Methods and compositions for messenger RNA purification

Abstract

To provide an effective, reliable and efficient method for producing high-quality RNA in a large scale for therapeutic use.SOLUTION: A method of capping and tailing a purified in vitro transcribed messenger RNA (mRNA) preparation, wherein the method comprises enzymatically capping and tailing the mRNA in a reaction-buffer comprising 1mM having a concentration of 0. 0mM to 1. MgCl2 and having a pH of 6.0 to 7.8.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 972,471, filed February 10, 2020, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Messenger RNA therapy (MRT) is a promising new approach for treating various diseases. MRT involves administering messenger RNA (mRNA) to patients in need of this therapy. The administered mRNA produces the protein or peptide encoded by the mRNA in the patient's body. mRNA is typically synthesized using an in vitro transcription system (IVT), which involves an enzymatic reaction using RNA polymerase. The IVT synthesis process usually involves reactions for adding a 5' cap (capping reaction) and a 3' polyA tail (polyadenylation).

[0003] Effective mRNA therapy requires efficient delivery of mRNA to patients and efficient production of the protein encoded by the mRNA within the patient's body. To optimize in vivo mRNA delivery and protein production, constructs typically require an appropriate cap at the 5' end, which protects the mRNA from degradation and promotes successful protein translation. The presence of a "tail" at the 3' end protects the mRNA from exonuclease degradation. New and improved methods are needed to achieve high RNA integrity while maintaining high capping and tailing efficiency at a manufacturing scale for therapeutic mRNAs. Summary of the Invention

[0004] The present invention provides an improved method for preparing in vitro transcribed (IVT) mRNA. This invention is based, in part, on the surprising discovery that capping and tailing mRNA under reaction conditions with low pH and low concentrations of magnesium chloride (MgCl2) significantly improves the RNA integrity of the mRNA product while maintaining all other important quality characteristics. Specifically, the capping and tailing reaction conditions disclosed herein successfully reduce degraded RNA species in the final mRNA product. This unique and advantageous capping and tailing reaction condition was not understood prior to the present invention and is truly unexpected, particularly because optimized cap and tail conditions can increase the RNA integrity of the mRNA product by at least approximately 25%. Based on this unexpected discovery, the inventors have successfully developed a large-scale production method for synthesizing and purifying mRNA molecules with high RNA integrity suitable for mRNA therapeutics. Thus, the present invention enables more efficient and reliable production of mRNA for therapeutic applications.

[0005] In one aspect, the invention provides a method of capping and tailing a purified in vitro transcribed messenger RNA (mRNA) preparation, the method comprising capping and tailing the mRNA in a reaction buffer that includes MgCl2 and has a pH below 8.0.

[0006] In some embodiments, the method comprises capping the mRNA in a reaction buffer comprising MgCl2 and having a pH below 8.0. In some embodiments, the method comprises capping the mRNA in a reaction buffer comprising MgCl2 and having a pH below 8.0. Typically, the steps of capping the mRNA in a reaction buffer containing MgCl2 and having a pH below 8.0 and tailing the mRNA in a reaction buffer containing MgCl2 and having a pH below 8.0 are performed separately. In some embodiments, the steps of capping the mRNA in a reaction buffer containing MgCl2 and having a pH below 8.0 and tailing the mRNA in a reaction buffer containing MgCl2 and having a pH below 8.0 are performed sequentially.

[0007] In some embodiments, the reaction buffer further comprises a salt. In some embodiments, the reaction buffer further comprises KCl. In some embodiments, the reaction buffer further comprises NaCl. In some embodiments, the reaction buffer further comprises CaCl2. In some embodiments, the reaction buffer further comprises LiCl. In some embodiments, the reaction buffer further comprises ammonium acetate. In some embodiments, the reaction buffer further comprises a combination of salts. In some embodiments, the reaction buffer comprises a salt concentration ranging from 0.1 mM to 100 mM. In some embodiments, the reaction buffer comprises a salt concentration ranging from 1 mM to 50 mM. In some embodiments, the reaction buffer comprises a salt concentration ranging from 1 mM to 10 mM. In some embodiments, the reaction buffer comprises a salt concentration ranging from 5 mM to 8 mM. In some embodiments, the reaction buffer comprises a salt concentration of 1 mM. In some embodiments, the reaction buffer comprises a salt concentration of 3 mM. In some embodiments, the reaction buffer comprises a salt concentration of 5 mM. In some embodiments, the reaction buffer comprises a salt concentration of 8 mM. In some embodiments, the reaction buffer contains salt at a concentration of 10 mM.

[0008] In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.10 mM to 1.25 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.75 mM to 1.25 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.50 mM to 1.0 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.75 mM to 1.0 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.25 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.5 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.7 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.75 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.8 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.9 mM. In some embodiments, the MgCl in the reaction buffer has a concentration of 1.0 mM. In some embodiments, the MgCl in the reaction buffer has a concentration of 1.10 mM. In some embodiments, the MgCl in the reaction buffer has a concentration of 1.20 mM.

[0009] In some embodiments, the reaction buffer comprises MnCl. In some embodiments, the reaction buffer comprises MgCl and MnCl.

[0010] In some embodiments, the MnCl2 in the reaction buffer has a concentration of about 0.10 mM to 1.25 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of about 0.75 mM to 1.25 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of about 0.50 mM to 1.0 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of about 0.75 mM to 1.0 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.25 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.5 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.7 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.75 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.8 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.8 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 0.9 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 1.0 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 1.10 mM. In some embodiments, the MnCl2 in the reaction buffer has a concentration of 1.20 mM.

[0011] In some embodiments, the pH of the reaction buffer is about 6.0 to 8.0. In some embodiments, the pH of the reaction buffer is about 6.5 to 8.0. In some embodiments, the pH of the reaction buffer is about 7.0 to 7.8. In some embodiments, the pH of the reaction buffer is about 7.2 to 7.7. In some embodiments, the pH of the reaction buffer is about 7.4 to 7.6. In some embodiments, the pH of the reaction buffer is about 7.0. In some embodiments, the pH of the reaction buffer is about 7.2. In some embodiments, the pH of the reaction buffer is about 7.3. In some embodiments, the pH of the reaction buffer is about 7.4. In some embodiments, the pH of the reaction buffer is about 7.5. In some embodiments, the pH of the reaction buffer is about 7.6. In some embodiments, the pH of the reaction buffer is about 7.7. In some embodiments, the pH of the reaction buffer is about 7.8. In some embodiments, the pH of the reaction buffer is about 8.0.

[0012] In some embodiments, the mRNA is on the order of 5 mg, 1 g, 15 g, 100 g, 250 g, 500 g, or 1 kg or more. In some embodiments, methods according to the invention yield at least 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 5 mg of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 100 mg of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 500 mg of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 1 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 5 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 10 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 15 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 50 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 100 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 250 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 500 g of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 1 kg of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 10 kg of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 50 kg of mRNA in a single batch. In some embodiments, methods according to the invention yield at least 100 kg of mRNA in a single batch.As used herein, the term "batch" refers to a quantity or amount of mRNA synthesized at one time, e.g., produced according to a single manufacturing setup. A batch may refer to the amount of mRNA synthesized in a single reaction, occurring via a single aliquot of enzyme and / or a single aliquot of DNA template for continuous synthesis under one set of conditions. mRNA synthesized in a single batch does not include mRNA synthesized at different times that are combined to achieve the desired amount.

[0013] In some embodiments, the tailing of the mRNA is between about 50 nucleotides and 1000 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of about 100 to 900 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of about 250 to 750 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 50 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 100 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 200 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 250 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 300 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 400 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length greater than about 500 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of greater than about 600 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of greater than about 750 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of greater than about 900 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of about 250 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of about 500 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of about 600 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of greater than about 700 nucleotides. In some embodiments, tailing the mRNA comprises adding a poly-A tail having a length of about 750 nucleotides.In some embodiments, tailing of the mRNA comprises the addition of a polyA tail having a length of about 900 nucleotides.

[0014] In some embodiments, the tailing of the mRNA has an efficiency of about 70% to 95%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 60%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 70%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 72%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 75%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 78%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 80%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 82%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 85%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 88%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 90%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 95%. In some embodiments, the tailing of the mRNA has an efficiency greater than about 97%. In some embodiments, the tailing of the mRNA has an efficiency of greater than about 99%. In some embodiments, the tailing of the mRNA has an efficiency of about 70%. In some embodiments, the tailing of the mRNA has an efficiency of about 72%. In some embodiments, the tailing of the mRNA has an efficiency of about 75%. In some embodiments, the tailing of the mRNA has an efficiency of about 78%. In some embodiments, the tailing of the mRNA has an efficiency of about 80%. In some embodiments, the tailing of the mRNA has an efficiency of about 82%. In some embodiments, the tailing of the mRNA has an efficiency of about 85%. In some embodiments, the tailing of the mRNA has an efficiency of about 88%. In some embodiments, the tailing of the mRNA has an efficiency of about 90%. In some embodiments In some embodiments, the tailing of the mRNA has an efficiency of about 95%. In some embodiments, the tailing of the mRNA has an efficiency of about 97%. In some embodiments, the tailing of the mRNA has an efficiency of about 99%. In some embodiments, the tailing of the mRNA has an efficiency of about 100%. In some embodiments, the tailing efficiency is assessed by capillary electrophoresis (CE) shift.

[0015] In some embodiments, capping and tailing mRNA in a reaction buffer having a pH below 8.0 results in capped and tailed mRNA with greater integrity compared to mRNA capped and tailed using a reaction buffer having a pH of 8.0 or greater.

[0016] In some embodiments, capping and tailing mRNA in a reaction buffer having an MgCl2 concentration of 1.0 mM or less results in capped and tailed mRNA with greater integrity compared to mRNA capped and tailed using a reaction buffer having an MgCl2 concentration greater than 1.0 mM.

[0017] In some embodiments, mRNA integrity is at least 60% or greater. In some embodiments, mRNA integrity is at least 65% or greater. In some embodiments, mRNA integrity is at least 70% or greater. In some embodiments, mRNA integrity is at least 75% or greater. In some embodiments, mRNA integrity is at least 80% or greater. In some embodiments, mRNA integrity is at least 85% or greater. In some embodiments, mRNA integrity is at least 90% or greater. In some embodiments, mRNA integrity is at least 92% or greater. In some embodiments, mRNA integrity is at least 95% or greater. In some embodiments, mRNA integrity is at least 99% or greater. In some embodiments, mRNA integrity is assessed by capillary electrophoresis (CE) smear. In some embodiments, mRNA integrity is assessed by CGE smear.

[0018] In some embodiments, the method has an mRNA capping efficiency of 70% or greater. In some embodiments, the method has an mRNA capping efficiency of 80% or greater. In some embodiments, the method has an mRNA capping efficiency of 85% or greater. In some embodiments, the method has an mRNA capping efficiency of 90% or greater. In some embodiments, the method has an mRNA capping efficiency of 95% or greater. In some embodiments, the method has an mRNA capping efficiency of 98% or greater. In some embodiments, the method has an mRNA capping efficiency of 80%. In some embodiments, the method has an mRNA capping efficiency of 85%. In some embodiments, the method has an mRNA capping efficiency of 90%. In some embodiments, the method has an mRNA capping efficiency of 95%. In some embodiments, the method has an mRNA capping efficiency of 97%. In some embodiments, the method has an mRNA capping efficiency of 98%. In some embodiments, the method has an mRNA capping efficiency of 99%. In some embodiments, the method has an mRNA capping efficiency of 100%.

[0019] In one aspect, the present invention provides, inter alia, a method for capping and tailing a purified in vitro transcribed messenger RNA (mRNA) preparation, the method comprising capping and tailing the mRNA in a reaction buffer comprising a pH of about 7.5 and a MgCl concentration of about 1.0 mM, wherein the capping and tailing of the mRNA has a capping and tailing efficiency of 80% or greater, and The mRNA obtained has at least 65% integrity.

[0020] The above and further features will be more clearly understood from the following detailed description when read in conjunction with the accompanying drawings, which, however, are for purposes of illustration only and not of limitation. [Brief explanation of the drawings]

[0021] [Figure 1] Capillary electrophoresis (CE) profiles of purified CFTR mRNA before capping and tailing (left graph), purified capped and tailed CFTR mRNA in reaction buffer containing 1.25 mM MgCl2 at pH 8.0 (middle graph), and purified capped and tailed CFTR mRNA in reaction buffer containing 1.0 mM MgCl2 at pH 7.5 (right graph) at a 5 mg scale are shown. The arrows indicate the shoulder representing degraded RNA species. [Figure 2] Capillary electrophoresis (CE) profiles of purified DNAH5 mRNA before capping and tailing (left graph), purified capped and tailed CFTR mRNA in reaction buffer containing 1.25 mM MgCl2 at pH 8.0 (middle graph), and purified capped and tailed CFTR mRNA in reaction buffer containing 1.0 mM MgCl2 at pH 7.5 (right graph) at a 5 mg scale are shown. The arrow indicates the shoulder representing degraded RNA species. [Figure 3] Capillary electrophoresis (CE) profile of purified capped and tailed CFTR mRNA in reaction buffer containing 1.0 mM MgCl2 at pH 7.5 at 1 gram scale, demonstrating the integrity of the capped and tailed mRNA under optimized reaction conditions. The arrow indicates the shoulder representing degraded RNA species. [Figure 4] Capillary electrophoresis (CE) profiles of purified CFTR mRNA before capping and tailing (left graph), purified capped and tailed CFTR mRNA in reaction buffer containing 1.25 mM MgCl2 at pH 8.0 (middle graph), and purified capped and tailed CFTR mRNA in reaction buffer containing 1.0 mM MgCl2 at pH 7.5 (right graph) at a 15 gram scale are shown. The arrow indicates the shoulder representing degraded RNA species. [Figure 5] Capillary electrophoresis (CE) profiles of purified CFTR mRNA before capping and tailing (left graph), purified capped and tailed CFTR mRNA in reaction buffer containing 1.25 mM MgCl2 at pH 8.0 (middle graph), and purified capped and tailed CFTR mRNA in reaction buffer containing 1.0 mM MgCl2 at pH 7.5 (right graph) at a 100 gram production scale are shown. The arrows indicate the shoulder representing degraded RNA species. [Figure 6] Capillary electrophoresis (CE) profiles of purified capped and tailed OTC mRNA at a 10 gram production scale in a reaction buffer containing 1.25 mM MgCl2 at pH 8.0 (left graph) and at a 250 gram production scale in a reaction buffer containing 1.0 mM MgCl2 at pH 7.5 (right graph) are shown. The arrows indicate the shoulder representing degraded RNA species.

[0022] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions of these terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide further details regarding its practice are incorporated herein by reference.

[0023] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. As used herein, a "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxy- and / or amino-terminal amino acids in a peptide, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. An amino acid can participate in a disulfide bond. An amino acid may include one or post-translational modifications, such as association with one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and can refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which it is used.

[0024] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less than) of the stated reference value, unless otherwise specified or otherwise clear from the context (except when such number exceeds 100% of the possible values).

[0025] Batch: As used herein, the term "batch" refers to the quantity or amount of mRNA synthesized at one time, e.g., produced according to a single production sequence during the same production cycle. A batch may refer to the amount of mRNA synthesized in a single reaction, occurring via a single aliquot of enzyme and / or a single aliquot of DNA template for sequential synthesis under one set of conditions. In some embodiments, a batch includes mRNA produced from a reaction in which all reagents and / or components are replenished and / or not replenished as the reaction progresses. The term "batch" does not refer to mRNA synthesized at different times that are combined to achieve a desired amount.

[0026] Biologically active: As used herein, the term "biologically active" refers to the characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.

[0027] Codon optimization: As used herein, the terms "codon optimization" and "codon-optimized" refer to the modification of the codon composition of a native or wild-type nucleic acid that encodes a peptide, polypeptide, or protein without changing its amino acid sequence, thereby improving protein expression of the nucleic acid. Such modifications to a native or wild-type nucleic acid may be performed in a manner that is compatible with the desired target sequence. This may be done to achieve as high a G / C content as possible, adjust codon usage to avoid rare or rate-limiting codons, remove destabilizing nucleic acid sequences or motifs, and / or remove pause sites or terminator sequences.

[0028] Contaminant: As used herein, the term "contaminant" refers to a substance in a limited amount of liquid, gas, or solid that differs from the chemical composition of the target substance or compound. Contaminants are also referred to as impurities. Examples of contaminants or impurities include buffers, proteins (e.g., enzymes), nucleic acids, salts, solvents, and / or wash solutions.

[0029] Dispersing agent: As used herein, the term "dispersing agent" refers to solid particles that reduce the likelihood of mRNA precipitates forming a hydrogel. Examples of dispersing agents include, but are not limited to, one or more of ash, clay, diatomaceous earth, filter media, glass beads, plastic beads, polymers, polypropylene beads, polystyrene beads, salts (e.g., cellulose salts), sand, and sugars. In embodiments, the dispersing agent is a polymer microsphere (e.g., poly(styrene-co-divinylbenzene) microsphere).

[0030] Delivery: As used herein, the term "delivery" encompasses both local delivery and systemic delivery. For example, the delivery of mRNA encompasses the situation where mRNA is delivered to a target tissue, its encoded protein is expressed, and the target tissue retains the protein (also referred to as "local distribution" or "local delivery"); the situation where mRNA is delivered to a target tissue, its encoded protein is expressed, and the protein is secreted into the patient's circulatory system (e.g., serum), and then distributed throughout the body and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery"). In some embodiments, delivery is pulmonary delivery, including, for example, nebulization.

[0031] Encapsulation: As used herein, the term "encapsulation," or grammatical equivalents, refers to the process of entrapment of nucleic acid molecules within nanoparticles.

[0032] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., antibody heavy or light chains) into an intact protein (e.g., an antibody), and / or the post-translational modification of the polypeptides or fully assembled protein (e.g., an antibody). In this application, the terms "expression" and "production" and grammatical equivalents are used interchangeably.

[0033] Full-length mRNA: As used herein, "full-length mRNA" is characterized when using a specific assay, such as detection using UV and UV absorption spectroscopy followed by separation by gel electrophoresis or capillary electrophoresis. The length of an mRNA molecule encoding a full-length polypeptide and obtained after any of the purification methods described herein is at least 50% of the length of the full-length mRNA molecule transcribed from the target DNA, e.g., at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.01%, 99.05%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the length of the full-length mRNA molecule transcribed from the target DNA and prior to purification by any of the methods described herein.

[0034] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0035] Half-life: As used herein, the term "half-life" refers to the life of a nucleic acid or protein It is the time required for a quantity, such as concentration or activity, to fall to half of its value measured at the beginning of a period.

[0036] Improve, increase, or decrease: As used herein, "improve," "increase," or "reduce," or grammatical equivalents, refer to a value compared to a baseline measurement, e.g., a measurement in the same individual before initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject suffering from the same form of disease as the subject being treated and who is approximately the same age as the subject being treated.

[0037] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0038] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0039] Isolated: As used herein, the term "isolated" refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) artificially produced, prepared, and / or manufactured. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of percent purity of isolated substances and / or entities should not include excipients (e.g., buffers, solvents, water, etc.).

[0040] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, liposomes used herein can be formed by mixing one or more lipids or by mixing one or more lipids with a polymer. In some embodiments, liposomes suitable for the present invention comprise a cationic lipid and optionally a non-cationic lipid, optionally a cholesterol-based lipid, and / or optionally a PEG-modified lipid.

[0041] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA can contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, or optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. The mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0042] mRNA integrity: As used herein, the term "mRNA integrity" generally refers to the quality of mRNA. In some embodiments, mRNA integrity refers to the proportion of mRNA that is not degraded after the purification process (e.g., the methods described herein). mRNA integrity can be determined using methods specifically described herein, such as TAE agarose gel electrophoresis, or by SDS-PAGE with silver staining, or by methods well known in the art, for example, by RNA agarose gel electrophoresis (e.g., Ausubel et al., John Wiley & Sons, Inc., 1997, Current Protocols in Molecular Biology).

[0043] N / P ratio: As used herein, the term "N / P ratio" refers to the molar ratio of the positively charged molecular units in the cationic lipid in the lipid nanoparticle to the negatively charged molecular units in the mRNA encapsulated in the lipid nanoparticle.Thus, N / P ratio is typically calculated as the ratio of the moles of amine groups in the cationic lipid in the lipid nanoparticle to the moles of phosphate groups in the mRNA encapsulated in the lipid nanoparticle.

[0044] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single- and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNA may contain introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, optionally purified, chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Nucleic acids are presented in a 5' to 3' orientation unless otherwise indicated.In some embodiments, nucleic acids are selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, , C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). In some embodiments, the present invention provides nucleic acids (e.g., nucleotides and / or nucleotides) that are not chemically modified to enhance or achieve delivery. The term "unmodified nucleic acids" refers to polynucleotides containing nucleotides T and U, and is specifically directed to "unmodified nucleic acids," which means polynucleotides containing nucleotides T and U (polynucleotides and residues containing nucleotides T and U). In some embodiments, the nucleotides T and U are used interchangeably in the sequence description.

[0045] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In certain embodiments, the patient is a human. Humans include prenatal and postnatal forms.

[0046] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, inflammatory irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0047] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or salts of amino groups formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts of amino groups formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Salts derived from appropriate bases include: lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-methyl-N ... + (C 1-4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations, and amine cations, formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, for example, alkyl halides, to form quaternized alkylated amino salts.

[0048] Systemic distribution or delivery: As used herein, the terms "systemic distribution," "systemic delivery," or grammatical equivalents refer to a delivery or distribution mechanism or approach that affects the entire body or the entire organism. Typically, systemic distribution or delivery is accomplished via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery."

[0049] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, a subject is a human. A subject may be a patient, and refers to a person who sees a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0050] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or reach completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention describes a method for preparing scalable quantities of pure, high-quality mRNA. mRNA is typically synthesized by in vitro transcription (IVT) using polymerases such as SP6 or T7 polymerase, followed by capping and tailing to generate full-length, in vivo translatable mRNA. Preparation of correctly capped RNA is essential for assessing mRNA function in a cellular context. Furthermore, modifying the cap structure has the potential to increase mRNA stability and translation efficiency. These two properties may provide the key to therapeutic applications of mRNA.

[0052] The present invention is based, at least in part, on the surprising and unexpected discovery that when a buffer having a lower than conventional pH and containing a low concentration of magnesium chloride (MgCl2) was used for the capping and tailing reactions, RNA integrity improved by more than 25% as assessed by capillary electrophoresis (CE) or capillary gel electrophoresis (CGE). The mRNA preparation method disclosed herein also maintained high capping and tailing efficiency. This improvement was translatable across different scales, demonstrating the scalability of the method and its suitability for use in mRNA production and therapeutics.

[0053] 5-Cap Typically, eukaryotic mRNA has a "cap" structure at the 5' end, which plays an important role in translation. For example, the cap plays an important role in mRNA metabolism, and is required to varying degrees for the processing and maturation of RNA transcripts in the nucleus, the transport of mRNA from the nucleus to the cytoplasm, the stability of mRNA, and the efficient translation of mRNA into protein. The 5' cap structure is involved in the initiation of protein synthesis of eukaryotic cell and eukaryotic virus mRNA, as well as the processing and stability of mRNA in vivo (see, for example, Shatkin, AJ, CELL, 9:645-653 (1976); Furuichi, et al., NATURE, 266:235 (1977); FEDERATION OF EXPERIMENTAL BIOLOGISTS SOCIETY LETTER 96:1-11 (1978); Sonenberg, N., PROG. NUC. ACID RES MOL BIOL, 35:173-207 (1988)). There are specific cap-binding proteins that are components of the machinery required for the initiation of mRNA translation (see, e.g., Shatkin, AJ, CELL, 40:223-24 (1985); Sonenberg, N., PROG. NUC. ACID RES MOL BIOL, 35:173-207 (1988)). The mRNA cap is recognized by the translation initiation factor eIF4E (see, e.g., Gingras, et al., ANN. REV. BIOCHEM. 68:913-963 (1999)). (See, e.g., Ross, J., MOL. BIOL. MED. 5:1-14 (1988); Green, MR et al., CELL, 32:681-694 (1983)). The benefit of the cap extends to stabilizing pre-mRNAs, as the primary transcripts of many eukaryotic and viral genes require processing to remove intervening sequences (introns) within the coding regions of these transcripts.

[0054] In vitro, capped RNAs have been reported to be translated more efficiently than uncapped transcripts in various in vitro translation systems, such as rabbit reticulocyte lysate or wheat germ translation systems (see, e.g., Shimotohno, K., et al., PROC. NATL. ACAD. SCI. USA, 74:2734-2738 (1977); Paterson and Rosenberg, NATURE, 279:692 (1979)). This effect is also thought to be due, in part, to protection of the RNA from exoribonucleases present in the in vitro translation system, as well as other factors.

[0055] The naturally occurring cap structure is linked to the 5' end of the first transcribed nucleotide by a triphosphate bridge, resulting in m 7 It contains a 7-methylguanosine, resulting in a dinucleotide cap of G(5')'ppp(5')'N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyltransferase. Addition of the cap to the 5' end of the RNA occurs immediately after transcription initiation. The terminal nucleoside is typically guanosine, in the reverse orientation relative to all other nucleotides, i.e., G(5')ppp(5')GpNpNp.

[0056] A common cap for mRNAs produced by in vitro transcription is m7G(5')ppp(5')G, which is used as a dinucleotide cap during in vitro transcription with T7 or SP6 RNA polymerase to obtain RNAs with a cap structure at their 5' end. A common in vitro method for capped mRNA synthesis employs a preformed dinucleotide with the form m7G(5')ppp(5')G ("m7GpppG") as the initiating factor for transcription. The disadvantage of using the pseudosymmetric dinucleotide m7G(5')ppp(5')G is the tendency of the 3'-OH of either the G or m7G moiety to serve as the initiating nucleophile for transcription elongation. In other words, the presence of a 3'-OH on both the m7G and G moieties leads to up to half of mRNAs incorporating the cap in an improper orientation. This leads to the synthesis of two isomeric RNAs, mG(5')pppG(pN)n and G(5')pppmG(pN)n, in approximately equal proportions, depending on the ionic conditions of the transcription reaction. Variations in isomeric forms can adversely affect in vitro translation and are undesirable for homogeneous therapeutics.

[0057] To date, the usual form of synthetic dinucleotide cap used in in vitro translation experiments is the anti-reverse cap analog ("ARCA"), which is a modified cap analog in which the 2' or 3' OH group is generally replaced with -OCH3. ARCA and triple-methylated cap analogs are incorporated in the forward direction, with m at either the 2' or 3' OH group of the ribose ring. 7 Chemical modification of G allows the cap to be incorporated only in the forward orientation, even though the 2'OH group is not involved in the phosphodiester bond (Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties", RNA, 9:1108-1122 (2003)). Selection procedures for methylation of guanosine in 5' diphosphate synthesis and methylation of guanosine in 5' diphosphate synthesis have been established (Kore, A. and Parmar, G. NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS, 25:337-340, (2006) and Kore, A.R., et al. NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS 25(3):307-14, (2006)).

[0058] Transcription of RNA typically begins with a nucleoside triphosphate (usually a purine, A or G). In vitro transcription typically involves a phage RNA polymerase such as T7, T3, or SP6, a DNA template containing a phage polymerase promoter, nucleotides (ATP, GTP, CTP, and UTP), and a buffer containing a magnesium salt. Synthesis of capped RNA involves the incorporation of a cap analog (e.g., m7GpppG) in the transcription reaction; in some embodiments, this is incorporated by the addition of recombinant guanylyltransferase. An excess of m7GpppG to GTP (4:1) increases the chance that each transcript will have a 5' cap. Kits for capping in vitro transcribed mRNA are commercially available, including the mMESSAGE mMACHINE® kit (Ambion, Inc., Austin, Tex.). These kits typically yield 80% capped RNA to 20% uncapped RNA, but total RNA yields are low as GTP concentration becomes rate-limiting because GTP is required for transcript elongation. In contrast, the method described herein yields capping efficiencies of over 90% and RNA integrity of over 70%.

[0059] In some embodiments, the inventive method of the present invention comprises a compound of Formula I: [ka] (In the formula, B is a nucleobase, R1 is selected from halogen, OH, and OCH3; R2 is selected from H, OH, and OCH3; R3 is CH3, CH2CH3, CH2CH2CH3, or a cavity; R4 is NH2, R5 is selected from OH, OCH3 and halogen; n is 1, 2, or 3; and M is a nucleotide of the mRNA).

[0060] In some embodiments, the nucleobase is guanine.

[0061] The 5' cap is typically added as follows: First, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphate groups. Guanosine triphosphate (GTP) is then added to the terminal phosphate via a guanylyltransferase, resulting in a 5'5'5 triphosphate linkage. The 7-nitrogen of the guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, mG(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G. Additional cap structures are described in published U.S. Patent Application Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0062] 3' polyA tail The presence of a "tail" at the 3' end serves to protect the mRNA from exonuclease degradation. The 3' tail may be added before, after, or simultaneously with the addition of the 5' cap.

[0063] In some embodiments, the polyA tail is 25 to 5,000 nucleotides in length. The tail structure typically comprises a poly(A) tail and / or a poly(C) tail (A: adenosine, C: cytosine). In some embodiments, the polyA tail or polyC tail on the 3' end of the mRNA comprises at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, or at least Each of the fragments contains 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotides, respectively.In some embodiments, the poly-A tail or poly-C tail each has between about 10 and 800 adenosine or cytosine nucleotides (e.g., between about 10 and 200 adenosine or cytosine nucleotides, between about 10 and 300 adenosine or cytosine nucleotides, between about 10 and 400 adenosine or cytosine nucleotides, between about 10 and 500 adenosine or cytosine nucleotides, between about 10 and 550 adenosine or cytosine nucleotides, between about 10 and 600 adenosine or cytosine nucleotides, between about 50 and 600 adenosine or cytosine nucleotides, between about 10 ... adenosine nucleotides or cytosine nucleotides, about 150 to 600 adenosine nucleotides or cytosine nucleotides, about 200 to 600 adenosine nucleotides or cytosine nucleotides, about 250 to 600 adenosine nucleotides or cytosine nucleotides, about 300 to 600 adenosine nucleotides or cytosine nucleotides, about 350 to 600 adenosine nucleotides or cytosine nucleotides, about 400 to 600 adenosine nucleotides or cytosine nucleotides, about 450 to 600 adenosine nucleotides or cytosine nucleotides, about 500 to 600 adenosine nucleotides or cytosine nucleotides, about 10 to 150 adenosine nucleotides or cytosine nucleotides. The tail structure may be adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides). In some embodiments, the tail structure comprises a combination of poly(A) tails and poly(C) tails of various lengths as described herein. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0064] Other capping and / or tailing methods are available in the art and can be used to practice the present invention.

[0065] As described herein, the addition of a 5' cap and / or 3' tail facilitates the detection of abortive transcripts generated during in vitro synthesis because, without capping and / or tailing, the size of these prematurely aborted mRNA transcripts may be too small to be detected. Thus, in some embodiments, a 5' cap and / or 3' tail is added to a synthetic mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, a 5' cap and / or 3' tail is added to a synthetic mRNA before the mRNA is purified as described herein. In some embodiments, a 5' cap and / or 3' tail is added to a synthetic mRNA after the mRNA is purified as described herein.

[0066] mRNA synthesis and purification Maintaining high RNA integrity during in vitro transcription synthesis and mRNA purification is important in the production of mRNA for therapeutic purposes. Furthermore, high capping and tailing efficiency of mRNA with a polyA tail of the desired length is an important attribute of mRNA quality. mRNA according to the present invention can be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Patent Application No. 2018 / 0258423 and can be used in practicing the present invention, all of which are incorporated herein by reference. For example, mRNA according to the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is often performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0067] In some embodiments, in vitro transcription occurs in a single batch. In some embodiments, the IVT reaction includes a capping and tailing reaction (C / T). In some embodiments, the capping and tailing reactions are performed separately from the IVT reaction. In some embodiments, mRNA is recovered from the IVT reaction, followed by a first precipitation and purification of the mRNA by methods known in the art. The recovered purified mRNA is then capped and tailed, and subjected to a second precipitation and purification.

[0068] In some embodiments, suitable mRNA sequences are those that encode proteins or peptides. In some embodiments, suitable mRNA sequences are those that encode proteins or peptides. Codon optimization is optimized for efficient expression. Codon optimization typically involves modifying a native or wild-type nucleic acid sequence encoding a peptide, polypeptide, or protein to achieve the highest possible G / C content, adjusting codon usage to avoid rare or rate-limiting codons, removing destabilizing nucleic acid sequences or motifs, and / or removing pause sites or terminator sequences without changing the amino acid sequence of the mRNA-encoded peptide, polypeptide, or protein. In some embodiments, a suitable mRNA sequence is a native or wild-type sequence. In some embodiments, a suitable mRNA sequence encodes a protein or peptide containing one or more mutations in the amino acid sequence.

[0069] The methods of the present invention can be used to prepare mRNAs of various lengths. In some embodiments, the present invention can be used to prepare in vitro synthesized mRNAs of lengths of about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 20 kb, 30 kb, 40 kb, or greater than 50 kb. In some embodiments, the present invention can be used to deliver in vitro synthesized mRNAs of lengths ranging from about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 50 kb. Thus, the methods of the present invention can be used to prepare mRNA for any gene of interest.

[0070] IVT reaction IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application. A suitable DNA template typically contains a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired nucleotide sequence of the desired mRNA and a termination signal. In some embodiments, the produced mRNA is codon-optimized.

[0071] In some embodiments, an exemplary IVT reaction mixture contains a linear double-stranded DNA template along with an SP6 polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs, 10 mM DTT, and reaction buffer (800 mM HEPES, 20 mM spermidine, 250 mM MgCl, pH 7.7 for 10x), and sufficient qs (QS) to bring to the desired reaction volume with RNase-free water, and then the reaction mixture is incubated at 37°C for 60 minutes. The polymerase reaction was then quenched by adding DNase I and DNase I buffer (100 mM Tris-HCl, 5 mM MgCl, and 25 mM CaCl, pH 7.6 for 10x) to facilitate digestion of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[0072] Other IVT methods are available in the art and can be used to practice the present invention.

[0073] Post-synthesis processing Typically, a 5' cap and / or a 3' tail may be added post-synthetically. The presence of a cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a "tail" serves to protect the mRNA from exonuclease degradation. Add.

[0074] Capping and Tailing (C / T) Reaction Typically in eukaryotes, mRNA processing involves adding a "cap" on the N-terminus (5') and a "tail" on the C-terminus (3'). A typical cap is a 7-methylguanosine cap, which is a guanosine linked via a 5'-5'-triphosphate linkage to the first transcribed nucleotide. In some embodiments, in vitro transcribed mRNA is subjected to 5'N cap addition using guanylate transferase. 7 -methylguanylate cap structure and Fechter, P.; Brownlee, GG "Recognition of mRNA cap structures" Cap analogs are enzymatically modified by the addition of a methyl group at the 2'O position of the penultimate nucleotide, resulting in a Cap1 structure, using a 2'O-methyltransferase, as described in "Capping RNA by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249. For capping as part of the IVT reaction, a cap analog can be incorporated as the first "base" of the nascent RNA strand. Cap analogs include Cap0, Cap1, Cap2, m6 A m , or a non-natural cap.

[0075] In some embodiments, the 5' cap is typically added as follows: First, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates. Then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyltransferase, resulting in a 5'-5'5 triphosphate linkage. The 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')G, G(5')ppp(5')A, and G(5')ppp(5')G. Briefly, purified IVT mRNA is typically mixed with GTP, S-adenosylmethionine, an RNase inhibitor, 2'-O methyltransferase, and guanylyltransferase in the presence of a reaction buffer containing Tris-HCl, MgCl2, and RNase-free HO, and then incubated at 37°C. A conventional capping reaction buffer contains 50 mM Tris-HCl pH 8.0 and 1.25 mM MgCl2.

[0076] In some embodiments, after the addition of the Cap1 structure, a polyadenylate tail is enzymatically added to the 3' end of the in vitro transcribed mRNA using poly-A polymerase. The tail is typically a polyadenylation event, which adds a polyadenylyl moiety to the 3' end of the mRNA molecule. In some embodiments, after the capping reaction incubation, the tailing reaction is initiated by adding a tailing buffer containing Tris-HCl, NaCl, MgCl2, ATP, poly-A polymerase, and RNase-free HO. The reaction is quenched by the addition of EDTA. A conventional tailing reaction buffer contains 50 mM Tris-HCl pH 8.0 and 1.25 mM MgCl2.

[0077] In some embodiments, the pH of the optimized reaction buffer of the present invention is about 6.0 to 8.0. In some embodiments, the pH of the reaction buffer is about 6.5 to 8.0. In some embodiments, the pH of the reaction buffer is about 7.0 to 7.8. In some embodiments, the pH of the reaction buffer is about 7.2 to 7.7. In some embodiments, the pH of the reaction buffer is about 7.4 to 7.6. In some embodiments, the pH of the reaction buffer is about 7.0. In some embodiments, the pH of the reaction buffer is about 7.2. In some embodiments, the pH of the reaction buffer is about 7.3. In some embodiments, the pH of the reaction buffer is about 7.4 ... H is about 7.5. In some embodiments, the pH of the reaction buffer is about 7.6. In some embodiments, the pH of the reaction buffer is about 7.7. In some embodiments, the pH of the reaction buffer is about 7.8. In some embodiments, the pH of the reaction buffer is about 8.0.

[0078] In some embodiments, the MgCl2 in the optimized reaction buffer of the present invention has a concentration of about 0.10 mM to 1.25 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.75 mM to 1.25 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.50 mM to 1.0 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of about 0.75 mM to 1.0 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.25 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.5 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.7 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.75 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.8 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 0.9 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 1.0 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 1.10 mM. In some embodiments, the MgCl2 in the reaction buffer has a concentration of 1.20 mM.

[0079] mRNA purification In some embodiments, mRNA before and after the capping and tailing reactions can be further purified. Various methods can be used to purify synthesized mRNA according to methods known in the art. For example, mRNA purification can be performed using centrifugation, filtration, and / or chromatography. In some embodiments, synthesized mRNA is purified by ethanol precipitation, filtration, chromatography, gel purification, or any other suitable means. In some embodiments, mRNA is purified by HPLC. In some embodiments, mRNA is extracted with a standard phenol:chloroform:isoamyl alcohol solution well known to those skilled in the art. In some embodiments, mRNA is purified using tangential flow filtration. Suitable purification methods include those described in Published U.S. Patent Application No. 2016 / 0040154, Published U.S. Patent Application No. 2015 / 0376220, Published U.S. Patent Application No. 2018 / 0251755, Published U.S. Patent Application No. 2018 / 0251754, U.S. Provisional Patent Application No. 62 / 757,612 filed November 8, 2018, and U.S. Provisional Patent Application No. 62 / 891,781 filed August 26, 2019, all of which are incorporated by reference herein and may be used to practice the present invention.

[0080] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified before and after capping and tailing. Generally, the purification steps described herein can be performed after each step of mRNA synthesis, optionally in conjunction with other purification processes such as dialysis.

[0081] In some embodiments, the mRNA is purified by centrifugation either before or after capping and tailing, or both before and after capping and tailing.

[0082] In some embodiments, the mRNA is purified by filtration either before or after capping and tailing, or both before and after capping and tailing. .

[0083] In some embodiments, mRNA is purified by tangential flow filtration (TFF) either before or after capping and tailing, or both before and after capping and tailing.

[0084] In some embodiments, the mRNA is purified by chromatography either before or after capping and tailing, or both before and after capping and tailing.

[0085] mRNA precipitation mRNA in impure preparations, such as in vitro synthesis reaction mixtures, may be precipitated using buffers and suitable conditions described in U.S. Provisional Patent Application No. 62 / 757,612, filed November 8, 2018, or U.S. Provisional Patent Application No. 62 / 891,781, filed August 26, 2019, and may be subsequently used to practice the present invention, following various purification methods known in the art. As used herein, the term "precipitation" (or any grammatical equivalent) refers to the formation of an insoluble material (e.g., a solid) in a solution. When used in reference to mRNA, the term "precipitation" refers to the formation of an insoluble or solid form of mRNA in a liquid.

[0086] Typically, mRNA precipitation is accompanied by denaturing conditions.As used herein, the term " denaturing conditions " refers to any chemical or physical conditions that can cause the destruction of the native conformation of mRNA.Because the native conformation of a molecule is usually the most water-soluble, destroying the secondary and tertiary structure of the molecule can cause changes in solubility, and can cause mRNA to precipitate from solution.

[0087] For example, a suitable method for precipitating mRNA from an impure preparation involves treating the impure preparation with a denaturing reagent so that mRNA precipitates.Examples of denaturing reagents suitable for the present invention include, but are not limited to, lithium chloride, sodium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, ammonium acetate, and combinations thereof.Suitable reagents can be provided in solid form or in solution.

[0088] In some embodiments, guanidinium salts are used in denaturing buffers for precipitating mRNA. Non-limiting examples of guanidinium salts include guanidinium chloride, guanidinium thiocyanate, or guanidinium isothiocyanate. Guanidinium thiocyanate, also known as guanidinium thiocyanate, can be used to precipitate mRNA. The present invention is based on the surprising discovery that mRNA precipitation buffers containing guanidinium salts, such as guanidinium thiocyanate, can be used at concentrations higher than those typically used in denaturing reactions, resulting in mRNA that is substantially free of protein contaminants. In some embodiments, solutions suitable for mRNA precipitation contain guanidinium thiocyanate at a concentration greater than 4M.

[0089] In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises greater than 4 M guanidine thiocyanate. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 5 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 5.5 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 6 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 6.5 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 7 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 7.5 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 8 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 8.5 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 9 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises about 10 M GSCN. In some embodiments, a buffer containing a denaturing reagent suitable for mRNA precipitation comprises more than 10 M GSCN.

[0090] In addition to the denaturing reagent, a solution suitable for mRNA precipitation may contain additional salts, detergents, and / or buffering agents. For example, a suitable solution may further contain sodium lauryl sarkosyl and / or sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation contains about 5 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation contains about 10 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation contains about 20 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation contains about 25 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation contains about 30 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation contains about 50 mM sodium citrate.

[0091] In some embodiments, a buffer suitable for mRNA precipitation comprises a detergent such as N-lauryl sarcosine (sarkosyl). In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.01% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.05% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.1% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.5% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises 1% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 1.5% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 2%, about 2.5%, or about 5% N-lauryl sarcosine.

[0092] In some embodiments, the solution suitable for mRNA precipitation contains a reducing agent. In some embodiments, the reducing agent is selected from dithiothreitol (DTT), beta-mercaptoethanol (b-ME), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), dithioerythritol (DTE), and dithiobutylamine (DTBA). In some embodiments, the reducing agent is dithiothreitol (DTT).

[0093] In some embodiments, DTT is present at a final concentration of greater than 1 mM and up to about 200 mM. In some embodiments, DTT is present at a final concentration of 2.5 mM to 100 mM. In some embodiments, DTT is present at a final concentration of 5 mM to 50 mM.

[0094] In some embodiments, DTT is present at a final concentration of 1 mM or greater. In some embodiments, DTT is present at a final concentration of 2 mM or greater. In some embodiments, DTT is present at a final concentration of 3 mM or greater. In some embodiments, DTT is present at a final concentration of 4 mM or greater. In some embodiments, DTT is present at a final concentration of 5 mM or greater. In some embodiments, DTT is present at a final concentration of 6 mM or greater. In some embodiments, DTT is present at a final concentration of 7 mM or greater. In some embodiments, DTT is present at a final concentration of 8 mM or greater. In some embodiments, DTT is present at a final concentration of 9 mM or greater. In some embodiments, DTT is present at a final concentration of 10 mM or greater. In some embodiments, DTT is present at a final concentration of 11 mM or greater. In some embodiments, DTT is present at a final concentration of 12 mM or greater. In some embodiments, DTT is present at a final concentration of 13 mM or greater. In some embodiments, DTT is present at a final concentration of 14 mM or greater. In some embodiments, DTT is present at a final concentration of 15 mM or greater. In some embodiments, DTT is present at a final concentration of 16 mM or greater. In some embodiments, DTT is present at a final concentration of 17 mM or greater. In some embodiments, DTT is present at a final concentration of 18 mM or greater. In some embodiments, DTT is present at a final concentration of 19 mM or greater. In some embodiments, DTT is present at a final concentration of about 20 mM.

[0095] In some embodiments, the denaturation buffer comprises 2M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises 3M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises 4M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises about 5M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises about 6M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises about 7M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises about 8M or more GSCN and DTT. In some embodiments, the denaturation buffer comprises about 9M or more GSCN and DTT.

[0096] In some embodiments, the denaturation buffer comprises a DTT concentration of 1 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 2 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 3 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 4 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 5 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 6 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 7 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 8 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 9 mM or more and a GSCN concentration of about 5M. In some embodiments, the denaturation buffer comprises a DTT concentration of 10 mM or more and a GSCN concentration of about 5M.

[0097] Protein denaturation can occur even at low concentrations of denaturing reagents, with or without reducing agents. The combination of high concentrations of GSCN and DTT in the denaturing solution to precipitate impure mRNA results in pure mRNA that is substantially free of protein contaminants. The mRNA precipitated in the buffer can be processed through a filter. In some embodiments, the eluate after filtration following a single precipitation using a buffer containing about 5 M GSCN and about 10 mM DTT is of high quality and purity, with no detectable protein impurities. Furthermore, this method is reproducible over a wide range of mRNA throughputs, including at scales of about 1 gram, about 10 grams, about 100 grams, about 500 grams, or about 1000 grams or more of mRNA, without impeding fluid flow through the filter.

[0098] In some embodiments, the buffer for the precipitation step further comprises alcohol. In some embodiments, the precipitation is carried out under conditions where the mRNA, denaturation buffer (comprising GSCN and a reducing agent, e.g., DTT), and alcohol are present in a volume ratio of 1:(5):(3). In some embodiments, the precipitation is carried out under conditions where the mRNA, denaturation buffer, and alcohol are present in a volume ratio of 1:(3.5):(2.1). In some embodiments, the precipitation is carried out under conditions where the mRNA, denaturation buffer, and alcohol are present in a volume ratio of 1:(4):(2). In some embodiments, the precipitation is carried out under conditions where the mRNA, denaturation buffer, and alcohol are present in a volume ratio of 1:(2.8):(1.9). In some embodiments, the precipitation is carried out under conditions where the mRNA, denaturation buffer, and alcohol are present in a volume ratio of 1:(2.3):(1.7). In some embodiments, the precipitation is carried out under conditions where the mRNA, denaturation buffer, and alcohol are present in a volume ratio of 1:(2.1):(1.5).

[0099] In some embodiments, it may be desirable to incubate the impure preparation with one or more denaturing reagents described herein for a period of time at a desired temperature that allows for precipitation of a substantial amount of mRNA. For example, the mixture of the impure preparation and denaturing agent may be incubated at room temperature or at ambient temperature for a period of time. In some embodiments, suitable incubation times are about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 minutes or more. In some embodiments, suitable incubation times are about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, or 5 minutes or less. In some embodiments, the mixture is incubated at room temperature for about 5 minutes. Typically, "room temperature" or "ambient temperature" refers to a temperature in the range of about 20-25°C, e.g., about 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. In some embodiments, the mixture of impure preparation and denaturant can be incubated at room temperature or above (e.g., about 30-37°C, or particularly about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C) or below room temperature (e.g., about 15-20°C, or particularly about 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C). The incubation period can be adjusted based on the incubation temperature. Typically, higher incubation temperatures require shorter incubation times.

[0100] Alternatively or additionally, a solvent may be used to promote mRNA precipitation. Suitable exemplary solvents include, but are not limited to, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, and combinations thereof. For example, a solvent (e.g., absolute ethanol) can be added to the impure preparation together with the denaturing reagent or after the addition of the denaturing reagent and the incubation described herein to further enhance and / or promote mRNA precipitation. Typically, after the addition of a suitable solvent (e.g., absolute ethanol), the mixture can be incubated at room temperature for another hour. Typically, the suitable incubation time is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 minutes or more. In some embodiments, the suitable incubation time is about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, or 5 minutes or less. Typically, the mixture is incubated at room temperature for about 5 minutes. Temperatures above or below room temperature can be used with appropriate adjustment of the incubation time. Alternatively, incubation can be performed at 4°C or -20°C for precipitation.

[0101] In some embodiments, the precipitation of mRNA into a suspension comprises one or more amphipathic polymers. In some embodiments, the precipitation of mRNA into a suspension comprises a PEG polymer. Various types of PEG polymers are recognized in the art, some of which have distinct geometric shapes. For example, suitable PEG polymers include PEG polymers having linear, branched, Y-shaped, or multi-armed shapes. In some embodiments, the PEG is in a suspension comprising one or more PEGs of distinct geometric shapes. In some embodiments, the precipitation of mRNA can be achieved by precipitating the mRNA using PEG-6000. In some embodiments, the precipitation of mRNA can be achieved by precipitating the mRNA using PEG-400. In some embodiments, In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using triethylene glycol (TEG). In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using triethylene glycol monomethyl ether (MTEG). In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using tert-butyl-TEG-O-propionate. In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using TEG-dimethacrylate. In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using TEG-dimethyl ether. In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using TEG-divinyl ether. In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using TEG-monobutyl ether. In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using TEG-methyl ether methacrylate. In some embodiments, mRNA precipitation can be achieved by precipitating the mRNA using TEG-monodecyl ether. In some embodiments, precipitation of mRNA can be achieved using TEG-dibenzoate to precipitate the mRNA. Any one of these PEG- or TEG-based reagents can be used in combination with guanidinium thiocyanate to precipitate the mRNA.

[0102] Many amphiphilic polymers are known in the art. In some embodiments, the amphiphilic polymer comprises pluronic, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or a combination thereof. In some embodiments, the amphiphilic polymer is selected from one or more of the following: PEG triethylene glycol, tetraethylene glycol, PEG 200, PEG 300, PEG 400, PEG 600, PEG 1,000, PEG 1,500, PEG 2,000, PEG 3,000, PEG 3,350, PEG 4,000, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, PEG 35,000, and PEG 40,000, or a combination thereof. In some embodiments, the amphiphilic polymer comprises a mixture of PEG polymers of two or more molecular weights. For example, in some embodiments, PEG polymers of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 molecular weights comprise the amphiphilic polymer. Thus, in some embodiments, the PEG solution comprises a mixture of one or more PEG polymers. In some embodiments, the mixture of PEG polymers comprises polymers with distinct molecular weights.

[0103] In some embodiments, the method for precipitating mRNA in a suspension comprises a PEG polymer, wherein the PEG polymer comprises a PEG-modified lipid. In some embodiments, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG-2K). In some embodiments, the PEG-modified lipid is a DOPA-PEG conjugate. In some embodiments, the PEG-modified lipid is a poloxamer-PEG conjugate. In some embodiments, the PEG-modified lipid comprises DOTAP. In some embodiments, the PEG-modified lipid comprises cholesterol.

[0104] In some embodiments, mRNA is precipitated in a suspension containing an amphiphilic polymer. In some embodiments, mRNA is precipitated in a suspension containing any of the aforementioned PEG reagents. In some embodiments, PEG is in the suspension at a concentration of about 10% w / v to about 100% w / v. For example, in some embodiments, PEG is in the suspension at a concentration of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100% w / v. %, 95%, 100% wt / vol, and any value therebetween. In some embodiments, PEG is present in the suspension at a concentration of about 5% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 6% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 7% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 8% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 9% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 10% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 12% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 15% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 18% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 20% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 25% wt / vol. In some embodiments, PEG is present in the suspension at a concentration of about 30% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 35% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 40% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 45% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 50% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 55% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 60% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 65% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 70% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 75% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 80% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 85% w / v. In some embodiments, PEG is present in the suspension at a concentration of about 90% weight / volume.In some embodiments, PEG is present in the suspension at a concentration of about 95% weight / volume, hi some embodiments, PEG is present in the suspension at a concentration of about 100% weight / volume.

[0105] In some embodiments, precipitating mRNA in suspension involves a volume:volume ratio of PEG to the total mRNA suspension volume of about 0.1 to about 5.0. For example, in some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0. Thus, in some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.1. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.2. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.3. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.4. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.6. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.7. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.8. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.9. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.0. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.75. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.0. In some embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 2.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.75. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.0. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.75. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 4.0. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 4.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 4.50. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 4.75. In some embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 5.0.

[0106] In some embodiments, the reaction volume for mRNA precipitation includes GSCN and PEG.

[0107] In some embodiments, the method for purifying mRNA does not include alcohol.

[0108] In some embodiments, a non-aqueous solvent (e.g., alcohol) is added to precipitate the mRNA. In some embodiments, the solvent can be isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, and combinations thereof. In embodiments, the solvent is an alcohol solvent (e.g., methanol, ethanol, or isopropanol). In embodiments, the solvent is a ketone solvent (e.g., acetone, methyl ethyl ketone, or methyl isobutyl ketone). In some embodiments, the non-aqueous solvent is mixed with the amphipathic solution.

[0109] In some embodiments, an aqueous solution is added to precipitate the mRNA. In some embodiments, the aqueous solution comprises a polymer. In some embodiments, the aqueous solution comprises a PEG polymer.

[0110] In some embodiments, the method further comprises adding one or more agents that denature the protein and / or maintain the protein soluble in the aqueous medium (e.g., RNA polymerase and DNase I added after transcription to remove the DNA template). In some embodiments, the one or more agents that denature the protein and / or maintain the protein soluble in the aqueous medium are salts, e.g., chaotropic salts.

[0111] In some embodiments, the precipitation step involves the use of a chaotropic salt (e.g., guanidine thiocyanate) and / or an amphiphilic polymer (e.g., polyethylene glycol or an aqueous solution of polyethylene glycol) and / or an alcoholic solvent (e.g., an aqueous solution of an alcohol, such as absolute ethanol or an aqueous ethanol solution). Thus, in some embodiments, the precipitation step involves the use of a chaotropic salt and an amphiphilic polymer, such as GSCN and PEG, respectively.

[0112] In some embodiments, the agent that promotes mRNA precipitation includes a denaturant or results from denaturing conditions. As used herein, the term "denaturing conditions" refers to any chemical or physical condition that can cause denaturation. Exemplary denaturing conditions include, but are not limited to, the use of chemical reagents, high temperatures, extreme pH, etc. In some embodiments, the denaturing conditions are achieved by adding one or more denaturants to an impure preparation containing the mRNA to be purified. In some embodiments, denaturants suitable for the present invention are protein and / or DNA denaturants. In some embodiments, Denaturing agents can be 1) enzymes (such as serine proteinases or DNases), 2) acids, 3) solvents, 4) cross-linking agents, 5) chaotropic agents, 6) reducing agents, and / or 7) high ionic strength via high salt concentrations. In some embodiments, a particular agent may fall into one or more of these categories.

[0113] nucleotide In some embodiments, the mRNA comprises naturally occurring nucleosides (or unmodified nucleosides, i.e., adenosine, guanosine, cytidine, and uridine) or consists of naturally occurring nucleosides. In some embodiments, the mRNA comprises one or more modified nucleosides (e.g., an adenosine analog, a guanosine analog, a cytidine analog, or a uridine analog). In some embodiments, the mRNA comprises both unmodified and modified nucleosides. In some embodiments, the one or more modified nucleosides are nucleoside analogs. In some embodiments, the one or more modified nucleosides comprise at least one modification selected from a modified sugar and a modified nucleobase. In some embodiments, the mRNA comprises one or more modified nucleotides.

[0114] In some embodiments, the one or more modified nucleosides are one of a nucleoside analog, e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine. For example, see U.S. Patent No. 8,278,036 or WO2011 / 012316 for a discussion of 5-methyl-cytidine, pseudouridine, and 2-thio-uridine and their incorporation into mRNA. In some embodiments, the mRNA can be RNA, in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings on the use of such modified RNA are disclosed in U.S. Patent Application Publication No. 2012 / 0195936 and International Publication No. 2011 / 012316, both of which are incorporated herein by reference in their entirety. In some embodiments, the presence of one or more nucleoside analogs can make the mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only naturally occurring nucleosides.

[0115] In some embodiments, one or more modified nucleosides comprise a modified nucleobase, e.g., a chemically modified base, a biologically modified base (e.g., a methylated base), or an intermediate base. In some embodiments, one or more modified nucleosides include, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5- Uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl- and modified nucleobases selected from modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), such as uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, beta-D-mannosyl-queosine, wybutoxosine, and phosphoramidites, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, inosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, diaminopurine, and 2-chloro-6-aminopurine cytosine. For example, the preparation of such modified nucleobases is known to those skilled in the art from, e.g., U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0116] In some embodiments, the mRNA contains one or more modified nucleotides. For example, one or more of the modified nucleotides used to produce the mRNA of the present invention may contain a modified phosphate group. Thus, in the mRNA, one or more phosphodiester bonds are replaced with another anionic, cationic, or neutral group. For example, in some embodiments, one or more modified nucleotides contain a modified phosphate group selected from methyl phosphonate, methyl phosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, and a positively charged guanidinium group. In some embodiments, one or more modified internucleoside linkages are phosphorothioate linkages. In some embodiments, one or more modified internucleoside linkages are 5'-N-phosphoramidite linkages.

[0117] In some embodiments, one or more modified nucleosides comprise a modified sugar. In some embodiments, one or more modified nucleosides comprise a modification to the furanose ring. In some embodiments, one or more modified nucleosides are selected from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligoribonucleotides, The modified nucleosides include modified sugars selected from 2'-deoxy-2'-C-alkyl oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-arauidine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate). In some embodiments, one or more modified nucleosides include modified sugars selected from 2'-O-alkyl modifications or locked nucleic acids (LNAs). In some embodiments, the sugar modification is a 2'-O-alkyl modification, such modifications can include, but are not limited to, 2'-deoxy-2'-fluoro modifications, 2'-O-methyl modifications, 2'-O-methoxyethyl modifications, and 2'-deoxy modifications. In some embodiments, the one or more modified nucleosides comprise a modified sugar selected from 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose.

[0118] In some embodiments, any of these modifications occurs in 0-100% of the nucleotides, e.g., 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 100%, 120%, 140%, 160%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280 There may be more than 90%, 95%, or 100% individually or in combination.

[0119] In some embodiments, the RNA may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs).

[0120] Purified mRNA product The method of capping and tailing in vitro transcribed purified mRNA according to the present invention results in high RNA integrity and capping / tailing efficiency. The purified capped mRNA produced according to the present invention is substantially free of contaminants, including short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvents, and / or residual salts.

[0121] The mRNA prepared according to the present invention encodes a protein or peptide. The mRNA prepared according to the present invention can encode any gene of interest, for example, as listed in published U.S. Patent Application No. 2017 / 0314041, the entire contents of which are incorporated herein by reference. In some embodiments, the mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR). In some embodiments, the mRNA encodes human phenylalanine hydroxylase (hPAH). In several embodiments, the mRNA encodes ornithine transcarbamylase (OTC) protein.

[0122] RNA Integrity: In some embodiments, assessment of mRNA purity includes assessment of mRNA integrity, capping and tailing efficiency, tri-tail length, assessment of residual plasmid DNA, and assessment of residual solvent.

[0123] In some embodiments, mRNA products capped and tailed by this method are significantly more uniform and homogeneous in full-length mRNA molecules than mRNA products capped and tailed by conventional methods, which have a more heterogeneous profile characterized by the presence of lower molecular weight pre-cleaved transcripts, as characterized by glyoxal agarose gel electrophoresis or capillary electrophoresis after capping and tailing. In particular, capping and tailing mRNA in reaction conditions containing Tris-HCl pH 7.5 buffer and 1.0 mM MgCl2 resulted in at least 70% RNA integrity. This unique and advantageous capping and tailing reaction condition was not understood prior to the present invention and is truly unexpected, especially since optimized capping and tailing conditions can increase RNA integrity by at least about 25%. Based on this unexpected discovery, the inventors have successfully developed a large-scale production method for preparing mRNA molecules with high RNA integrity suitable for mRNA therapeutics.

[0124] In various embodiments, the purified mRNA of the present invention maintains a high degree of integrity. As used herein, the term "mRNA integrity" generally refers to the quality of mRNA after purification. mRNA integrity can be determined by methods well known in the art, such as RNA agarose gel electrophoresis. In some embodiments, mRNA integrity can be determined by the band pattern of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present invention shows little or no banding compared to the reference band of RNA agarose gel electrophoresis.

[0125] In some embodiments, an acceptable level of mRNA integrity is determined by agarose gel electrophoresis. The purity of the purified mRNA is assessed by capillary gel electrophoresis. The gel is analyzed to determine whether the band pattern and apparent nucleotide length match those of analytical reference standards. Additional methods for assessing RNA integrity include, for example, evaluating purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, acceptable purity of purified mRNA as determined by CGE is that the purified mRNA composition has about 55% or less long abortive / degraded species.

[0126] In some embodiments, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the mRNA products are full length. In some embodiments, the mRNA products are substantially full length.

[0127] In some embodiments, the mRNA composition contains less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% abortive transcripts. In some embodiments, the mRNA compositions according to the present invention are substantially free of abortive transcripts.

[0128] In some embodiments, full-length or abortive transcripts of mRNA are detected by gel electrophoresis (e.g., agarose gel electrophoresis), where the mRNA is denatured with glyoxal before agarose gel electrophoresis (glyoxal agarose gel electrophoresis). The mRNA synthesized according to the methods of the present invention contains undetectable amounts of abortive transcripts on glyoxal agarose gel electrophoresis.

[0129] In some embodiments, full-length or aborted transcripts of mRNA are detected by capillary electrophoresis, for example, capillary electrophoresis coupled with fluorescence-based detection or capillary electrophoresis coupled with UV absorption spectroscopy detection. When detection is by capillary electrophoresis coupled with fluorescence-based detection or capillary electrophoresis coupled with UV absorption spectroscopy, the relative amounts of full-length or aborted transcripts of synthetic mRNA are determined by the relative peak areas corresponding to the full-length or aborted transcripts.

[0130] Full-length or abortive transcripts of mRNA may be detected prior to capping and / or tailing the synthetic mRNA.

[0131] In some embodiments, the method further comprises capping and / or tailing the synthetic mRNA. Full-length or abortive transcripts of the mRNA may be detected after capping and / or tailing the synthetic mRNA.

[0132] In some embodiments, the full-length mRNA molecule is at least 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 8 kb, 10 kb, 12 kb, 14 kb, 15 kb, 18 kb, or 20 kb in length.

[0133] In some embodiments, at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 150 g, 200 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more of mRNA is synthesized and purified in a single batch.

[0134] In some embodiments, purified mRNA is evaluated for one or more of appearance, identity, quantity, concentration, presence of impurities, microbiological evaluation, pH level, and activity. In some embodiments, acceptable appearance includes a clear, colorless solution essentially free of visible particles. In some embodiments, mRNA identity is evaluated by sequencing methods. In some embodiments, concentration is evaluated by a suitable method, such as UV spectrophotometry. In some embodiments, a suitable concentration is approximately 90%-110% nominal (0.9-1.1 mg / mL).

[0135] In some embodiments, assessing mRNA purity includes assessing mRNA integrity, residual plasmid DNA, and residual solvents. In some embodiments, an acceptable level of mRNA integrity is assessed by agarose gel electrophoresis. The gel is analyzed to determine whether the band pattern and apparent nucleotide length match an analytical reference standard. Additional methods for assessing RNA integrity include, for example, evaluating purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, acceptable purity of purified mRNA as determined by CGE is that the purified mRNA composition has about 70% or less long abortive / degraded species. In some embodiments, residual plasmid DNA is assessed by methods known in the art, such as using qPCR. In some embodiments, less than 10 pg / mg (e.g., less than 10 pg / mg, less than 9 pg / mg, less than 8 pg / mg, less than 7 pg / mg, less than 6 pg / mg, less than 5 pg / mg, less than 4 pg / mg, less than 3 pg / mg, less than 2 pg / mg, or less than 1 pg / mg) is an acceptable level of residual plasmid DNA. In some embodiments, acceptable residual solvent levels are 10,000 ppm or less, 9,000 ppm or less, 8,000 ppm or less, 7,000 ppm or less, 6,000 ppm or less, 5,000 ppm or less, 4,000 ppm or less, 3,000 ppm or less, 2,000 ppm or less, or 1,000 ppm or less. Thus, in some embodiments, acceptable residual solvent levels are 10,000 ppm or less. In some embodiments, acceptable residual solvent levels are 9,000 ppm or less. In some embodiments, the acceptable residual solvent level is 8,000 ppm or less. In some embodiments, the acceptable residual solvent level is 7,000 ppm or less. In some embodiments, the acceptable residual solvent level is 6,000 ppm or less. In some embodiments, the acceptable residual solvent level is 5,000 ppm or less. In some embodiments, the acceptable residual solvent level is 4,000 ppm or less.In some embodiments, the acceptable residual solvent level is 3,000 ppm or less. In some embodiments, the acceptable residual solvent level is 2,000 ppm or less. In some embodiments, the acceptable residual solvent level is 1,000 ppm or less.

[0136] In some embodiments, microbiological testing is performed on the purified mRNA, including, for example, evaluation of bacterial endotoxin. In some embodiments, the bacterial endotoxin is <0.5 EU / mL, <0.4 EU / mL, <0.3 EU / mL, <0.2 EU / mL, or <0.1 EU / mL. Thus, in some embodiments, the bacterial endotoxin in the purified mRNA is <0.5 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.4 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.3 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.1 EU / mL. In some embodiments, the purified mRNA has a bacterial endotoxin content of 1 CFU / 10 mL or less, 1 CFU / 25 mL or less, The mRNA concentration is 1 CFU / 50 mL or less, 1 CFU / 75 mL or less, or 1 CFU / 100 mL or less. Thus, in some embodiments, the purified mRNA is 1 CFU / 10 mL or less. In some embodiments, the purified mRNA is 1 CFU / 25 mL or less. In some embodiments, the purified mRNA is 1 CFU / 50 mL or less. In some embodiments, the purified mRNA is 1 CFU / 75 mL or less. In some embodiments, the purified mRNA has 1 CFU / 100 mL.

[0137] In some embodiments, the pH of the purified mRNA is evaluated. In some embodiments, an acceptable pH for purified mRNA is between 5 and 8. Thus, in some embodiments, the purified mRNA has a pH of about 5. In some embodiments, the purified mRNA has a pH of about 6. In some embodiments, the purified mRNA has a pH of about 7. In some embodiments, the purified mRNA has a pH of about 7. In some embodiments, the purified mRNA has a pH of about 8.

[0138] In some embodiments, the translation fidelity of purified mRNA is evaluated.Translation fidelity can be evaluated by various methods, for example, includes transfection and Western blot analysis.Acceptable characteristics of purified mRNA include the band pattern on Western blot that migrates at a similar molecular weight to that of reference standard.

[0139] In some embodiments, the purified mRNA is assessed for conductance. In some embodiments, acceptable characteristics of the purified mRNA include a conductance of about 50% to 150% of the reference standard.

[0140] The purified mRNA is also evaluated for cap percentage and poly-A tail length. In some embodiments, acceptable cap percentages include Cap1, % Area: NLT90. In some embodiments, acceptable poly-A tail lengths are approximately 100-1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides).

[0141] In some embodiments, the purified mRNA is also assessed for any residual PEG. In some embodiments, the purified mRNA has less than 10 ng PEG / mg purified mRNA to less than 1000 ng PEG / mg mRNA. Thus, in some embodiments, the purified mRNA has less than about 10 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 100 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 250 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 500 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 750 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 1000 ng PEG / mg purified mRNA.

[0142] Various methods for detecting and quantifying mRNA purity are known in the art. For example, such methods include blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. In some embodiments, the mRNA is first denatured with glyoxal dye before gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, the synthetic mRNA is characterized before capping or tailing. In some embodiments, the synthetic mRNA is characterized after capping and tailing. Be appreciated.

[0143] Capping and tailing efficiency The purified mRNA is also evaluated for cap percentage and poly-A tail length. In some embodiments, acceptable cap percentages include Cap1, % area: NLT90. Various methods known in the art can be used to evaluate capping and tailing efficiency and tail length. In some embodiments, capping efficiency is evaluated by UPLC-MS cap assay. In some embodiments, tailing efficiency is evaluated by capillary electrophoresis (CE) shift. In some embodiments, RNA tail length is evaluated by CE shift. In some embodiments, RNA tail length is evaluated by agarose gel electrophoresis.

[0144] In some embodiments, acceptable poly-A tail lengths are between about 100 and 1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides). Thus, in some embodiments, an acceptable poly-A tail length is about 100 nucleotides. In some embodiments, the poly-A tail length is about 200 nucleotides. In some embodiments, the poly-A tail length is about 250 nucleotides. In some embodiments, the poly-A tail length is about 300 nucleotides. In some embodiments, the poly-A tail length is about 350 nucleotides. In some embodiments, the poly-A tail length is about 400 nucleotides. In some embodiments, the poly-A tail length is about 450 nucleotides. In some embodiments, the poly-A tail length is about 500 nucleotides. In some embodiments, the poly-A tail length is about 550 nucleotides. In some embodiments, the poly-A tail length is about 600 nucleotides. In some embodiments, the poly-A tail length is about 650 nucleotides. In some embodiments, the poly-A tail length is about 700 nucleotides. In some embodiments, the poly-A tail length is about 750 nucleotides. In some embodiments, the poly-A tail length is about 800 nucleotides. In some embodiments, the poly-A tail length is about 850 nucleotides. In some embodiments, the poly-A tail length is about 900 nucleotides. In some embodiments, the poly-A tail length is about 950 nucleotides. In some embodiments, the poly-A tail length is about 1000 nucleotides. In some embodiments, the poly-A tail length is about 1100 nucleotides. In some embodiments, the poly-A tail length is about 1200 nucleotides. In some embodiments, the poly-A tail length is about 1300 nucleotides. In some embodiments, the polyA tail length is about 1400 nucleotides. In some embodiments, the polyA tail length is about 1500 nucleotides.

[0145] scale A particular advantage provided by the present invention is the ability to purify mRNA, particularly in vitro synthesized mRNA, on a large or commercial scale. For example, in some embodiments, in vitro synthesized mRNA is prepared at a scale of about 100 milligrams, 1 gram, 10 grams, 50 grams, 150 grams, 100 grams, 150 grams, 200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 450 grams, 500 grams, 550 grams, 600 grams, 650 grams, 700 grams, 750 grams, 800 grams, 850 grams, 900 grams, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1 metric ton, 10 metric ton or more per batch. In embodiments, in vitro synthesized mRNA is prepared at a scale of about 1 kg or more.

[0146] In one specific embodiment, in vitro synthesized mRNA is prepared at a 10 gram scale per batch. In one specific embodiment, in vitro synthesized mRNA is prepared at a 20 gram scale per batch. In one specific embodiment, in vitro synthesized mRNA is prepared at a 25 gram scale per batch. In one specific embodiment, in vitro synthesized mRNA is prepared at a 50 gram scale per batch. In another specific embodiment, in vitro synthesized mRNA is prepared at a 100 gram scale per batch. In another specific embodiment, in vitro synthesized mRNA is prepared at a 250 gram scale per batch. In yet another specific embodiment, in vitro synthesized mRNA is prepared at a 1 kg scale per batch. In yet another specific embodiment, in vitro synthesized mRNA is prepared at a 10 kg scale per batch. In yet another specific embodiment, in vitro synthesized mRNA is prepared at a 100 kg scale per batch. In yet another specific embodiment, the in vitro synthesized mRNA is prepared at a scale of 1,000 kg per batch. In yet another specific embodiment, the in vitro synthesized mRNA is prepared at a scale of 10,000 kg per batch.

[0147] In some embodiments, mRNA is prepared at a scale of 1 gram, 5 gram, 10 gram, 15 gram, 20 gram, 25 gram, 30 gram, 35 gram, 40 gram, 45 gram, 50 gram, 75 gram, 100 gram, 150 gram, 200 gram, 250 gram, 300 gram, 350 gram, 400 gram, 450 gram, 500 gram, 550 gram, 600 gram, 650 gram, 700 gram, 750 gram, 800 gram, 850 gram, 900 gram, 950 gram, 1 kg, 2.5 kg, 5 kg, 7.5 kg, 10 kg, 25 kg, 50 kg, 75 kg, 100 kg or more per batch.

[0148] In some embodiments, the solution containing mRNA contains at least 1 gram, 10 grams, 100 grams, 1 kilogram, 10 kilograms, 100 kilograms, 1 metric ton, 10 metric ton, or more of mRNA, or any amount therebetween. In some embodiments, the methods described herein are used to prepare mRNA in an amount that is at least about 250 mg of mRNA. In one embodiment, the methods described herein are used to prepare mRNA in an amount that is at least about 250 mg of mRNA, about 500 mg of mRNA, about 750 mg of mRNA, about 1000 mg of mRNA, about 1500 mg of mRNA, about 2000 mg of mRNA, or about 2500 mg of mRNA. In embodiments, the methods described herein are used to prepare mRNA in an amount that is at least about 250 mg of mRNA to about 500 g of mRNA. In embodiments, the methods described herein are used to prepare amounts of mRNA that are at least about 500 mg to about 250 g of mRNA, about 500 mg to about 100 g of mRNA, about 500 mg to about 50 g of mRNA, about 500 mg to about 25 g of mRNA, about 500 mg to about 10 g of mRNA, or about 500 mg to about 5 g of mRNA. In embodiments, the methods described herein are used to prepare amounts of mRNA that are at least about 100 mg to about 10 g of mRNA, about 100 mg to about 5 g of mRNA, or about 100 mg to about 1 g of mRNA.

[0149] yield In some embodiments, the methods described herein provide a recovery (or yield) of purified mRNA that is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. Thus, in some embodiments, the recovery of purified mRNA is about 40%. In some embodiments, the recovery of purified mRNA is about 40%. RNA recovery is about 45%. In some embodiments, purified mRNA recovery is about 50%. In some embodiments, purified mRNA recovery is about 55%. In some embodiments, purified mRNA recovery is about 60%. In some embodiments, purified mRNA recovery is about 65%. In some embodiments, purified mRNA recovery is about 70%. In some embodiments, purified mRNA recovery is about 75%. In some embodiments, purified mRNA recovery is about 75%. In some embodiments, purified mRNA recovery is about 80%. In some embodiments, purified mRNA recovery is about 85%. In some embodiments, purified mRNA recovery is about 90%. In some embodiments, purified mRNA recovery is about 91%. In some embodiments, purified mRNA recovery is about 92%. In some embodiments, purified mRNA recovery is about 93%. In some embodiments, the recovery of purified mRNA is about 94%. In some embodiments, the recovery of purified mRNA is about 95%. In some embodiments, the recovery of purified mRNA is about 96%. In some embodiments, the recovery of purified mRNA is about 97%. In some embodiments, the recovery of purified mRNA is about 98%. In some embodiments, the recovery of purified mRNA is about 99%. In some embodiments, the recovery of purified mRNA is about 100%.

[0150] purity The mRNA compositions described herein are substantially free of contaminants, including short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptases, residual solvents, and / or residual salts.

[0151] The mRNA compositions described herein have a purity of about 60% to about 100%. Thus, in some embodiments, the purified mRNA has a purity of about 60%. In some embodiments, the purified mRNA has a purity of about 65%. In some embodiments, the purified mRNA has a purity of about 70%. In some embodiments, the purified mRNA has a purity of about 75%. In some embodiments, the purified mRNA has a purity of about 80%. In some embodiments, the purified mRNA has a purity of about 85%. In some embodiments, the purified mRNA has a purity of about 90%. In some embodiments, the purified mRNA has a purity of about 91%. In some embodiments, the purified mRNA has a purity of about 92%. In some embodiments, the purified mRNA has a purity of about 93%. In some embodiments, the purified mRNA has a purity of about 94%. In some embodiments, the purified mRNA has a purity of about 95%. In some embodiments, the purified mRNA has a purity of about 96%. In some embodiments, the purified mRNA has a purity of about 97%. In some embodiments, the purified mRNA has a purity of about 98%. In some embodiments, the purified mRNA has a purity of about 99%. In some embodiments, the purified mRNA has a purity of about 100%.

[0152] In some embodiments, the mRNA compositions described herein have less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, and / or 0.1% impurities other than full-length mRNA. Impurities include IVT contaminants such as proteins, enzymes, DNA templates, free nucleotides, residual solvents, residual salts, double-stranded RNA (dsRNA), premature termination RNA sequences ("shortmers" or short abortive RNA species), and / or long abortive RNA species. In some embodiments, the purified mRNA is substantially free of processing enzymes.

[0153] In some embodiments, the residual plasmid DNA in the purified mRNA of the present invention is less than about 1 pg / mg, less than about 2 pg / mg, less than about 3 pg / mg, less than about 4 pg / mg, less than about 5 pg / mg, less than about 6 pg / mg, less than about 7 pg / mg, less than about 8 pg / mg, less than about 9 pg / mg, less than about 10 pg / mg, less than about 11 pg / mg, or less than about 12 pg / mg. Thus, the residual plasmid DNA in the purified mRNA is less than about 1 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 2 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 3 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 4 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 5 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 6 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 7 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 8 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 9 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 10 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 11 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 12 pg / mg.

[0154] In some embodiments, the methods of the present invention remove more than about 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or substantially all premature termination RNA sequences. In some embodiments, the mRNA composition is substantially free of premature termination RNA sequences. In some embodiments, the mRNA composition contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) premature termination RNA sequences. In some embodiments, the mRNA composition contains less than about 1% (e.g., less than about 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) premature termination RNA sequences. In some embodiments, the mRNA composition contains undetectable premature abort RNA sequences, as determined, for example, by high-performance liquid chromatography (HPLC) (e.g., a shoulder or separate peak), ethidium bromide, Coomassie staining, capillary electrophoresis, or glyoxal gel electrophoresis (e.g., the presence of a distinct lower band). As used herein, the terms "shortmer," "short abortive RNA species," "prematurely abortive RNA sequences," or "long abortive RNA species" refer to any transcript that is shorter than full length. In some embodiments, a "shortmer," "short abortive RNA species," or "prematurely abortive RNA sequence" is less than 100 nucleotides in length, less than 90 nucleotides in length, less than 80 nucleotides in length, less than 70 nucleotides in length, less than 60 nucleotides in length, less than 50 nucleotides in length, less than 40 nucleotides in length, less than 30 nucleotides in length, less than 20 nucleotides in length, or less than 10 nucleotides in length. In some embodiments, shortmers are detected or quantified after the addition of a 5' cap and / or a 3' polyA tail.In some embodiments, the prematurely terminated RNA transcript is comprised of fewer than 15 bases (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or fewer than 3 bases). In some embodiments, the prematurely terminated RNA transcript contains about 8-15 bases, 8-14 bases, 8-13 bases, 8-12 bases, 8-11 bases, or 8-10 bases.

[0155] In some embodiments, the purified mRNA of the present invention is purified using T7 RNA polymerase. The purified mRNA is substantially free of enzymatic reagents used in in vitro synthesis, including, but not limited to, DNAse I, pyrophosphatase, and / or RNAse inhibitors. In some embodiments, purified mRNA according to the present invention contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of the enzymatic reagents used in in vitro synthesis. In some embodiments, purified mRNA contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the enzymatic reagents used in in vitro synthesis. In some embodiments, purified mRNA contains undetectable enzymatic reagents used in in vitro synthesis, including, for example, as determined by silver staining, gel electrophoresis, high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), and / or capillary electrophoresis, ethidium bromide and / or Coomassie staining.

[0156] Therapeutic Uses of the Composition The mRNA prepared according to the method of the present invention can be used as a pharmaceutical for therapeutic applications. In particular, the mRNA prepared according to the method of the present invention can be delivered to a subject requiring in vivo protein production. To promote in vivo mRNA expression, a delivery vehicle such as a liposome may be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or may be formulated into a pharmacological composition mixed with a suitable excipient. Techniques for formulation and drug administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. (latest edition).

[0157] In some embodiments, the composition comprises mRNA encapsulated or complexed with a delivery vehicle, hi some embodiments, the delivery vehicle is selected from the group consisting of a liposome, a lipid nanoparticle, a solid lipid nanoparticle, a polymer, a virus, a sol-gel, and a nanogel.

[0158] In some embodiments, a suitable delivery vehicle is a liposome delivery vehicle, such as a lipid nanoparticle. As used herein, a liposome delivery vehicle, such as a lipid nanoparticle, is generally characterized as a microvesicle having an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of a liposome is typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of a liposome may also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, a liposome delivery vehicle typically serves to transport a desired nucleic acid (e.g., mRNA or MCNA) to a target cell or tissue.

[0159] In some embodiments, the nanoparticle delivery vehicle is a liposome. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids. A typical liposome for use in the present invention is composed of four lipid components: a cationic lipid, a non-cationic lipid (e.g., DOPE or DEPE), a cholesterol-based lipid (e.g., cholesterol), and a PEG-modified lipid (e.g., DMG-PEG2K). In some embodiments, the liposome comprises three or fewer distinct lipid components. In some embodiments, one distinct lipid component is a sterol-based cationic lipid. An exemplary liposome is composed of three lipid components: a sterol-based cationic lipid, a non-cationic lipid (e.g., DOPE or DEPE), and a PEG-modified lipid (e.g., DMG-PEG2K).

[0160] Various methods for encapsulating mRNA are described in published U.S. patent application Ser. No. 2011 / 0244026, published U.S. patent application Ser. No. 2016 / 0038432, published U.S. patent application Ser. No. 2018 / 0153822, published U.S. patent application Ser. No. 2018 / 0125989, and U.S. provisional patent application Ser. No. 62 / 877,597, filed July 23, 2019, and can be used to practice the present invention, all of which are incorporated herein by reference. [Example]

[0161] Example 1. Synthesis and analysis of capped and tailed mRNA In vitro transcription mRNA synthesis In the following examples, unless otherwise noted, mRNA was synthesized via in vitro transcription (IVT) using either T7 polymerase or SP6 polymerase. Any method of IVT synthesis known in the art can be used to practice the present invention. In vitro transcribed mRNA was purified and concentrated via ultrafiltration / diafiltration (UFDF) prior to the cap / tail reaction.

[0162] The purified mRNA product from the in vitro transcription step described above was capped with Cap1 and tailed. The reaction mixture was treated with a portion of GTP (1.0 mM), S-adenosylmethionine, RNAse inhibitor, 2'-O-methyltransferase, and guanylyltransferase, and mixed with reaction buffer (10x, 500 mM Tris-HCl (pH 8.0 or pH 7.5), 60 mM KCl, 12.5 or 10.0 mM MgCl). The combined solution was incubated at 37°C for a set time ranging from 30 to 90 minutes. Upon completion, ATP (2.0 mM), polyA polymerase, and an aliquot of tailing reaction buffer were added, and the total reaction mixture was further incubated at 37°C for a time ranging from 20 to 45 minutes. Upon completion, the final reaction mixture was quenched and purified accordingly.

[0163] RNA integrity analysis (fragment analyzer-capillary electrophoresis). RNA integrity and tail length were assessed using a CE fragment analyzer and a commercially available RNA detection kit. Analysis of the peak profile of integrity and size shift of tail length was performed on raw data as well as normalized data sets.

[0164] mRNA cap species analysis (HPLC / MS) The cap species present in the final purified mRNA product were quantified using the chromatographic method described in U.S. Patent No. 9,970,047. This method can accurately quantify uncapped mRNA as a percentage of total mRNA. This method can also quantify the amount of specific cap structures, such as the amount of CapG, Cap0, and Cap1, which can be reported as a percentage of total mRNA.

[0165] Example 2. Optimizing cap and tail reaction conditions increases CFTR mRNA integrity This example demonstrates that the capping and tailing reaction conditions of the present invention result in increased mRNA integrity suitable for therapeutic use. The increase in mRNA integrity was independent of the size or nucleotide composition of the mRNA construct.

[0166] CFTR mRNA (approximately 4,600 nt) and DNAH5 mRNA (approximately 14,000 nt) were synthesized via IVT synthesis and purification as described in Example 1. Prior to the capping and tailing reaction, the purified mRNA was analyzed using CE. Five mg batches of purified and concentrated IVT mRNA were then analyzed under two different conditions, as shown in Table 1. The capped and tailed mRNA was capped and tailed via an enzymatic process at 20°C. Other than the concentration of MgCl and pH, the remaining reaction condition variables remained the same. The integrity and polyA tail length of the purified capped and tailed mRNA were assessed by CE as described in Example 1. [Table 1]

[0167] For both CFTR and DNAH5 mRNA, the optimized capping and tailing reaction conditions resulted in increased mRNA integrity compared to the control. As shown in Figure 1, the final product of capping / tailing CFTR mRNA under the optimized conditions (Sample B) has a clearly defined peak at a tail length within the target range. Sample B, capped and tailed under reaction conditions containing 1.0 mM MgCl2 and 50 mM Tris at pH 7.5, contained substantially no "shoulder" (indicated by the arrow in Figure 1). Similarly, Figure 2 shows that the final product of Sample D has a clearly defined peak at a tail length within the target range. Notably, DNAH5 mRNA capped and tailed under reaction conditions containing 1.0 mM MgCl2 and 50 mM Tris at pH 7.5 exhibited a more intense and sharp peak corresponding to the full-length product and contained substantially no "shoulder." The results demonstrated that the optimized capping and tailing reaction conditions of the present invention resulted in increased RNA integrity regardless of the construct size or nucleotide composition.

[0168] Example 3. Optimized Capping and Tail Reactions at 1 Gram and 15 Gram Scales This example demonstrates that the optimized capping and tailing reaction conditions of the present invention can be used to cap and tail mRNA at the scale and quality required for therapeutic applications. mRNA purified at 1 gram and 15 gram scales according to the methods described herein resulted in high RNA integrity, capping and tailing efficiency, and desired tail length, demonstrating the scalability of the method.

[0169] One batch of CFTR mRNA was synthesized at a 1 gram scale, and two batches of CFTR mRNA were synthesized at a 15 gram scale via IVT synthesis as described in Example 1. The resulting 1 gram IVT mRNA sample was then capped and tailed via an enzymatic process under reaction conditions including 50 mM Tris pH 7.5 and 1.0 mM MgCl. For the 15 gram scale, 50 mM Tris pH 8.0 and 1.25 mM MgCl (conventional conditions). Alternatively, capping and tailing reactions were performed under reaction conditions containing 50 mM Tris pH 7.5 and 1.0 mM MgCl (optimized conditions). The integrity, tailing efficiency, and polyA tail length of the purified capped and tailed mRNA were assessed by CE. Capping efficiency was also assessed by UPLC-MS as described in Example 1. [Table 2]

[0170] As shown in Table 2, optimized capping and tailing reaction conditions containing Tris pH 7.5 and 1.0 mM MgCl2 resulted in increased CFTR mRNA integrity and high capping and tailing efficiency at the 1 gram scale. Figure 3 shows that the final capped / tailed CFTR mRNA product had a well-defined, sharp peak and was substantially free of a "shoulder," corresponding to a full-length product with a tail length within the target range. [Table 3]

[0171] Figure 4 shows that the final CFTR mRNA product has a clearly defined peak with a tail length within the target range at the 15-gram scale. Notably, CFTR mRNA capped and tailed under reaction conditions containing 1.0 mM MgCl and 50 mM Tris at pH 7.5 (optimized conditions) displayed a more intense and sharper peak corresponding to the full-length product, without the "shoulder" that was still present in CFTR mRNA capped and tailed under historical conditions (1.25 mM MgCl at pH 8.0). Analysis also shows that, as shown in Table 3, the optimized cap and tail reaction conditions containing Tris pH 7.5 and 1.0 mM MgCl resulted in increased CFTR mRNA integrity and high capping and tailing efficiency at the 15-gram scale. Notably, RNA integrity was greater than 70% as measured by CE smear or CGE smear. A polyA tail length of 387 nt was observed, which was within the target range of 500 nt. The optimized reaction conditions also resulted in a tailing efficiency of over 75% and a capping efficiency of 100%. Furthermore, the capping reaction yielded the desired cap species, 100% Cap1 (Table 4). [Table 4]

[0172] Together, the data demonstrate the scalability of the optimized capping and tailing reaction conditions for mRNA preparation at the scale and quality required for clinical therapeutic applications. mRNA capped and tailed at 1 and 15 gram scales by the methods described herein resulted in high mRNA integrity while maintaining all other important quality attributes, demonstrating the method's use in mRNA therapeutics.

[0173] Example 4. Optimized Capping and Tail Reaction at 100 Gram Manufacturing Scale This example demonstrates that mRNA can be capped and tailed at a manufacturing scale with high RNA integrity using the optimized capping and tailing reaction conditions of the present invention. mRNA purified at 1 gram and 15 gram scales according to the methods described herein resulted in high integrity, capping and tailing efficiency, and desired tail length, demonstrating the scalability of the method.

[0174] Two batches of CFTR mRNA were synthesized on a 100-gram scale via IVT synthesis as described in Example 1. The resulting 100-gram IVT mRNA samples were then capped and tailed via an enzymatic process under reaction conditions including 50 mM Tris pH 8.0 and 1.25 mM MgCl2 or 50 mM Tris pH 7.5 and 1.0 mM MgCl2. The integrity, tailing efficiency, and polyA tail length of the purified capped and tailed mRNA were assessed by CE. Capping efficiency was also assessed by UPLC-MS as described in Example 1.

[0175] Figure 5 shows that the final CFTR mRNA product has a well-defined peak with a tail length within the target range at the 100 gram scale. Notably, CFTR mRNA capped and tailed under reaction conditions containing 1.0 mM MgCl2 and 50 mM Tris at pH 7.5 (optimized conditions) showed a more intense and sharper peak corresponding to the full-length product and did not contain a "shoulder," whereas a shoulder was still seen in CFTR mRNA capped and tailed under historical conditions (1.25 mM MgCl2 at pH 8.0). This indicates a significant reduction in degraded RNA species for the final mRNA product capped and tailed under the optimized reaction conditions.

[0176] Overall, the data demonstrate the scalability of the optimized capping and tailing reaction conditions for mRNA synthesis at a manufacturing scale, resulting in the high quality required for clinical therapeutic applications. The capping and tailing of mRNA at the 100 gram scale by the method described herein resulted in high mRNA integrity while maintaining all other important quality attributes, demonstrating the method's use in mRNA manufacturing and therapeutics.

[0177] Example 5. Optimized Capping and Tail Reaction at 250 Gram Manufacturing Scale This example demonstrates that the optimized capping and tailing reaction conditions of the present invention can be used to cap and tail mRNA at a manufacturing scale with high RNA integrity. 1, 15 gram, 100 gram, and 25 gram capping reactions can be performed according to the methods described herein. Purified mRNA at a 0 gram scale yielded high integrity, capping and tailing efficiency, and desired tail length, demonstrating the scalability of the method.

[0178] OTC mRNA was synthesized on a 250 gram scale via IVT synthesis as described in Example 1. The resulting 250 gram IVT mRNA sample was then diluted in 50 mM Tris The capping and tailing was performed via an enzymatic process under reaction conditions including pH 7.5 and 1.0 mM MgCl2. Another 10 gram IVT mRNA sample was synthesized via the IVT synthesis described in Example 1 and capped and tailed via an enzymatic process under reaction conditions including 50 mM Tris pH 8.0 and 1.25 mM MgCl2. The integrity, tailing efficiency, and polyA tail length of the purified capped and tailed mRNA were evaluated by CE. Capping efficiency was also evaluated by UPLC-MS as described in Example 1.

[0179] Figure 6 shows that the final OTC mRNA product has a well-defined peak with a tail length within the target range at the 250 gram scale. Notably, OTC mRNA capped and tailed under reaction conditions containing 1.0 mM MgCl2 and 50 mM Tris at pH 7.5 (optimized conditions) displayed a more intense and sharper peak corresponding to the full-length product and did not contain a "shoulder," whereas a shoulder was still seen in 10 gram OTC mRNA capped and tailed under historical conditions (1.25 mM MgCl2 at pH 8.0). These results demonstrated a significant reduction in degraded RNA species for the final mRNA product capped and tailed under optimized reaction conditions.

[0180] Overall, the data demonstrated the scalability of the optimized capping and tailing reaction conditions for mRNA synthesis at a manufacturing scale, with the high quality required for clinical therapeutic applications. The mRNA capped and tailed at a 250 gram scale by the method described herein resulted in high mRNA integrity while maintaining all other important quality attributes, demonstrating the method's use in mRNA manufacturing and therapeutics.

[0181] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method for capping and tailing a purified in vitro transcribed messenger RNA (mRNA) preparation, the method comprising: ２ and capping and tailing the mRNA in a reaction buffer having a pH below 8.

0.

2. The method of claim 1 , wherein the reaction buffer further comprises KCl.

3. The MgCl in the reaction buffer ２ 10. The method of any one of the preceding claims, wherein has a concentration of about 0.75 mM to 1.25 mM.

4. The MgCl in the reaction buffer ２ The method of claim 3 , wherein the β-glucan has a concentration of about 1.0 mM.

5. The MgCl in the reaction buffer ２ 10. The method of any one of the preceding claims, wherein said ATP has a concentration of about 1.0 mM.

6. 10. The method of any one of the preceding claims, wherein the pH of the reaction buffer is about 7.2 to 7.

7.

7. 10. The method of any one of the preceding claims, wherein the pH of the reaction buffer is about 7.

5.

8. 10. The method of any one of the preceding claims, wherein the mRNA is on the scale of 5 mg, 1 g, 15 g, 100 g, 250 g, 500 g, or 1 kg or more.

9. 9. The method of claim 8, wherein the mRNA is at a 100 g scale.

10. 10. The method of any one of the preceding claims, wherein tailing the mRNA comprises adding a poly-A tail having a length of about 250 to 750 nucleotides.

11. 11. The method of claim 10, wherein tailing the mRNA comprises adding a polyA tail having a length of about 500 nucleotides.

12. 12. The method of claim 10 or 11, wherein tailing the mRNA has an efficiency of about 70% to 95%.

13. 13. The method of claim 12, wherein tailing the mRNA has an efficiency of about 80%.

14. 10. The method of any one of the preceding claims, wherein capping the mRNA has an efficiency of 90% or more.

15. 10. The method of any one of the preceding claims, wherein capping the mRNA has an efficiency of about 100%.

16. Capping and tailing the mRNA in a reaction buffer having a pH below 8.0 results in capping and tailing with greater integrity compared to mRNA capped and tailed using a reaction buffer having a pH of 8.0 or greater.

10. The method of any one of the preceding claims, resulting in tailed mRNA.

17. ≤1.0 mM MgCl ２ capping and tailing the mRNA in a reaction buffer having a concentration greater than 1.0 mM MgCl ２ 10. The method of any one of the preceding claims, wherein a reaction buffer having a concentration results in capped and tailed mRNA with greater integrity compared to capped and tailed mRNA.

18. 18. The method of claim 16 or 17, wherein the integrity of the mRNA is at least 65% or greater.

19. 19. The method of claim 18, wherein the integrity of the mRNA is at least 75% or greater.

20. 20. The method of any one of claims 16 to 19, wherein the method has an mRNA capping efficiency of 80% or more.

21. 21. The method of claim 20, wherein the mRNA capping efficiency is about 90% or more.

22. 1. A method for capping and tailing a purified in vitro transcribed messenger RNA (mRNA) preparation, said method comprising capping and tailing a purified in vitro transcribed messenger RNA (mRNA) preparation at a pH of about 7.5 and about 1.0 mM MgCl ２ 10. The method of claim 1, further comprising capping and tailing the mRNA in a reaction buffer comprising a concentration of 0.1% or more of a nucleotide sequence, wherein the capping and tailing of the mRNA has a capping and tailing efficiency of 80% or more and the capped and tailed mRNA has an integrity of at least 65% or more.

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