WO2023118571A1

WO2023118571A1 - Methods and systems for capping nucleic acid

Info

Publication number: WO2023118571A1
Application number: PCT/EP2022/087737
Authority: WO
Inventors: Kristof VANDEKERCKHOVE; Grégory GODEFROI; Blandine DAVID
Original assignee: Quantoom Biosciences SA
Current assignee: Quantoom Biosciences SA
Priority date: 2021-12-23
Filing date: 2022-12-23
Publication date: 2023-06-29
Anticipated expiration: 2024-06-23

Abstract

Described herein are systems for capping nucleic acid. Also described herein are methods for capping nucleic acid with the systems described herein.

Description

METHODS AND SYSTEMS FOR CAPPING NUCLEIC ACID

BACKGROUND

In vitro nucleic acid processing is widely used in biomedical or bioscience fields. One of the in vitro nucleic acid processing or manufacturing methods involves capping of messenger RIMA (mRNA) for manufacturing of mRNA or peptide encoded by the mRNA in industrial quantities. Two main strategies are currently used for production of 5'- capped mRNA: co-transcriptional capping, whereby a synthetic oligonucleotide integrating the cap structure is incorporated during transcription of the template DNA strand; and post-transcriptional capping, whereby the biosynthesis of the cap structure and associated reactions is enzymatically catalyzed.

Co-transcriptional capping is described by Whitley et al., 2021. In post- transcriptional capping, the capping reaction is usually inhibited by the by-products of IVT and thus prior to the capping the mRNA is usually purified. WO201815714, and Fuchs et al., 2016, describe mRNA purification methods prior to capping. These purification steps are usually time-consuming and can result in loss of a significant part of the sample.

The current invention aims to develop a simplified post-transcriptional capping method, that overcomes at least some of the above mention drawbacks.

SUMMARY

Efficient post-transcriptional capping of mRNA requires prior treatment of the reaction harvest obtained after in vitro transcription (IVT). The traditional method of pretreatment involves at least one purification operation between the IVT step and the enzymatic capping step in order to ensure sufficient capping efficiency. Such steps increase process duration and complexity and decreases the overall capped RNA molecule yield. Accordingly, systems and methods for simple, inexpensive and fast treatment of mRNA reaction harvest to ensure efficient enzymatic capping downstream are of interest. Described herein, in some aspects, is a method for producing at least one capped ribonucleic acid (RNA) molecule, comprising : providing a plurality of uncapped RNA molecules in a first solution; diluting the first solution by at least 4-fold to form a second solution containing the plurality of uncapped RNA molecules; contacting the second solution with a plurality of capping enzyme molecules; and adding a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule. In some embodiments, diluting the first solution comprises adding a volume of a diluent to said first solution. In some embodiments, the first solution is diluted by between about 4 and 1000-fold. In some embodiments, the first solution is diluted by at least about 200-fold. In some embodiments, the first solution is diluted by at least about 50-fold. In some embodiments, the first solution is diluted by at least about 10-fold. In some embodiments, the method further comprises removing an excess volume of the second solution. In some embodiments, removing the excess volume comprises ultrafiltration, microfiltration, tangential flow filtration, or a combination thereof. In some embodiments, diluting the first solution does not comprise an additional purifying step. In some embodiments, the additional purifying step comprises a chromatography. In some embodiments, diluting the first solution occurs in a same vessel or reactor as the plurality of uncapped RNA molecules generated via an in vitro transcription (IVT) reaction. In some embodiments, diluting the first solution occurs in a different vessel or reactor as the plurality of uncapped RNA molecules generated via an IVT reaction. In some embodiments, the IVT reaction occurs in a continuous reactor or a batch reactor. In some embodiments, the plurality of capping enzyme is selected from the group consisting of Cap-specific mRNA (nucleoside-2'-O-)- methyltransferase, Vaccinia capping enzyme (VCE), Bluetongue Virus capping enzyme, Chlorella Virus capping enzyme, S. cerevisiae capping enzyme, Mimivirus capping enzyme, African swine fever virus capping enzyme, and Avian Reovirus capping enzyme. In some embodiments, adding the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 87%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. In some embodiments, adding the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of about 100%.

In some embodiments, the RNA comprises a nucleic acid sequence encoding a peptide or protein. In some embodiments, the RNA polymerase is selected from the group consisting of a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a RNA polymerase I, a RNA polymerase II, a RNA polymerase III, a RNA polymerase IV, a RNA polymerase V, and a single subunit RNA polymerase. In some embodiments, the method further comprises synthesizing a peptide or protein utilizing the at least one capped mRNA molecule. In some embodiments, diluting the first solution comprises decreasing a concentration of a plurality of molecules that inhibits a capping reaction of adding a capping structure to a 5' end of an uncapped RIMA molecule. In some embodiments, the concentration of the plurality of molecules is decreased to a level that the capping reaction is no longer inhibited. In some embodiments, diluting the first solution comprises decreasing a concentration of the plurality of uncapped RNA molecules. In some embodiments, the concentration of the plurality of uncapped RNA molecules is decreased to a level that the capping reaction is no longer inhibited.

Described herein, in some aspects, is a pharmaceutical composition obtained using a method described herein. In some embodiments, the pharmaceutical composition is a vaccine and a pharmaceutical acceptable carrier.

Described herein, in some aspects, is a peptide or protein obtained using a method described herein. In some embodiments, the peptide or protein is produced in vivo. In some embodiments, the peptide or protein is produced in vitro. In some embodiments, the peptide or protein is a prophylactic or a therapeutic peptide or protein.

Described herein, in some aspects, is a composition comprising a plurality of 5' capped RNA molecules, wherein said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping occurred post-transcriptionally, said composition comprises reagents for in vitro transcription, and wherein the concentration of RNA in said composition is less than 20 mg/ml.

Described herein, in some aspects, is a composition comprising a plurality of 5'- capped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping reaction occurred post- transcriptionally, wherein the capping reaction efficiency is at least 75% without utilizing chromatography.

Described herein, in some aspects, is a composition comprising a plurality of 5'- capped and uncapped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping occurred post- transcriptionally, said composition comprises reagents for in vitro transcription wherein a ratio between the plurality of uncapped RNA molecules and the plurality of capped RNA molecules is between 0.0001 to 1. Descried herein, in some aspects, is a system for producing at least one capped RNA molecule. The system comprises a bioreactor configured to contain a first solution containing a plurality of uncapped RNA molecules, wherein the first solution is diluted by at least 4-fold to form a second solution, wherein the second solution comprises the plurality of uncapped RNA molecules; and the bioreactor or a second bioreactor configured to add a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule by contacting the second solution with a plurality of capping enzyme molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Fig. 1 illustrates a non-limiting example of the system described herein for capping nucleic acid. The system comprises a first batch reactor (101) comprising a first solution containing uncapped nucleic acid such as uncapped RNA molecules. The first solution may be passed through a continuous flow reactor (line marked with arrow; 104) where it is mixed with a suitable diluent through an inlet at a defined flow rate (q_d il ution) for achieving the target dilution. The diluted solution may also be passed through a filtration device (103) prior to arrive at a second batch reactor (102), from which it is passed on to the enzymatic capping reactor. Because the concentration of a plurality of molecules that negatively influence the capping reaction is decreased by diluting the first solution (to obtain a second solution), such dilution may enhance 5' capping of the in vitro transcribed (IVT) RNA molecules. The diluted first solution, i.e., the second solution containing the nucleic acid may be followed by ultrafiltration or any other suitable methods to form a third solution. Ultrafiltration or any other suitable methods decreases the total volume of solution to be processed downstream and permits adjustment of the concentration of uncapped mRNA to a concentration that is optimal for the next operation downstream (e.g. the enzymatic capping reaction). The capped nucleic acid may be further processed downstream, such as by chromatography, tangential flow filtration or other suitable purification methods.

Fig. 2 illustrates a non-limiting example of experimental protocols, where a concentration of the uncapped RNA may be diluted 10 fold (lOx) in certain embodiments. Fig. 3 illustrates a non-limiting example of an embodiment of the system, which comprises at least one or a first container (301) comprising a first solution (302) comprising at least one uncapped nucleic acid. The first solution (302) may be diluted directly in the first container (301) to obtain a second solution (304). The system may further comprise at least one additional container (e.g., a second container, 303) for diluting the first solution (302) to obtain a second solution (304) in the second container (303). Due to the decreased plurality of molecules that negatively influence the capping reaction in the second solution (304), the capping agents, (e.g., capping enzyme(s)) then caps the nucleic acid (capped nucleic acid, 305). The capped nucleic acid (305) may be further processed, for example, to change the concentration (306) of the capped nucleic acid (305) or exchange buffer systems, before further downstream processing.

The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

DETAILED DESCRIPTION

Overview

Efficient post-transcriptional capping of messenger RNA produced by in vitro transcription (IVT) is only possible after an intermediate treatment of the IVT reaction product. This intermediate treatment typically involves a purification operation (such as chromatography), usually in combination with a tangential flow filtration step. This suggests that the in vitro transcription reaction product contains substances that interfere with the capping reagents. Enzymatic capping immediately following IVT, without intermediate treatment of the reaction product, produces reduced amounts or approaching 0% capped mRNA molecules. When adding the capping reagents to the IVT for "concurrent transcription and capping" (one-pot reaction), marginal capping may be achieved (less than 10%). This confirms that successful capping requires some intermediate treatment of the IVT product. A conventional intermediate treatment, as mentioned above, involves one or several purification steps, which comes with several disadvantages: inevitable loss of product (decreased process yield); increased process complexity requiring development of extra steps; the need for additional equipment; and increased cost due to product loss and the cost of the extra operation steps. The need for additional equipment also leads to increased operational cost, increased system footprint, and extended process duration.

Described herein are systems and methods for dilution of the IVT product for decreasing the concentration of interfering agents to a level that no longer interfere with the enzymatic capping reaction of adding cap structures to uncapped RNA molecules. The decreased concentration of uncapped RNA molecules prior to conducting capping reaction might also contribute to achieve an efficient capping reaction for adding a cap structure to an uncapped RNA molecule. As demonstrated herein, a dilution of the IVT reaction product may yield a capping efficiency of at least about 90% to 99.9%. This is comparable or substantially similar to the capping efficiency that was achieved in a control condition, whereby the IVT was purified using a nucleic acid purification kit. Achieving substantially similar high mRNA capping efficiency compared to using the currently conventional methods in industry, the current systems and methods also allow for a significantly reduced operational cost and improved process yield. Further, by using the current systems and methods, it is much simpler to scale up the production of capped mRNA molecules.

In some embodiments, the systems and methods described herein provide dilution of a first solution comprising uncapped RNA molecules to form a second solution, which comprises a decreased concentration of a plurality of molecules that negatively influence or inhibit the enzymatic capping reaction and the resulting capping efficiency. In some embodiments, the second solution is contacted with a plurality of capping enzymes and other reagents (e.g., guanosine-5'-triphosphate, S- adenosylmethionine) to produce capped RNA molecules. In some cases, at least one type of capping enzyme is used. In some cases, more than one type of capping enzymes are used. In some embodiments, the dilution factor used for diluting the first solution to obtain the second solution is sufficient for decreasing the inhibition of capping reaction exerted by the plurality of the molecules in the second solution. Fig. 1 provides a non-limiting example of a system described herein. In this nonlimiting example, the 5' capping reaction of an mRNA molecule may be inhibited by the presence of a plurality of molecules in a first solution. By diluting the first solution, a second solution is obtained, where the concentration of the plurality of the molecules that negatively influences or inhibits the capping reaction is decreased. In some embodiments, the second solution further goes through a filtration step to remove volume and reconcentrate the plurality of the uncapped RNA molecules. Such decrease in concentration or removal of the plurality of the molecules allows the capping reaction to occur efficiently. In some embodiments, the second solution may undergo buffer exchange by dialysis or other means for exchanging the buffer of the uncapped mRNA before proceeding with the capping reaction. In some embodiments, the solution containing the capped mRNA molecules may be further purified. In some embodiments, the capped mRNA molecules obtained from the systems and methods described herein may be utilized for purposes such as manufacturing pharmaceuticals or diagnostic compositions.

Systems

Described herein are systems for processing nucleic acids with the methods described below, such as processing solutions containing uncapped RNA molecules derived from in vitro transcription (IVT) reactions to avoid inhibition of capping reactions related to adding a cap structure to an uncapped RNA molecule. The system may comprise an upstream portion directed to provide IVT reaction mixtures containing the uncapped RNA molecules. In some cases, the system may comprise a downstream portion for further processing of capped RNA molecules, such as purification to remove undesired substances and tangential flow filtration to modify the composition and the concentration of the solution of capped RNA molecules. In some embodiments, the system may be further configured to manufacture compounds, biomolecules, or pharmaceutical compositions using the capped RNA as input. For example, the system described herein may synthesize or increase yield of synthesizing an antigen encoded by the capped RNA or capped mRNA, where the antigen may be further formulated into a vaccine. In some embodiments, the system comprises components or devices for initiating or maintaining biological reactions. In some embodiments, the system may be configured to effect any sort of appropriate process. Non-limiting examples of processes for which systems disclosed herein may be suited include production of a biological compound; production of a pharmaceutical or biopharmaceutical compound; RNA synthesis, including IVT, post- transcriptional processes and RNA purification; protein synthesis, including celldependent protein synthesis and cell-free protein synthesis (CFPS); or a combination thereof.

In some embodiments, the system described herein is modular, where each component of the system may be independently assembled or disassembled based on the functionality needed. In some embodiments, the system may be an open system or a closed system such that the system comprises a continuous reactor or a batch reactor. In some cases, the system may comprise a continuous reactor. The system may be operated in a continuous mode. In other cases, the system may comprise a batch reactor. The system may be operated in a non-continuous mode or a batch reaction mode. In other cases, the system may comprise a combination of a continuous reactor and a batch reactor and the system may be operated in a semi- continuous mode.

In some embodiments, the system as illustrated in FIG. 1 comprises at least two containers, where a first container 101 holds a first solution comprising the uncapped RIMA molecules. In some embodiments, the first solution may be diluted for obtaining a second solution. For example, a fraction of the first solution may be transferred to a second container for diluting the first solution into the second solution. In some cases, this transferring and diluting process is a continuous process. The flow rate of the first solution transferring to a second solution is regulated and controlled depending on a speed of the dilution process. In some cases, after diluting at least a portion of the first solution containing the uncapped RNA molecules, the process further comprises mixing the diluted first solution before transferring the diluted first solution to a next step. In some cases, the transferring and diluting process is a non- continuous process or a batch process. In some cases, the transferring and diluting process is a combination of a non-continuous process and/or a continuous process.

In some cases, the mixing step is conducted by a mixing system, which may comprise a magnetic object, an impeller, a baffle, a bead, or any other suitable objects that can be used to mix a solution.

After diluting or additionally mixing, the concentration of the plurality of molecules that inhibits the capping reaction is decreased, which leads to capping of the uncapped RNA molecules in the second solution. In some embodiments, the dilution factor for diluting the first solution to form the second solution is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, at least 20- fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70- fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 1000-fold, or any numerical numbers in between the aforementioned dilution factors.

As illustrated in FIG. 1, the system may comprise a filtration device 103 that is configured to filter the diluted first solution to re-concentrate the plurality of the uncapped RNA molecules before entering batch reactor 102. In some embodiments, the system does not comprise a filtration device 103 or the diluted first solution does not go through the filtration device 103 before entering batch reactor 102. The batch reactor 101, the container for diluting the first reaction to obtain the second solution, the mixing device, the filtration device 103, and the batch reactor 102 are in fluid communication with each other. In some embodiments, the capping reaction of adding a cap structure to an uncapped RNA molecule is conducted in batch reactor 102. Any necessary reagents, such as capping enzymes as described herein, guanosine-5'-triphosphate, buffers and salts, methyl donor (e.g. S- adenosylmethionine), and other necessary reagents may be added to batch reactor 102 via a valve or an opening.

In some embodiments, the system may comprise a filtration unit. The filtration units may comprise a dead-end filtration unit, a spin filtration unit, a tangential flow filtration (TFF) unit, an alternating tangential flow (ATF) filtration unit, a microfiltration unit, an ultrafiltration unit or any other suitable filtration unit known in the art.

The solution may be transported from one part of the system to another (e.g., from one segment to another) or into or out of the system by the opening or closing of valves. Valves may be directed to open or close at certain times by the system. The system may further comprise pumps or other means, which are additionally directed by the system, for transporting the solution.

In some embodiments, the system may comprise a purification component or device for purifying the biomolecule synthesized or present in the solution (e.g., the capped RNA molecule or polypeptide encoded from the capped RNA molecule) and preparing for any suitable downstream reactions. Non-limiting example of the purification component or device may include chromatography or filtration.

Methods

Described herein are methods for capping RNA molecules (including mRNA molecules, for example) synthesized from IVT reaction. In some embodiments, the method increases capping efficiency of RNA molecules synthesized from an IVT reaction. In some embodiments, the method removes an inhibition to a capping reaction of adding a cap structure to an uncapped RNA molecule. In some embodiments, the method utilizes the systems described herein for diluting the plurality of molecules that inhibits the capping reaction. In some embodiments, the method comprises diluting a first solution containing the uncapped RNA molecules or uncapped mRNA molecules to form a second solution, where the dilution of the plurality of molecules that inhibits the capping reaction allows post-transcriptional capping of the uncapped RIMA or mRNA molecules in the second solution. In some embodiments, the capping reaction is initiated by contacting the second solution with a plurality of capping enzymes and other reagents to form at least one capped RNA molecule. In some embodiments, the diluting of the first solution allows the post- transcriptional capping efficiency for producing capped RNA molecules to reach at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 87%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of capped RNA molecules, or any percentages in between the aforementioned percentages.

In some preferred embodiments, the method comprises the steps of:

- providing a plurality of uncapped RNA molecules, obtained via an in vitro transcription (IVT) reaction, in a first solution, wherein said first solution comprises reagents for said IVT reaction;

- diluting the first solution by at least 4-fold to form a second solution containing the plurality of uncapped RNA molecules;

- contacting the second solution with a plurality of capping enzyme molecules;

- adding a cap structure to a 5' end of an uncapped RNA molecule capping reaction to form at least one capped RNA molecule.

In a preferred embodiment, the diluting step occurs in the same vessel or reactor as the plurality of uncapped RNA molecules were generated via the (IVT) reaction.

Alternatively, the uncapped RNA molecules are transferred after the IVT reaction to another vessel where the dilution occurs.

The method prevents the by-products and reagents of the IVT reaction to interfere with the capping process. In a preferred embodiment, the IVT solution does not undergo an addition purification step prior to dilution. It is preferred that the dilution of the first solution is the unique step between the IVT reaction and capping. This method simplifies the protocol of RNA processing after the IVT reaction and prior to the capping reaction, while providing high capping efficiencies and is as a result time- and cost-effective compared with state-of-the-art mRNA transcription and capping methods. The solution provided by the method disclosed herein does not require intermediate purification steps of RNA before performing the enzymatic capping and simplify therefore the manufacturing process while providing high yields of in vitro transcribed and capped RNA.

In a preferred embodiment of the method as disclosed herein, the first solution prior to the dilution step did not undergo a purification step. In a further embodiment, the capping step is performed on the diluted second solution.

In some embodiments, after in vitro transcription reaction (IVT) to obtain a plurality of uncapped RNA molecules, the DNA template used in the IVT reaction may be removed. For example, the DNA template may be removed by DNase treatment. After DNase treatment, the IVT reaction may be terminated by heating, cooling, or contacting the solution containing the RNA molecules with a chelating agent. Nonlimiting example of the chelating agent may include 8-hydroxyquinoline, carboplatin, EDTA, EGTA, hyxadecylpyridinum bromide, or sodium tartrate. Before capping, the uncapped RNA molecules may be denatured (e.g., by heating to 65°C for 5 minutes). The temperature for denaturing and the length for denaturing may vary. In some embodiments, the denaturation of the uncapped RNA molecules is not conducted before the capping reaction.

An IVT reaction typically comprises nucleotide triphosphates (NTPs), a Rnase inhibitor and a DNA-dependent RNA polymerase in a transcription buffer. The NTPs may be naturally occurring NTPs and/or modified NTPs. The DNA -dependent RNA polymerase may be selected from but is not limited to T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase or mutant polymerases thereof.

In some embodiments, the capping efficiency may be determined by dividing the amount of uncapped RNA molecules by the amount of capped RNA molecules for obtaining a ratio of uncapped RNA/capped RNA. For example, but not meant to be limiting, liquid chromatography coupled with UV absorbance measurement and mass spectrometry (LC-UV-MS) may be used to assess capped or uncapped RNA molecule concentrations. In some embodiments, the concentrations of the uncapped RNA molecules and capped RNA molecules are calculated based on the absorbance readings of the eluted molecules as identified by in-line mass spectrometry. The capping efficiency is calculated based on the calculated concentrations. In some embodiments, the capping efficiency is calculated directly based on the absorbance readings of the capped RNA molecules and uncapped RNA molecules. In some embodiments, the diluting of the first solution allows capping of the uncapped RNA, where after the capping reaction the ratio of uncapped RNA/capped RNA is at most 1.0, at most 0.8, at most 0.6, at most 0.4, at most 0.2, at most 0.1, at most 0.09, at most 0.08, at most 0.07, at most 0.06, at most 0.05, at most 0.04, at most 0.03, at most 0.02, at most 0.01, at most 0.001, or at most 0.0001. In some embodiments, the ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is between about 0.001 to about 10. In some embodiments, the ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is between about 0.001 to about 0.002, about 0.001 to about 0.005, about 0.001 to about 0.01, about 0.001 to about 0.02, about 0.001 to about 0.05, about 0.001 to about 0.1, about 0.001 to about 0.2, about 0.001 to about 0.5, about 0.001 to about 1, about 0.001 to about 5, about 0.001 to about 10, about 0.002 to about 0.005, about 0.002 to about 0.01, about 0.002 to about 0.02, about 0.002 to about 0.05, about 0.002 to about 0.1, about 0.002 to about 0.2, about 0.002 to about 0.5, about 0.002 to about 1, about 0.002 to about 5, about 0.002 to about 10, about 0.005 to about 0.01, about 0.005 to about 0.02, about 0.005 to about 0.05, about 0.005 to about 0.1, about 0.005 to about 0.2, about 0.005 to about 0.5, about 0.005 to about 1, about 0.005 to about 5, about 0.005 to about 10, about 0.01 to about 0.02, about 0.01 to about 0.05, about 0.01 to about 0.1, about 0.01 to about 0.2, about 0.01 to about 0.5, about 0.01 to about 1, about 0.01 to about 5, about 0.01 to about 10, about 0.02 to about 0.05, about 0.02 to about 0.1, about 0.02 to about 0.2, about 0.02 to about 0.5, about 0.02 to about 1, about 0.02 to about 5, about 0.02 to about 10, about 0.05 to about 0.1, about 0.05 to about 0.2, about 0.05 to about 0.5, about 0.05 to about 1, about 0.05 to about 5, about 0.05 to about 10, about 0.1 to about 0.2, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 5, about 0.1 to about 10, about 0.2 to about 0.5, about 0.2 to about 1, about 0.2 to about 5, about 0.2 to about 10, about 0.5 to about 1, about 0.5 to about 5, about 0.5 to about 10, about 1 to about 5, about 1 to about 10, or about 5 to about 10. In some embodiments, the ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is about 0.001, about 0.002, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.2, about 0.5, about 1, about 5, or about 10. In some embodiments, the ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is at least about 0.001, about 0.002, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.2, about 0.5, about 1, or about 5. In some embodiments, the ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is at most about 0.002, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.2, about 0.5, about 1, about 5, or about 10.

In some embodiments, the capping reaction can be conducted at a reduced or absence of inhibition by diluting the first solution to form the second solution, where no additional purification or filtration is needed. For example, the capping reaction is performed by the method described herein without the need of utilizing chromatography to first purify the uncapped RIMA. In some embodiments, the IVT reaction mixture (e.g., the first solution prior to dilution) is not purified or filtered (e.g., by conventional chromatography or filtration) before the capping reaction. In some embodiments, the second solution before being contacted with the capping enzyme and reagents for the capping reaction) is filtered to reconcentrate the plurality of uncapped RNA molecules. In some embodiments, the second solution before conducting the capping reaction is not filtered or purified. In some embodiments, the solution after the capping reaction containing a plurality of capped RNA molecules is not purified or filtered (e.g., by conventional chromatography or filtration) before being formulated into a composition or pharmaceutical composition described herein or being utilized for any other application where a capped nucleic may be used. In some embodiments, the solution containing a plurality of capped RNA molecules after the capping reaction is purified or filtered.

In some embodiments, the first solution may be diluted by at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold to form a second solution, where the capping reaction can occur in the second solution after contacting the uncapped RNA molecules with capping agents, for example capping enzymes, in the presence of, for example, enzyme substrates, capping reaction buffers, salts, and other reagents. In some embodiments, the first solution is diluted by about 1-fold to about 100-fold. In some embodiments, the first solution is diluted by about 1-fold to about 5-fold, about 1-fold to about 10-fold, about 1-fold to about 20-fold, about 1-fold to about 30-fold, about 1-fold to about 40-fold, about 1-fold to about 50-fold, about 1-fold to about 60-fold, about 1-fold to about 70-fold, about 1-fold to about 80-fold, about 1-fold to about 100-fold, about 2-fold to about 5-fold, about 2-fold to about 10-fold, about 2- fold to about 20-fold, about 2-fold to about 30-fold, about 2-fold to about 40-fold, about 2-fold to about 50-fold, about 2-fold to about 60-fold, about 2-fold to about 70-fold, about 2-fold to about 80-fold, about 2-fold to about 100-fold, about 5-fold to about 10-fold, about 5-fold to about 20-fold, about 5-fold to about 30-fold, about 5-fold to about 40-fold, about 5-fold to about 50-fold, about 5-fold to about 60-fold, about 5-fold to about 70-fold, about 5-fold to about 80-fold, about 5-fold to about 100-fold, about 10-fold to about 20-fold, about 10-fold to about 30-fold, about 10- fold to about 40-fold, about 10-fold to about 50-fold, about 10-fold to about 60-fold, about 10-fold to about 70-fold, about 10-fold to about 80-fold, about 10-fold to about 100-fold, about 20-fold to about 30-fold, about 20-fold to about 40-fold, about 20- fold to about 50-fold, about 20-fold to about 60-fold, about 20-fold to about 70-fold, about 20-fold to about 80-fold, about 20-fold to about 100-fold, about 30-fold to about 40-fold, about 30-fold to about 50-fold, about 30-fold to about 60-fold, about 30-fold to about 70-fold, about 30-fold to about 80-fold, about 30-fold to about 100- fold, about 40-fold to about 50-fold, about 40-fold to about 60-fold, about 40-fold to about 70-fold, about 40-fold to about 80-fold, about 40-fold to about 100-fold, about 50-fold to about 60-fold, about 50-fold to about 70-fold, about 50-fold to about 80- fold, about 50-fold to about 100-fold, about 60-fold to about 70-fold, about 60-fold to about 80-fold, about 60-fold to about 100-fold, about 70-fold to about 80-fold, about 70-fold to about 100-fold, or about 80-fold to about 100-fold. In some embodiments, the first solution is diluted by about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60- fold, about 70-fold, about 80-fold, or about 100-fold. In some embodiments, the first solution is diluted by at least about 1-fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70- fold, or about 80-fold. In some embodiments, the first solution is diluted by at most about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 40- fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, or about 100-fold.

In some embodiments, the first solution may be diluted in the same container as where the IVT and the capping reaction occurs. For example, a volume of diluent may be added to the first solution in the same container to form a second (diluted) solution. In some embodiments, any diluent that is inert and does not interfere with capping reaction of the uncapped RIMA or mRNA molecules may be used. In some embodiments, the diluent is water.

In some embodiments, the first solution may be diluted by mixing a portion of the first solution with a diluent in a second container to form the second (diluted) solution. In some instances, the second solution may undergo buffer exchange to a suitable buffer or solution conditions for further processing to occur. In some embodiments, the second solution may be further purified (e.g., by chromatography) or concentrated (e.g., by filtration or ultrafiltration) prior to the capping reaction. In some embodiments, the volume of the second solution may be decreased prior to the capping reaction (e.g., by filtration or ultrafiltration). For example, the second solution may be re-concentrated by contacting with a filter membrane (e.g., a filter membrane with a 10 kDa cut-off) to remove excess fluid. In some embodiments, the membrane may have a molecular weight cut-off (MWCO) of at least IkDa, 3kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 750kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, or any MWCO between any two of ranges described here. In some embodiments, the MWCO is at least about 30kDa to at least about 100 kDa. In some embodiments, the MWCO is at least about 30kDa to at least about 500 kDa. In some embodiments, the MWCO is at least about 30kDa to at least about 1000 kDa. In some embodiments, the second solution may be purified or re-concentrated before the capping reaction. In some embodiments, the second solution may be purified and re-concentrated before the capping reaction. In some embodiments, the second solution may only be purified before the capping reaction. In some embodiments, the second solution may only be concentrated before the capping reaction.

In some embodiments, the dilution of the first solution to form the second solution creates increased volume, where the second solution may be concentrated (e.g., by filtration or ultrafiltration) to remove excess volume prior to the capping reaction. In some embodiments, the dilution of the first solution to form the second solution does not create excess volume according to the needs of downstream reactions and the capping reaction may be initiated directly in the second solution. In some embodiments, the capped RIMA molecules may then be purified from the second solution and further formulated (e.g., in a pharmaceutical composition described herein) via various downstream processes. In some embodiments, additional processes may be carried out after the capping reaction to produce a final pharmaceutical composition. For example, the capped RNA or mRNA molecules function as templates for peptide or protein synthesis. The capped RNA molecules may also be encapsulated into or adsorbed onto lipid nanoparticles (LNPs) or other RNA delivery systems for production of mRNA vaccines. In some embodiments, the method comprises first synthesizing the uncapped RIMA or mRNA molecules. For example, the uncapped RNA molecules may be synthesized from in vitro transcription (IVT). In some embodiments, the IVT reaction may be carried out in any one of the containers of the system described herein. In some embodiments, the IVT reaction occurs in a continuous reactor. In some embodiments, the IVT reaction occurs in a batch reactor. In some embodiments, the IVT reaction may be terminated by inactivating RNA polymerase in the solution. RNA polymerase may be inactivated by heating, cooling, addition of chelator (e.g., EDTA), or a combination thereof. Non-limiting examples of RNA polymerase that may be used to synthesize the uncapped RNA molecules via IVT may include T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase. In some embodiments, the method comprises degrading the DNA template after IVT.

In some embodiments, the method comprises contacting the second solution (comprising the diluted plurality of molecules that inhibits capping reaction), with capping enzyme and other reagents for initiating the capping reaction in the second solution. Non-limiting examples of capping enzyme include Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase, Vaccinia capping enzyme (VCE), Bluetongue Virus capping enzyme, Chlorella Virus capping enzyme, S. cerevisiae capping enzyme, Mimivirus capping enzyme, African swine fever virus capping enzyme, or Avian Reovirus capping enzyme. Non-limiting examples of other reagents may include Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase, 2 '-O- Methyltransferase, a magnesium salt, guanosine-5'-triphosphate, S- adenosylmethionine, buffering agents, RNase inhibitor. Non-limiting examples of capping structures formed during the capping reaction include GpppN, m7GpppN (Cap 0), m7Gpppm6A, m7GpppmlA, m7GpppNm (Cap 1), m2,7GpppNm, m2,2,7GpppNm, m7Gpppm6Am, m7GpppmlAm, m7GpppNmpNm (Cap 2), m7GpppNmpNmpNm (Cap 3), m7GpppNmpNmpNmpNm (Cap 4), where N stands for any nucleotide, A for adenosine, G for guanosine, m for a methyl group and p for a phosphate group. In some embodiments, the capping structure comprises chemically modified nucleotide. In some embodiments, the capping reaction described herein yields a majority of one species of capped structure (e.g., Cap 1). In some embodiments, the capping reaction described herein yields other minor cap structures such as unmethylated cap, Cap 0, Cap 2, or other.

In some embodiments, the first solution comprises uncapped RNA molecules. In some embodiments, the uncapped RNA molecules may include long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), self-amplifying mRNA (SAM), Trans amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), transcription start site-associated (TSSa-)RNAs, upstream antisense (ua) RNAs, promoter upstream transcripts (PROMPTS), or a combination thereof. In some embodiments, the uncapped RNA molecules comprise at least one chemical modification comprising backbone modification, sugar modification, or base modification. In this context, a modified RNA molecule comprises nucleotide modifications, e.g. backbone modifications, sugar modifications or base modifications. A sugar modification in connection with the present disclosure is a chemical modification of the sugar of the nucleotides of the RNA molecule. Furthermore, a base modification in connection with the present disclosure is a chemical modification of the base moiety of the nucleotides of the RNA molecule. In this context, nucleotide modifications are selected from nucleotide modifications that are applicable for transcription and/or translation. In further embodiments, the modified RNA comprises nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, o-thio-cytidine, pseudo-iso-cytidine, 5- aminoallyl-uridine, 5-iodo-uridine, Nl-methyl-pseudouridine, 5,6-dihydrouridine, o- thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5- methyl-uridine, pyrrolo-cytidine, inosine, o-thio-guanosine, 6-methyl-guanosine, 5- methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, Nl-methyl-adenosine, 2- amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro- purine, N6-methyl-adenosine, o-thio-adenosine, 8-azido-adenosine, 7-deaza- adenosine, and a combination thereof.

In some embodiments, the method comprises diluting IVT reaction mixture for conducting capping reaction without inhibition. In some embodiments, the IVT reaction mixture (e.g., the first solution comprising a plurality of uncapped RNA molecules prior to dilution) is not purified (e.g., by conventional chromatography or other suitable purification methods) before the capping reaction. In some embodiments, the solution before or after capping reaction (e.g., the second solution before or after being contacted with the capping enzyme and other reagents for the capping reaction) is not purified (e.g., by conventional chromatography or other purification methods). In some embodiments, the diluted second solution does not go through a filtration step or any other suitable step to remove increased volume. In some embodiments, the capping reaction efficiency of the second solution is increased due to diluting of the IVT reaction mixture and not due to additional purification of the second solution. In some embodiments, the diluting of the IVT reaction mixture decreases the concentration of the plurality of molecules that inhibits the capping reaction, thus increasing the capping reaction efficiency in the diluted IVT reaction mixture. In some embodiments, the diluted solution, after capping reaction, may be re-concentrated or purified before formulated into a composition or pharmaceutical composition described herein or being utilized for any other application where a capped RNA may be used.

In some embodiments, the method comprises diluting the IVT reaction mixture followed by removing excess fluid volume by filtration or other suitable methods to concentrate the uncapped RNA molecule for downstream processing. In some embodiments, the diluting or the removing of excess fluid volume (via filtration or other suitable methods) decreases the concentration of or removes the majority of the plurality of molecules that inhibits the capping reaction, thus increasing the capping reaction efficiency. In some embodiments, the diluted then re-concentrated (e.g., via filtration) second solution, after capping reaction, can be further purified, concentrated and/or filtered before going through a series of downstream process steps to be formulated into a composition or pharmaceutical composition described herein or being utilized for any other application where a capped nucleic may be used.

In some embodiments, the method comprises diluting the IVT reaction mixture followed by removing increased fluid volume by ultrafiltration or microfiltration to concentrate the uncapped nucleic acid molecule (e.g., uncapped RNA molecule) for downstream processing. In some embodiments, the method comprises diluting the IVT reaction mixture followed by removing increased fluid volume by tangential flow filtration to concentrate the uncapped RNA molecules. In some embodiments, the diluting or the removing of excess fluid volume (via ultrafiltration using a membrane capable of retaining uncapped RNA molecules) decreases the concentration of the plurality of molecules that inhibits the capping reaction, thus increasing the capping reaction efficiency in the IVT reaction mixture. In some embodiments, the diluted then re-concentrated (e.g., via ultrafiltration) IVT reaction mixture, after capping reaction, can be further purified, concentrated and/or filtered before being formulated into a composition or pharmaceutical composition described herein or being utilized for any other application where a capped RNA may be used. Biomolecule production

In some cases of the present disclosure, the systems and methods described herein are designed to accommodate a reaction/process or part of a reaction/ process taking place in the system. In some cases, the reaction relates to processing a plurality of uncapped RNA molecules so that a capping reaction of adding a cap structure to an uncapped RNA molecule can carry on without interference or inhibition. In some cases, the process also comprises steps pertaining to in vitro (cell-free) translation of RNA to protein. In some cases, the reaction pertains to a combination of both processes, i.e., from DNA to RNA through transcription and from RNA to protein through translation.

In some cases, the in vitro transcription relates to a process in which RNA is synthesized in a cell-free system (/n vitro). In some cases, cloning vectors DNA, particularly plasmid DNA vectors are applied as template for the generation of RNA transcripts following linearization of circular plasmid DNA. These cloning vectors are generally designated as transcription vector. RNA may be obtained by DNA- dependent in vitro transcription of an appropriate DNA template. A promoter for controlling RNA in vitro transcription may be any promoter for any DNA-dependent RNA polymerase. In some embodiments, a viral RNA polymerase binds a viral promoter which is at least one promoter selected from the list consisting of T7, T3, T7lac, SP6, pL, pR, CMV, SV40, and CaMV35S. Alternately or in combination, the nucleic acid fragment comprising promoter sequence comprises a bacterial promoter. In some cases, a bacterial RNA polymerase binds a bacterial promoter which is at least one promoter selected from the list consisting of araBAD, trp, lac, and Ptac. In some cases, the nucleic acid fragment comprising promoter sequence comprises a eukaryotic promoter. In some cases, the eukaryotic RNA polymerase binds a eukaryotic promoter which is at least one promoter selected from the list consisting of EFla, PGK1, Ubc, beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, ALB, GALI, GAL10, TEF1, GDS, ADH1, Ubi, Hl, and U6. In some cases, the eukaryotic promoter is at least one promoter selected from the list consisting of an RNA pol I promoter, an RNA pol II promoter and an RNA pol III promoter.

In some cases, the DNA-dependent RNA polymerases comprises at least one of a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a RNA polymerase I, a RNA polymerase II, a RNA polymerase III, a RNA polymerase IV, a RNA polymerase V, and a single subunit RNA polymerase. The DNA template for in vitro RNA transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RIMA to be in vitro transcribed, and introducing it into an appropriate vector for RNA in vitro transcription, for example in circular plasmid DNA which is introduced in a host such as a bacterium. The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis or by amplification reactions, including polymerase chain reaction (PCR).

In some cases, the DNA template relates to a DNA molecule comprising a nucleic acid sequence encoding the RNA sequence. The template DNA is used as a template for RNA in vitro transcription in order to produce the RNA encoded by the template DNA. Therefore, the template DNA comprises all elements necessary for RNA in vitro transcription, particularly a promoter element for binding of a DNA dependent RNA polymerase as e.g. T3, T7 and SP6 RNA polymerases 5' of the DNA sequence encoding the target RNA sequence. The poly(A) tail may be either encoded into the DNA template or added enzymatically to RNA in a separate step after in vitro transcription. In some cases, the template DNA comprises primer binding sites 5' and/or 3' of the DNA sequence encoding the target RNA sequence to determine the identity of the DNA sequence encoding the target RNA sequence e.g. by PCR or DNA sequencing. In some cases, the DNA template comprises a 5' UTR or a 3' UTR. In some cases, the DNA template comprises a DNA vector, such as a plasmid DNA, which comprises a nucleic acid sequence encoding the RNA sequence. In some cases, the DNA template comprises a linear or a circular DNA molecule.

In some cases of the present disclosure, a DNA template encodes a different RNA molecule species. In some cases, the DNA template contains a sub-genomic promoter and a large ORF encoding for non-structural proteins which, following delivery of the biopharmaceutical into the cytosol, are transcribed in four functional components (nsPl, nsP2, nsP3, and nsp4) by the encoded RNA-dependent RNA polymerase (RDRP). RDRP than produces a negative-sense copy of the genome which serves as a template for two positive-strand RNA molecules: the genomic mRNA and a shorter sub-genomic mRNA. This sub-genomic mRNA is transcribed at very high levels, allowing the amplification of mRNA encoding the antigen of choice. A different RNA molecule species may encode an antigen of different serotypes or strains of a pathogen, a different allergen, a different autoimmune antigen, a different antigen of a pathogen, different adjuvant proteins, a different isoform or variant of a cancer or tumor antigen, a different tumor antigen of one patient, one antibody among a group of antibodies which target different epitopes of a protein or of a group of proteins, different proteins of a metabolic pathway, a single protein among a group of proteins which are defect in a subject, or a different isoform of a protein for molecular therapy.

In some embodiments, the RIMA molecules capped by the method described herein comprises a coding region of a peptide or protein. In such case, the capped RNA molecules serve as a template for peptide or protein synthesis. For example, the capped RNA molecules may be further formulated into a composition or a pharmaceutical composition to be administered to a subject, where the synthesis of the peptide or protein occurs in vivo. In some embodiments, the peptide or protein may be synthesized directed from the capped RNA molecules either in the same or different container of the system. The in vitro synthesized peptide or protein may then be formulated into a composition or a pharmaceutical composition. In some embodiments, the peptide or protein encoded by the capped RNA molecules may be prophylactic. In some embodiments, the peptide or protein encoded by the capped RNA molecules may be therapeutic. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) of about 1 kDa to about 1,000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) of about 10 kDa to about 20 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 200 kDa, about 10 kDa to about 300 kDa, about 10 kDa to about 400 kDa, about 10 kDa to about 500 kDa, about 10 kDa to about 600 kDa, about 10 kDa to about 700 kDa, about 10 kDa to about 800 kDa, about 10 kDa to about 1,000 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 100 kDa, about 20 kDa to about 200 kDa, about 20 kDa to about 300 kDa, about 20 kDa to about 400 kDa, about 20 kDa to about 500 kDa, about 20 kDa to about 600 kDa, about 20 kDa to about 700 kDa, about 20 kDa to about 800 kDa, about 20 kDa to about 1,000 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 200 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 400 kDa, about 50 kDa to about 500 kDa, about 50 kDa to about 600 kDa, about 50 kDa to about 700 kDa, about 50 kDa to about 800 kDa, about 50 kDa to about 1,000 kDa, about 100 kDa to about 200 kDa, about 100 kDa to about 300 kDa, about 100 kDa to about 400 kDa, about 100 kDa to about 500 kDa, about 100 kDa to about 600 kDa, about 100 kDa to about 700 kDa, about 100 kDa to about 800 kDa, about 100 kDa to about 1,000 kDa, about 200 kDa to about 300 kDa, about 200 kDa to about 400 kDa, about 200 kDa to about 500 kDa, about 200 kDa to about 600 kDa, about 200 kDa to about 700 kDa, about 200 kDa to about 800 kDa, about 200 kDa to about 1,000 kDa, about 300 kDa to about 400 kDa, about 300 kDa to about 500 kDa, about 300 kDa to about 600 kDa, about 300 kDa to about 700 kDa, about 300 kDa to about 800 kDa, about 300 kDa to about 1,000 kDa, about 400 kDa to about 500 kDa, about 400 kDa to about 600 kDa, about 400 kDa to about 700 kDa, about 400 kDa to about 800 kDa, about 400 kDa to about 1,000 kDa, about 500 kDa to about 600 kDa, about 500 kDa to about 700 kDa, about 500 kDa to about 800 kDa, about 500 kDa to about 1,000 kDa, about 600 kDa to about 700 kDa, about 600 kDa to about 800 kDa, about 600 kDa to about 1,000 kDa, about 700 kDa to about 800 kDa, about 700 kDa to about 1,000 kDa, or about 800 kDa to about 1,000 kDa. In some embodiments, the peptide or protein encoded by the capped RIMA molecules comprises a molecular weight (MW) of about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, or about 1,000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) of at least about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, or about 800 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) of at most about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, or about 1,000 kDa.

In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) at most about 10 kDa to about 1,000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) at most about 10 kDa to about 20 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 200 kDa, about 10 kDa to about 300 kDa, about 10 kDa to about 400 kDa, about 10 kDa to about 500 kDa, about 10 kDa to about 600 kDa, about 10 kDa to about 700 kDa, about 10 kDa to about 800 kDa, about 10 kDa to about 1,000 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 100 kDa, about 20 kDa to about 200 kDa, about 20 kDa to about 300 kDa, about 20 kDa to about 400 kDa, about 20 kDa to about 500 kDa, about 20 kDa to about 600 kDa, about 20 kDa to about 700 kDa, about 20 kDa to about 800 kDa, about 20 kDa to about 1,000 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 200 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 400 kDa, about 50 kDa to about 500 kDa, about 50 kDa to about 600 kDa, about 50 kDa to about 700 kDa, about 50 kDa to about 800 kDa, about 50 kDa to about 1,000 kDa, about 100 kDa to about 200 kDa, about 100 kDa to about 300 kDa, about 100 kDa to about 400 kDa, about 100 kDa to about 500 kDa, about 100 kDa to about 600 kDa, about 100 kDa to about 700 kDa, about 100 kDa to about 800 kDa, about 100 kDa to about 1,000 kDa, about 200 kDa to about 300 kDa, about 200 kDa to about 400 kDa, about 200 kDa to about 500 kDa, about 200 kDa to about 600 kDa, about 200 kDa to about 700 kDa, about 200 kDa to about 800 kDa, about 200 kDa to about 1,000 kDa, about 300 kDa to about 400 kDa, about 300 kDa to about 500 kDa, about 300 kDa to about 600 kDa, about 300 kDa to about 700 kDa, about 300 kDa to about 800 kDa, about 300 kDa to about 1,000 kDa, about 400 kDa to about 500 kDa, about 400 kDa to about 600 kDa, about 400 kDa to about 700 kDa, about 400 kDa to about 800 kDa, about 400 kDa to about 1,000 kDa, about 500 kDa to about 600 kDa, about 500 kDa to about 700 kDa, about 500 kDa to about 800 kDa, about 500 kDa to about 1,000 kDa, about 600 kDa to about 700 kDa, about 600 kDa to about 800 kDa, about 600 kDa to about 1,000 kDa, about 700 kDa to about 800 kDa, about 700 kDa to about 1,000 kDa, or about 800 kDa to about 1,000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) at most about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, or about 1,000 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) at most at least about 10 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, or about 800 kDa. In some embodiments, the peptide or protein encoded by the capped RNA molecules comprises a molecular weight (MW) at most at most about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, or about 1,000 kDa.

Compositions

Described herein is a composition comprising an agent or a composition described herein (e.g., the uncapped RNA molecules after processing as described herein). In some embodiments, the composition comprises substances with a MW of 400 kDa or less at a concentration of less than 15 v/v %. In some embodiments, the substances comprise an MW of about 20 kDa, about 50 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, or about 1,000 kDa. In some embodiments, the substances comprise a concentration of less than 30 v/v%, less than 25 v/v%, less than 20 v/v%, less than 19 v/v%, less than 18 v/v%, less than 17 v/v%, less than 16 v/v%, less than 15 v/v%, less than 14 v/v%, less than 13 v/v%, less than 12 v/v%, less than 11 v/v%, less than 10 v/v%, less than 9 v/v%, less than 8 v/v%, less than 7 v/v%, less than 6 v/v%, less than 5 v/v%, less than 4 v/v%, less than 3 v/v%, less than 2 v/v%, or less than 1 v/v%.

In some embodiments, the composition comprises a plurality of 5' uncapped RIMA molecules, wherein said uncapped RNA molecules are obtained by means of an in vitro transcription reaction, said composition comprises reagents for in vitro transcription, and wherein the concentration of uncapped RNA in said composition is less than 20 mg/ml. In some embodiments, the concentration of the uncapped RNA in said composition comprises a range between about 0.01 mg/ml to about 20 mg/ml. In some embodiments, the concentration of the uncapped RNA in said composition comprises a range between about 0.01 mg/ml to about 0.02 mg/ml, about 0.01 mg/ml to about 0.05 mg/ml, about 0.01 mg/ml to about 0.1 mg/ml, about 0.01 mg/ml to about 0.2 mg/ml, about 0.01 mg/ml to about 0.5 mg/ml, about 0.01 mg/ml to about 1 mg/ml, about 0.01 mg/ml to about 2 mg/ml, about 0.01 mg/ml to about 5 mg/ml, about 0.01 mg/ml to about 10 mg/ml, about 0.01 mg/ml to about 20 mg/ml, about 0.02 mg/ml to about 0.05 mg/ml, about 0.02 mg/ml to about 0.1 mg/ml, about 0.02 mg/ml to about 0.2 mg/ml, about 0.02 mg/ml to about 0.5 mg/ml, about 0.02 mg/ml to about 1 mg/ml, about 0.02 mg/ml to about 2 mg/ml, about 0.02 mg/ml to about 5 mg/ml, about 0.02 mg/ml to about 10 mg/ml, about 0.02 mg/ml to about 20 mg/ml, about 0.05 mg/ml to about 0.1 mg/ml, about 0.05 mg/ml to about 0.2 mg/ml, about 0.05 mg/ml to about 0.5 mg/ml, about 0.05 mg/ml to about 1 mg/ml, about 0.05 mg/ml to about 2 mg/ml, about 0.05 mg/ml to about 5 mg/ml, about 0.05 mg/ml to about 10 mg/ml, about 0.05 mg/ml to about 20 mg/ml, about 0.1 mg/ml to about 0.2 mg/ml, about 0.1 mg/ml to about 0.5 mg/ml, about 0.1 mg/ml to about 1 mg/ml, about 0.1 mg/ml to about 2 mg/ml, about 0.1 mg/ml to about 5 mg/ml, about 0.1 mg/ml to about 10 mg/ml, about 0.1 mg/ml to about 20 mg/ml, about 0.2 mg/ml to about 0.5 mg/ml, about 0.2 mg/ml to about 1 mg/ml, about 0.2 mg/ml to about 2 mg/ml, about 0.2 mg/ml to about 5 mg/ml, about 0.2 mg/ml to about 10 mg/ml, about 0.2 mg/ml to about 20 mg/ml, about 0.5 mg/ml to about 1 mg/ml, about 0.5 mg/ml to about 2 mg/ml, about 0.5 mg/ml to about 5 mg/ml, about 0.5 mg/ml to about 10 mg/ml, about 0.5 mg/ml to about 20 mg/ml, about 1 mg/ml to about 2 mg/ml, about 1 mg/ml to about 5 mg/ml, about 1 mg/ml to about 10 mg/ml, about 1 mg/ml to about 20 mg/ml, about 2 mg/ml to about 5 mg/ml, about 2 mg/ml to about 10 mg/ml, about 2 mg/ml to about 20 mg/ml, about 5 mg/ml to about 10 mg/ml, about 5 mg/ml to about 20 mg/ml, or about 10 mg/ml to about 20 mg/ml. In some embodiments, the concentration of the uncapped RNA in said composition comprises a range between about 0.01 mg/ml, about 0.02 mg/ml, about 0.05 mg/ml, about 0.1 mg/ml, about 0.2 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 5 mg/ml, about 10 mg/ml, or about 20 mg/ml. In some embodiments, the concentration of the uncapped RIMA in said composition comprises a range between at least about 0.01 mg/ml, about 0.02 mg/ml, about 0.05 mg/ml, about 0.1 mg/ml, about 0.2 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 5 mg/ml, or about 10 mg/ml. In some embodiments, the concentration of the uncapped RNA in said composition comprises a range between at most about 0.02 mg/ml, about 0.05 mg/ml, about 0.1 mg/ml, about 0.2 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 5 mg/ml, about 10 mg/ml, or about 20 mg/ml.

In some embodiments, the composition comprises a plurality of 5'-capped RNA molecules, wherein a capping reaction occurs post-transcriptionally and wherein a ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is between 0.0001 and 1. In some embodiments, the composition comprises a plurality of 5'-capped RNA molecules, wherein a capping reaction occurs post-transcriptionally and wherein a ratio between a plurality of uncapped RNA molecules and the plurality of capped RNA molecules is between 0.01 and 0.5. In some embodiments, the composition comprises the capped RNA molecules or peptide encoded by the capped RNA molecules. In some embodiments, the capped RNA molecules or peptide encoded by the capped RNA molecules may encode an antigen for vaccine formulation. In some embodiments, the capped RNA molecules are further processed for formulation into a vaccine composition.

In some embodiments, the vaccine composition comprises at least one 5' capped RNA molecule, where the 5' capped RNA molecule is a 5' capped mRNA molecule. In some embodiments, the at least one 5' capped mRNA molecule can be encapsulated in a nanoparticle to form a pharmaceutical composition. The nanoparticle can comprise lipids, carbohydrates, polypeptides, polymers formed from one or more monomers, or any combination thereof (including molecular combinations of these substances). In some embodiments, the at least one 5' capped RNA molecules is complexed with a charged polymer, e.g. by electrostatic interaction

In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule can be formulated with a charged lipid or an amino lipid. In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule can be formulated by complexing with lipids, liposomes, or lipoplexes. For example, the complexing can include contacting the at least one 5' capped mRNA molecule with a PEG-lipid or a zwitterionic lipid comprising a headgroup, where the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the head group. In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule can be formulated with a lipid bilayer carrier.

In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule comprises can be formulated with a natural or synthetic polymer. Non-limiting examples of such polymers can include chitosan or cyclodextrin. In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule comprises can be formulated in a polymeric formulation comprising polymer such polyethenes, polyethylene glycol (PEG), poly(l- lysine)(PLL), cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, acrylic polymers, amine-containing polymer, dextran polymer, dextran polymer derivative, or a combination thereof. In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule comprises can be formulated in a polyplex with one or more polymers commonly used in pharmaceutical formulation. In some embodiments, the polyplex comprises two or more cationic polymers such as poly(ethylene imine) (PEI). In some embodiments, the pharmaceutical composition comprising the at least one 5' capped mRNA molecule comprises can be formulated as a nanoparticle using a combination of polymers, lipids, or other biodegradable agents. In some embodiments, the lipid nanoparticles may comprise a core of the 5' capped mRNA described herein and a polymer shell. The polymer shell can be any of the polymers known in pharmaceutical formulation.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable: carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition described herein includes at least one additional active agent described herein. In some embodiments, the at least one additional active agent is a chemotherapeutic agent, cytotoxic agent, cytokine, growth- inhibitory agent, anti-hormonal agent, anti-angiogenic agent, or checkpoint inhibitor. In some embodiments, the at least one additional active agent is an adjuvant for increasing effectiveness of vaccination. In practicing the methods of treatment or use provided herein, therapeutically effective amount of pharmaceutical composition described herein is administered to a mammal having a disease, disorder, or condition to be treated. In some embodiments, the mammal is a human. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used and other factors. The therapeutic agents, and in some cases, compositions described herein, may be used singly or in combination with one or more therapeutic agents as components of mixtures.

The pharmaceutical composition described herein may be administered to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The composition described herein may include, but not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended-release formulations, pulsatile release formulations, multi-particulate formulations, and mixed immediate and controlled release formulations.

The pharmaceutical composition including a therapeutic agent may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, chaotic mixing, laminar mixing, dissolving, encapsulating or other processes.

The pharmaceutical composition may include at least an exogenous therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these biomolecules having the same type of activity. In some embodiments, therapeutic agents exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents are also considered to be disclosed herein.

In certain embodiments, the pharmaceutical composition provided herein includes one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetyl pyridinium chloride.

Use of absolute or sequential terms, for example, "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "subsequently," "before," "after," "lastly," and "finally," are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

As used herein, the phrases "at least one", "one or more", and "and/or" are open- ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C", "at least one of A, B, or C", "one or more of A, B, and C", "one or more of A, B, or C" and "A, B, and/or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

As used herein, "or" may refer to "and", "or," or "and/or" and may be used both exclusively and inclusively. For example, the term "A or B" may refer to "A or B", "A but not B", "B but not A", and "A and B". In some cases, context may dictate a particular meaning.

Any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as "first" and "second" do not necessarily imply priority, order of importance, or order of acts.

The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term "about" refers to ±10% of a stated number or value. The terms "increased", "increasing", or "increase" are used herein to generally mean an increase by a statically significant amount. In some embodiments, the terms "increased," or "increase," mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

The terms "decreased", "decreasing", or "decrease" are used herein generally to mean a decrease by a statistically significant amount. In some embodiments, "decreased" or "decrease" means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

The terms "RIMA" or "RNA molecule" are used herein to generally refer to any type RNA. Non-limiting example of RNA includes long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single stranded RNA (ssRNA), double stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), selfamplifying mRNA (SAM), Trans amplifying mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR/Cas9 guide RNAs, riboswitches, immunostimulating RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), transcription start site-associated (TSSa-)RNAs, upstream antisense (ua) RNAs, promoter upstream transcripts (PROMPTS), or a combination thereof. While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

The following illustrative examples are representative of embodiments of the systems and methods described herein and are not meant to be limiting in any way.

Example 1. Capping diluted nucleic acid

Uncapped RNA transcribed in vitro can be diluted before the capping reaction. Such dilution decreases the concentration of a plurality of molecules that inhibits capping reaction. The diluted uncapped RNA can then be contacted with capping reagents for initiating the capping reaction. Fig. 2 illustrates a non-limiting example for diluting and capping the in vitro transcribed (IVT) RNA. The solution containing the uncapped RNA molecules can be diluted. IVT reagents and reactions can be used to synthesize uncapped IVT RNA. The solution containing the uncapped IVT RNA can be diluted prior to the addition of capping reagents for capping reactions. The capping efficiency is then calculated as the proportion of capped RNA relative to the sum of uncapped and capped RNA. The capped RNA can also be purified for additional downstream processing. Example 2. Method of producing capped RNA

IVT Reaction

A linearized DNA template of a gene of interest was transcribed into mRNA using an IVT reaction. The IVT reaction comprised nucleotide triphosphates (NTPs), a RNase inhibitor and a DNA-dependent RNA polymerase in a transcription buffer. The IVT reaction was carried out at 37°C for 1 to 3 hours. mRNA sample dilution

After the completion of the IVT reaction, the IVT reaction units were placed on ice to cool and the IVT reaction liquid was transferred to new tubes where RNase-free water was added to obtain a dilution factor of 4x and one of lOx. An undiluted control was also prepared. In an alternative embodiment, the RNase-free water, is directly incorporated to the IVT reaction unit as well. mRNA capping

The diluted mRNA samples were heated at 65°C for 5 minutes and then cooled on ice. The enzymatic capping reaction was carried out with a NEB® kit (New England BioLabs) according to the supplier protocol. Briefly, the NEB® kit components, namely, lOx capping buffer, GTP 10 mM, SAM (4 mM) and Vaccinia capping enzyme (10 U/pl) were added to the reaction unit, together with mRNA Cap 2 '-O- Methyltransferase (50 U/pl) and a RNase inhibitor, not included in the kit. The capping reaction was carried out at 37°C for 60 minutes.

Capping efficiency and analysis of capped mRNA species

The capped mRNA was incubated with RNase A and probes to protect the 5' end from degradation. The capped mRNA was isolated and purified with magnetic beads. After elution, the small RNA fragments were analyzed by denaturing ion-paired reversed- phase high performance liquid chromatography (IP-RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) to determine the percentages of cap 1, cap 0, unmethylated cap and uncapped RNA in the sample.

Results: The capping efficiencies of the mRNA diluted directly after the IVT reaction and capped afterward are shown in Table 1. The results show that the enzymatic capping was achieved for both dilution conditions (4- and 10-fold) and efficiencies above 95% were obtained when the capping was performed on a 10-fold diluted IVT reaction product. Concluding, mRNA can be efficiently capped directly after its in vitro transcription with a capping efficiency that is correlated to the dilution step of the IVT reaction product. The dilution of the IVT solution is mandatory as the control condition (i.e., without any dilution) failed to be capped.

Table 1. Capping efficiency of mRNA capped after dilution

Capping unmethylated cap

Conditions cap 0 % cap 1 %

Control: undiluted IVT

RNA followed by

0.0% 0.0% 0.0% 0.0% enzymatic capping process

Condition 1: 4-fold dilution of IVT RNA reaction product 77.7% 34.3% 2.6% 40.9% followed by enzymatic capping process

95.3% 14.7% 1.1% 77.5%

Condition 2: 10-fold dilution of IVT RNA 94.6% 14.2% 0.0% 80.3% reaction product t 98.6% 4.3% 0.0% 94.3% followed by enzymatic capping process

98.7% 4.1% 0.0% 94.6%

While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail may be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually and separately indicated to be incorporated by reference for all purposes.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

34 CLAIMS

1. A method for producing at least one capped ribonucleic acid (RNA) molecule, comprising: a. providing a plurality of uncapped RNA molecules in a first solution; b. diluting the first solution by at least 4-fold to form a second solution containing the plurality of uncapped RNA molecules; c. contacting the second solution with a plurality of capping enzyme molecules; and d. adding a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule.

2. The method of claim 1, wherein the first solution prior to the dilution step did not undergo a purification step.

3. The method of any of claims 1 or 2, wherein the capping step is performed on the diluted second solution.

4. The method of any of claims 1 to 3, wherein diluting the first solution comprises adding a volume of a diluent to said first solution.

5. The method of claims 1-4, further comprising removing an excess volume of the second solution.

6. The method of claim 5, wherein removing the excess volume comprises ultrafiltration, microfiltration, tangential flow filtration, or a combination thereof.

7. The method of claim 5 or 6, wherein diluting the first solution does not comprise an additional purifying step.

8. The method of claim 7, wherein the additional purifying step comprises chromatography.

9. The method of any of claims 1-10, wherein diluting the first solution occurs in a same vessel or reactor as the plurality of uncapped RNA molecules generated via an in vitro transcription (IVT) reaction.

10. The method of any of claims 1-8, wherein diluting the first solution occurs in a different vessel or reactor as the plurality of uncapped RNA molecules generated via an IVT reaction.

11. The method of claims 1 to 10, wherein the IVT reaction occurs in a continuous reactor or a batch reactor.

12. The method of any of claims 1-11, wherein the plurality of capping enzyme is selected from the group consisting of Cap-specific mRNA (nucleoside-2'-O-)- methyltransferase, Vaccinia capping enzyme (VCE), Bluetongue Virus capping enzyme, Chlorella Virus capping enzyme, S. cerevisiae capping enzyme, Mimivirus 35 capping enzyme, African swine fever virus capping enzyme, and Avian Reovirus capping enzyme.

13. The method of any of claims 1-12, wherein the adding the cap structure to the 5' end of the uncapped RNA molecule occurs at an efficiency of at least 70%, at least 75%, at least 77%, at least 80%, at least 85%, at least 87%, at least 90%, at least

95%, at least 97%, at least 98%, at least 99%, or 100%.

14. The method of any of claims 1-13, wherein the RNA comprises a nucleic acid sequence encoding a peptide or protein.

15. The method of any of claims 1-14, wherein in the IVT reaction a RNA polymerase is used, and wherein the RNA polymerase is selected from the group consisting of a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, a RNA polymerase I, a RNA polymerase II, a RNA polymerase III, a RNA polymerase IV, a RNA polymerase V, and a single subunit RNA polymerase.

16. The method of any of claims 1-15, further comprising synthesizing a peptide or protein utilizing the at least one capped mRNA molecule.

17. The method of any of claims 1-16, wherein diluting the first solution comprises decreasing a concentration of a plurality of molecules that inhibits a capping reaction of adding a capping structure to a 5' end of an uncapped RNA molecule.

18. The method of claim 17, wherein the concentration of the plurality of molecules is decreased to a level that the capping reaction is no longer inhibited.

19. The method of any of claims 1-18, wherein diluting the first solution comprises decreasing a concentration of the plurality of uncapped RNA molecules.

20. The method of claim 19, wherein the concentration of the plurality of uncapped RNA molecules is decreased to a level that the capping reaction is no longer inhibited.

21. A pharmaceutical composition obtained using the method of any of claims 1-20.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is a vaccine and a pharmaceutical acceptable carrier.

23. A peptide or protein obtained using the method of any of claims 1-20.

24. The peptide or protein of claim 23, wherein the peptide or protein is produced in vivo.

25. The peptide or protein of claim 23, wherein the peptide or protein is produced in vitro.

26. The peptide or protein of claims 23-25, wherein the peptide or protein is a prophylactic or a therapeutic peptide or protein.

27. A composition comprising a plurality of 5' capped RNA molecules, wherein said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping occurred post-transcriptionally, said composition comprises reagents for in vitro transcription, and wherein the concentration of RIMA in said composition is less than 20 mg/ml.

28. A composition comprising a plurality of 5'-capped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping reaction occurred post-transcriptionally, wherein the capping reaction efficiency is at least 75% without utilizing chromatography.

29. A composition comprising a plurality of 5'-capped and uncapped RNA molecules, said RNA molecules are obtained by means of an in vitro transcription reaction and wherein said capping occurred post-transcriptionally, said composition comprises reagents for in vitro transcription wherein a ratio between the plurality of uncapped RNA molecules and the plurality of capped RNA molecules is between 0.0001 to 1.

30. A system for producing at least one capped ribonucleic acid (RNA) molecule, comprising: a bioreactor configured to contain a first solution containing a plurality of uncapped RNA molecules, wherein: the first solution is diluted by at least 4-fold to form a second solution, wherein the second solution comprises the plurality of uncapped RNA molecules; and the bioreactor or a second bioreactor configured to add a cap structure to a 5' end of an uncapped RNA molecule to form at least one capped RNA molecule by contacting the second solution with a plurality of capping enzyme molecules.