produced by the methods described herein comprises at least 80% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 85% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 90% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 95% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 96% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 97% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 98% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises at least 99% of the mRNA that was present in the composition prior to precipitation. In some embodiments, the purified mRNA composition comprises up to 100% of the mRNA that was present in the composition prior to precipitation.

[0116] In some embodiments, the purified mRNA composition produced by the methods described herein has a lower dsRNA concentration than the dsRNA concentration of a purified mRNA composition produced by purifying a similar initial mRNA composition by chromatography. Methods for measuring the presence and / or amount of dsRNA in a composition are known in the art. Non-limiting examples of methods for measuring the dsRNA content of a sample include ELISA and immunoblotting using antibodies specific for dsRNA. In addition, the total mass of RNA in a sample can be measured using techniques such as spectroscopy (NanoDrop), qRT-PCR, and / or ddPCR, and the mass of dsRNA can be measured using an intercalating agent that fluoresces when bound to dsRNA, such as acridine orange, and the dsRNA concentration is calculated by division. In some embodiments, the concentration of double-stranded RNA in a composition comprising RNA is 5% (% w / w) or less, 4% or less, 3% or less, 2.5% or less, 2% or less, 1.75% or less, 1.5% or less, 1.25% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.175% or less, 0.15% or less, 0.125% or less, or 0.1% or less. In some embodiments, the concentration of double-stranded RNA in a composition comprising RNA is 1% (% w / w) or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less. In some embodiments, the concentration of double-stranded RNA in a composition comprising RNA is 0.05% (% w / w) or less, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less.

[0117] In some embodiments, the percentage of RNA that is dsRNA in a purified composition produced by the methods described herein is lower than the percentage of dsRNA in a purified mRNA composition produced by purifying a similar initial mRNA composition by chromatography. In some embodiments, 1% or less of the RNA in the composition is dsRNA. In some embodiments, 0.9% or less of the RNA in the composition is dsRNA. In some embodiments, 0.8% or less of the RNA in the composition is dsRNA. In some embodiments, 0.7% or less of the RNA in the composition is dsRNA. In some embodiments, 0.6% or less of the RNA in the composition is dsRNA. In some embodiments, 0.5% or less of the RNA in the composition is dsRNA. In some embodiments, 0.4% or less of the RNA in the composition is dsRNA. In some embodiments, 0.3% or less of the RNA in the composition is dsRNA. In some embodiments, 0.2% or less of the RNA in the composition is dsRNA. In some embodiments, 0.1% or less of the RNA in the composition is dsRNA. In some embodiments, 0.05% or less of the RNA in the composition is dsRNA. In some embodiments, 0.04% or less of the RNA in the composition is dsRNA. In some embodiments, 0.03% or less of the RNA in the composition is dsRNA. In some embodiments, 0.02% or less of the RNA in the composition is dsRNA. In some embodiments, 0.0% or less of the RNA in the composition is dsRNA.

[0118] In some embodiments, the purified mRNA composition produced by the methods described herein has a protein concentration that is lower than the protein concentration of a purified mRNA composition produced by purifying a similar initial mRNA composition by chromatography. In some embodiments, the concentration of protein in the RNA composition produced by the methods is 1.0% (% w / w) or less, 0.8% or less, 0.6% or less, 0.4% or less, or 0.2% or less. Methods for measuring the amount of protein in a sample include colorimetric assays (e.g., Bradford assay), spectroscopy, ELISA, polyacrylamide gel electrophoretic electropherogram analysis, and chromatographic analysis. See, e.g., Sapan et al., Biotechnol Appl Biochem. 1999.29(2):99-108). In some embodiments, the concentration of protein in the RNA composition is 1.0% or less. In some embodiments, the concentration of protein in the RNA composition is 0.9% or less. In some embodiments, the concentration of protein in the RNA composition is 0.8% or less. In some embodiments, the concentration of protein in the RNA composition is 0.6% or less. In some embodiments, the concentration of protein in the RNA composition is 0.5% or less. In some embodiments, the concentration of protein in the RNA composition is 0.4% or less. In some embodiments, the concentration of protein in the RNA composition is 0.3% or less. In some embodiments, the concentration of protein in the RNA composition is 0.2% or less.

[0119] In some embodiments, the purified mRNA composition produced by the methods described herein has a lower DNA concentration than the DNA concentration of a purified mRNA composition produced by purifying a similar initial mRNA composition by chromatography. Methods for measuring the presence and / or amount of DNA in a composition are known in the art. Non-limiting examples of methods for measuring the dsRNA content of a sample include gel electrophoresis using an intercalating agent (e.g., ethidium bromide) that fluoresces when bound to DNA, spectroscopy (NanoDrop), qPCR, and / or ddPCR. In some embodiments, the concentration of DNA in the RNA composition is 1.0% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.9% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.8% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.6% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.5% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.4% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.3% or less. In some embodiments, the concentration of DNA in the RNA composition is 0.2% or less. EXAMPLES

[0120] Example 1: Impurity removal by precipitation and washing for mRNA capture. The mRNA composition containing impurities is purified by (i) precipitating the mRNA, (ii) removing the aqueous phase, or a portion thereof, containing soluble impurities, (iii) washing the precipitated mRNA by adding (a) a wash solution, (iii) (b) removing the wash solution, or a portion thereof, and (iv) resuspending the precipitated mRNA. The method described in this example can be used to purify mRNA produced by cell-free processes such as in vitro transcription, mRNA isolated from cells, or mRNA obtained from commercial suppliers.

[0121] A solution containing an appropriate salt at a concentration sufficient to bring the composition to the desired concentration of salt is mixed with the mRNA composition to promote nucleation and aggregation and precipitation of the mRNA. After salt addition, a volume-excluding agent such as alcohol (e.g., ethanol or isopropanol) is added to promote further aggregation of the mRNA and increase the rate of precipitation. Nucleic acids are less soluble in alcohols such as isopropanol, so the addition of alcohol promotes interactions between the mRNA in the aqueous portion of the composition. Precipitation is performed at room temperature to reduce the amount of solutes (e.g., sucrose or sodium chloride) that co-precipitate with the mRNA. During precipitation, the turbidity, osmolality, and particle size distribution of the mRNA composition are monitored in-line using dynamic light scattering and microflow imaging to assess the extent of precipitation over time.

[0122] After precipitation, the aqueous phase containing dissolved impurities is separated by filtration using a hollow fiber membrane with a pore size of 0.2 μm. This step removes many soluble impurities, and the remaining impurities are then removed by washing the precipitate with an alcohol solution (e.g., 70% ethanol or isopropanol) and passing the wash solution:precipitation mixture across a tangential flow filtration (TFF) membrane with a molecular weight cutoff of 30 kDa to remove the wash solution containing the dissolved impurities. This wash step is then repeated, which serves to further dilute and remove the remaining impurities before the precipitated mRNA is redissolved. Compared to using an equivalent total volume of wash solution in a single wash step, successively diluting and removing impurities in this manner increases the efficiency of the purification.

[0123] After impurities are removed by washing, a resuspension solution of 10 mM Tris-HCl, pH 5.5, is added to the precipitate to dissolve the mRNA. The resulting mRNA composition is then analyzed to determine the presence of protein, DNA, and dsRNA impurities. The amount of mRNA in the resuspended mRNA composition is quantified by HPLC or spectroscopy to determine the mRNA concentration for use in downstream applications. This amount of mRNA is then compared to the amount of mRNA that was present in the mRNA composition before precipitation to determine the percent yield of the purification process. The mRNA purity (e.g., the percentage of mRNA with the expected size and / or containing a poly(A) tail) is also measured, such as by HPLC, to confirm the suitability of the mRNA for downstream applications.

[0124] Equivalents and Scope While several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or obtaining one or more advantages described herein, and each such variation and / or improvement is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and equivalents thereto, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more of such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0125] All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0126] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0127] As used herein, the indefinite articles "a" and "an" in the specification and claims should be understood to mean "at least one," unless clearly indicated to the contrary.

[0128] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and conjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted similarly, i.e., "one or more" of the elements. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether or not associated with the specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" may refer in some embodiments to only A (optionally including elements other than B), in other embodiments to only B (optionally including elements other than A), in yet other embodiments to both A and B (optionally including other elements), and so forth. As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., including at least one, but also including more than one, and optionally including items not in the list. Only terms clearly indicating the contrary, such as "only one" or "exactly one," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a plurality or list of elements. In general, as used herein, the term "or" shall be construed to indicate exclusive alternatives (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either," "one," "only one," or "exactly one." "Consisting essentially," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0129] As used herein in the specification and claims, the phrase "at least one" should be understood in reference to a list of one or more elements to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that there may optionally be elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified element. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in some embodiments to at least one, optionally including multiple, A and no B (and optionally including elements other than B), in other embodiments to at least one, optionally including multiple, B and no A (and optionally including elements other than A), in yet other embodiments to at least one, optionally including multiple, A, and at least one, optionally including multiple, B (and optionally including other elements), etc. Each possibility represents a separate embodiment of the present invention.

[0130] Unless expressly indicated to the contrary, disclosure of numerical values ​​and numerical ranges herein should be understood to include both i) the exact value(s) or range specified, and ii) values ​​"about" the specified value(s) or range, as would be understood by one of ordinary skill in the art (e.g., values ​​or ranges that fall within a reasonable range, e.g., about 10% similar).

[0131] It is also to be understood that, unless expressly indicated to the contrary, in any method that includes two or more steps or acts disclosed herein, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are disclosed.

[0132] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to mean open-ended, i.e., including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the U.S. Patent and Trademark Office Guidelines, Section 2111.03.

Claims

1. 1. A continuous method for removing one or more impurities from a composition comprising messenger ribonucleic acid (mRNA), the continuous method comprising: (a) precipitating mRNA from the composition to produce a precipitated mRNA composition; (b) removing one or more impurities from the precipitated mRNA composition; and then (c) resuspending the precipitated mRNA.

2. 2. The continuous method of claim 1, wherein said precipitating comprises adding a high salt buffer and / or alcohol to said composition comprising said mRNA to produce said precipitated mRNA composition.

3. 3. The continuous method of claim 1 or 2, wherein removing the one or more impurities comprises contacting the precipitated mRNA composition with a filter membrane.

4. 4. The continuous method of any one of claims 1 to 3, wherein removing the one or more impurities comprises contacting the precipitated mRNA composition with a wash solution.

5. 5. The continuous method of any one of claims 1 to 4, wherein removing the one or more impurities comprises contacting the precipitated mRNA composition with a second filter membrane.

6. 6. The continuous method of any one of claims 1 to 5, wherein removing the one or more impurities comprises contacting the precipitated mRNA composition with a second wash solution.

7. The method further comprising: (i) adding a high salt buffer to the mRNA composition to produce a precipitated mRNA composition; (ii) contacting the precipitated mRNA composition with a filter membrane to remove one or more impurities from the precipitated mRNA composition; (iii)(a) contacting the precipitated mRNA composition with a wash solution; (iii)(b) contacting the precipitated mRNA composition with a second filter membrane to remove one or more impurities from the precipitated mRNA composition; (iv) contacting the precipitated mRNA composition with a resuspension solution to resuspend the mRNA.

8. 8. The method of claim 2 or 7, wherein the high salt buffer comprises a salt selected from the group consisting of lithium chloride, lithium acetate, lithium sulfate, sodium chloride, sodium acetate, sodium sulfate, ammonium chloride, ammonium acetate, and ammonium sulfate.

9. 9. The method of any one of claims 2 or 7-8, wherein the high salt buffer comprises ammonium sulfate.

10. 10. The method of any one of claims 2 or 7-9, wherein the high salt buffer has a salt concentration of about 0.1 M to about 5.0 M.

11. 11. The method of any one of claims 2 or 7-10, wherein the high salt buffer has a conductivity of 5 mS / cm or greater, optionally the high salt buffer has a conductivity of about 5 mS / cm to about 300 mS / cm.

12. The method of any one of claims 1 to 11, wherein the precipitation step further comprises adding an alcohol to the mRNA composition.

13. 13. The method of claim 12, wherein the alcohol is ethanol or isopropanol.

14. 14. The method of any one of claims 3 or 7 to 13, wherein the filter membrane is a tangential flow filtration (TFF) membrane.

15. The method of any one of claims 3 or 7 to 14, wherein the filter membrane is a hollow fiber membrane.

16. 16. The method of any one of claims 3 or 7-15, wherein the filter membrane has a molecular weight cutoff of about 20 kDa to about 150 kDa.

17. The method of any one of claims 4 or 7 to 16, wherein the wash solution comprises a detergent, a salt, DNase, and / or RNase III.

18. The method of any one of claims 4 or 7 to 17, wherein the cleaning solution comprises a surfactant.

19. The method of claim 18 , wherein the cleaning solution comprises a detergent.

20. 20. The method of claim 19, wherein the detergent is Triton X-100 detergent.

21. The method of any one of claims 4 or 7 to 20, wherein the washing solution comprises a salt.

22. 22. The method of claim 21 , wherein the salt comprises a monovalent or divalent cation.

23. 22. The method of claim 21 , wherein the monovalent or divalent cation is selected from the group consisting of lithium, sodium, ammonium, calcium, and magnesium.

24. 24. The method of any one of claims 21 to 23, comprising an anion selected from the group consisting of chloride, sulfate, and acetate.

25. The method of any one of claims 4 or 7 to 24, wherein the cleaning solution comprises sodium chloride, calcium chloride, and / or ammonium sulfate.

26. The method of any one of claims 21 to 25, wherein the salt concentration in the wash solution is from about 50 mM to about 800 mM.

27. The method of any one of claims 4 or 7 to 26, wherein the wash solution comprises a Tris buffer.

28. The method of any one of claims 4 or 7-27, wherein the wash solution comprises EDTA.

29. The method of any one of claims 4 or 7-28, wherein the resuspension solution comprises an RNase inhibitor.

30. 30. The method of any one of claims 4 or 7-29, wherein the wash solution comprises DNase.

31. The method of claim 30, wherein the DNase is DNase I.

32. The method of any one of claims 4 or 7-31, wherein the wash solution comprises RNase III.

33. The method of any one of claims 4 or 7 to 32, wherein the wash solution comprises a protease.

34. 34. The method of claim 33, wherein the protease is proteinase K.

35. 35. The method of any one of claims 5 or 7-34, wherein the second filter membrane is a tangential flow filtration (TFF) membrane.

36. The method of any one of claims 5 or 7-34, wherein the second filter membrane is a hollow fiber membrane.

37. 37. The method of any one of claims 5 or 7-36, wherein the second filter membrane has a molecular weight cutoff of about 20 kDa to about 150 kDa.

38. The method of any one of claims 7 to 37, further comprising repeating steps (iii)(a) and (iii)(b).

39. 39. The method of claim 38, wherein steps (iii)(a) and (iii)(b) are each performed 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more times prior to step (iv).

40. 40. The method of claim 38 or 39, wherein one or more repetitions of step (iii)(a) use a different wash solution than the first repetition of step (iii)(a).

41. 41. The method of any one of claims 1 to 40, wherein the method comprises contacting the precipitated mRNA composition with a salt, a detergent, a protease, a DNase, and an RNase III prior to resuspending the mRNA.

42. 42. The method of any one of claims 7 to 41, wherein the resuspension solution has a salt concentration of 20 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less.

43. The method of any one of claims 7 to 42, wherein the resuspension solution comprises Tris.

44. The method of any one of claims 7 to 43, wherein the resuspension solution comprises EDTA.

45. The method of any one of claims 7 to 44, wherein the resuspension solution comprises an RNase inhibitor.

46. 46. ​​The method of any one of claims 7 to 45, wherein the resuspension solution has a pH of about 5.0 to about 7.

5.

47. 47. The method of any one of claims 7 to 46, wherein the resuspension solution has a conductivity of 10 mS / cm or less, 8 mS / cm or less, 6 mS / cm or less, 5 mS / cm or less, 4 mS / cm or less, 3 mS / cm or less, 2.5 mS / cm or less, 2.0 mS / cm or less, 1.5 mS / cm or less, 1.0 mS / cm or less, or 0.5 mS / cm or less.

48. 48. A purified mRNA composition produced by the method of any one of claims 1 to 47, comprising resuspended mRNA, said resuspended mRNA comprising an open reading frame encoding a vaccine antigen or a therapeutic protein.

49. 49. The purified mRNA composition of Claim 48, wherein the purified mRNA composition has a salt concentration of 20 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less.

50. 50. The purified mRNA composition of claim 48 or 49, wherein less than 1%, less than 0.8%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% of the mRNA molecules in the purified mRNA composition are double-stranded RNA (dsRNA) molecules.

51. 51. The purified mRNA composition of any one of claims 48-50, wherein the concentration of double-stranded RNA (dsRNA) in the isolated mRNA composition is 1% (w / w) or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, or 0.008% or less, 0.006% or less, 0.004% or less, 0.002% or less, or 0.001% or less.

52. 52. The purified mRNA composition of any one of claims 48-51, wherein the purified mRNA composition has a protein concentration of 1% (% w / w) or less, 0.8% or less, 0.6% or less, 0.4% or less, or 0.2% or less.

53. 53. The purified mRNA composition of any one of claims 48-52, wherein the purified mRNA composition has a DNA concentration of 1% (% w / w) or less, 0.8% or less, 0.6% or less, 0.4% or less, or 0.2% or less.

54. 54. The purified mRNA composition of any one of claims 48-53, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the mRNA in the purified mRNA composition comprises a poly(A) tail.

Images