Fed-batch in vitro transcription process

Abstract

To provide empirically-balanced fed-batch in vitro transcription (IVT) methods and compositions that enable, inter alia, high yield, high integrity transcription of ribonucleic acid (RNA), such as messenger RNA (mRNA).SOLUTION: The present disclosure provides methods of in vitro transcribing a ribonucleic acid (RNA) of interest, comprising determining consumption rates of nucleoside triphosphates (NTPs).SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 816,734, filed March 11, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] In vitro transcription (IVT) uses bacteriophage DNA-dependent ribonucleic acid (RNA) polymerases (e.g., SP6, T3, and T7) to synthesize template-directed mRNA transcripts. IVT reactions are generally "batch" reactions in that several reagents, including nucleoside triphosphates (NTPs), magnesium, RNA polymerase, deoxyribonucleic acid (DNA), and pyrophosphatase, are mixed at the beginning of the reaction. These components are then incubated, and the reaction proceeds until at least one of the nucleotides is depleted. Thus, the reaction has at least one limiting reagent, which can cause low yields of RNA transcripts (products). Other potential drawbacks of IVT reactions include, for example, abortive (truncated) transcripts, run-on transcripts, poly-A tail variants that result in 3' heterogeneity, mutated transcripts, and / or double-stranded contaminants generated during the reaction. Summary of the Invention

[0003] The present disclosure provides, in some embodiments, empirically balanced fed-batch in vitro transcription (IVT) methods and compositions that, among other things, enable high-yield, reliable transcription of ribonucleic acid (RNA), e.g., messenger RNA (mRNA). Surprisingly, in some embodiments, RNA transcripts having lengths of at least 2000 nucleotides or more can be synthesized with yields at least 100% higher than the yields of RNA transcripts produced using known IVT methods, at least 90% of which are correctly capped using co-transcriptional capping. In certain embodiments, the empirically balanced fed-batch IVT reactions of the present disclosure include four nucleotides, namely CTP, GTP, UTP, and ATP, the relative molar ratios of each of which can be varied to maximize reactant usage and / or alter the attributes of the RNA product. Importantly, the ratios of these four nucleotides are balanced according to their rates of consumption, so that no single nucleotide is rate-limiting during the IVT reaction. Advantageously, and unlike previously described fed-batch methods, the present process does not require prior knowledge of the RNA product sequence, nor does it rely on the known sequence of the target RNA product.

[0004] The process, in some embodiments, involves performing an initial batch of IVT reactions with DNA encoding an RNA of interest using known initial concentrations of CTP, GTP, UTP, and ATP (e.g., equimolar concentrations, e.g., 5 mM), and measuring the rate at which the concentration of at least one of these NTPs decreases below a threshold value between 20 and 40 minutes over the course of the actual reaction. The collected measured concentrations and times can then be used to calculate the CTP, GTP, UTP, and ATP consumption rates (individual NTP consumption rates) during the reaction. For multiple time points, this can be the slope of a linear approximation of the data (see, e.g., FIG. 1A). The individual NTP consumption rates (for CTP, GTP, UTP, and ATP) are summed to determine the consumption rate of all nucleotides (total NTP consumption rate) (see, e.g., FIG. 1B). A percentage (%) value is then determined for each NTP by dividing the individual NTP consumption rate by the total NTP consumption rate.

[0005] The percent consumption values ​​can be used to formulate the initial "master" reaction mixture for the IVT reaction and the "feedstock" mixture. For example, to allow for unlimited consumption of all NTPs in a batch reaction, the initial reaction mixture can contain NTPs in molar ratios equal to the calculated percent consumption (%) value for each NTP (ATP, UTP, GTP, and CTP).

[0006] In some embodiments, the initial NTP concentrations comprise equimolar NTP concentrations of each [ATP], [CTP], [UTP], and [GTP] (eg, a molar ratio of 1:1:1:1).

[0007] In some embodiments, the initial NTP concentration includes non-equimolar NTP concentrations of each [ATP], [CTP], [UTP], and [GTP] (e.g., a molar ratio of 2:1:1:4). In some embodiments, the molar ratio of [ATP]:[UTP]:[CTP]:[GTP] is 1:1:1:1 to 2:1:1:4. In some embodiments, the molar ratio of [ATP]:[CTP] is 2:1. In some embodiments, the molar ratio of [ATP]:[UTP] is 2:1. In some embodiments, the molar ratio of [ATP]:[GTP] is 1:2. In some embodiments, the molar ratio of [CTP]:[UTP] is 1:1. In some embodiments, the molar ratio of [CTP]:[GTP] is 1:4. In some embodiments, the molar ratio of [UTP]:[GTP] is 1:4. In some embodiments, the initial NTP concentration comprises an [ATP]:[UTP] ratio of 1:1 to 2:1 and / or an [GTP]:[CTP] ratio of 1:1 to 2:1. In some embodiments, the initial NTP concentration comprises an [ATP]:[UTP] ratio of 2:1 to 4:1 and / or an [GTP]:[CTP] ratio of 1:1 to 4:1. In some embodiments, the initial NTP concentration comprises an [ATP]:[UTP] ratio of 2:1 and / or an [GTP]:[CTP] ratio of 4:1.

[0008] These % consumption values ​​are empirically determined and are specific to the RNA of interest. During the IVT reaction, the reaction mixture may be supplemented with a feed mixture, the feed mixture comprising NTPs, each NTP being present in the feed mixture in a molar ratio based on the % consumption value calculated for each NTP. In some embodiments, the feed mixture is supplemented in an amount that maintains the total NTP concentration in the reaction mixture greater than 0 mM, but at least 5%-50% of the initial NTP concentration. In some embodiments, the feed mixture is supplemented in an amount that maintains the total NTP concentration in the reaction mixture greater than 0 mM, but at least 5%-100% or at least 5%-200% of the initial NTP concentration. In some embodiments, the feed mixture is supplemented in an amount that maintains the total NTP concentration in the reaction mixture greater than 0 mM, but at least 5%-100% or at least 5%-200% of the initial NTP concentration. In some embodiments, the feed mixture is supplemented in an amount that maintains the total NTP concentration in the reaction mixture between 5 mM and 20 mM, and / or within 5%-75% of the initial NTP concentration. In some embodiments, the feed mixture is supplemented with an amount to maintain a ratio of [ATP]:[UTP] between 1:1 and 4:1, optionally between 1:1 and 2:1 (e.g., 2:1). In some embodiments, the feed mixture is supplemented with an amount to maintain a ratio of [GTP]:[CTP] between 1:1 and 4:1 (e.g., 4:1). Improvements in the IVT reaction have led to the development and incorporation of cap analogs called trinucleotides (see PCT / US2018 / 046989, incorporated herein by reference). This allows for co-transcriptional capping of the RNA product, eliminating the need for a subsequent processing step to cap the RNA. The trinucleotides are preferentially incorporated at the 5' end of the RNA product over mononucleotides in the reaction mixture, although some purines (ATP and GTP) may be incorporated in place of the cap analog. Such events lead to the generation of uncapped RNA products, which are undesirable variants of the inactive product. Our testing shows that maintaining a trinucleotide to purine ratio of 1 or greater allows for an acceptable concentration of capped RNA relative to uncapped RNA in the product.

[0009] When performing an IVT reaction using co-transcriptional capping, the consumption of cap analogs relative to the total nucleotides in the reaction to generate mRNA is extremely low. Only one mole of cap analog is consumed per mole of capped RNA product, whereas up to 5,000 or more moles of nucleotides are consumed per mole of capped RNA product (actual consumption varies depending on the sequence and length of the product). As a result, a large excess of expensive cap analogs remains unconsumed during the IVT reaction after NTPs are consumed. In batch reactions, this low utilization of cap analogs (e.g., trinucleotide capping reagents) contributes to a more expensive and wasteful process.

[0010] The fed-batch process of the present disclosure improves utilization of plasmid DNA, cap analogs (e.g., trinucleotide or tetranucleotide capping reagents) and other expensive reagents while maintaining high capping % RNA production and mRNA yield. For example, in the present disclosure, NTPs and magnesium can be added to active IVT reactions to prevent reactant depletion and improve RNA product yield without supplying additional DNA or cap analogs (e.g., trinucleotides). In another example, adding NTPs at a lower initial concentration and maintaining this low level throughout the reaction may allow for the use of lower concentrations of cap analog without compromising capping efficiency. Compared to the IVT batch and fed-batch processes described previously, the empirically determined consumption % balanced fed-batch IVT process provided herein showed a greater than 2-fold improvement in capped RNA yield relative to the amount of cap analog used in some embodiments involving co-transcriptional capping. Additionally, the molar ratio of generated RNA to input DNA used in the fed-batch IVT process provided herein is significantly increased. In some embodiments, the molar ratio of produced RNA to input DNA used in the fed-batch IVT processes provided herein is increased by 2- or even 3-fold relative to non-fed-batch or batch processes.

[0011] Thus, provided herein, in some aspects, is a method for determining the NTP consumption percentage (%) of an IVT reaction, the method comprising: (a) performing an IVT reaction in a reaction mixture comprising a known initial NTP concentration, a DNA encoding an mRNA of interest, an RNA polymerase, and a pyrophosphatase; and (b) calculating a consumption percentage (%) value for each NTP in the reaction mixture. In some embodiments, step (b) comprises (i) calculating the individual NTP consumption rate (individual NTP concentration / time) / total NTP consumption rate (total NTP concentration / time). In some embodiments, the known initial NTP concentration is an equimolar NTP concentration.

[0012] Also provided herein, in some aspects, is a method for fed-batch IVT of an RNA of interest, comprising: (a) performing an IVT reaction in a reaction mixture comprising DNA encoding the RNA of interest, an RNA polymerase, and NTPs (and optionally magnesium and / or pyrophosphatase); and (b) supplying the IVT reaction mixture with a feed mixture comprising NTPs (and optionally magnesium and / or pyrophosphatase) over time, each NTP being present in a molar ratio based on a calculated % consumption value for each NTP in a particular RNA of interest, the % consumption value being specific to the RNA of interest, and the feed mixture being supplied in an amount that maintains the total NTP concentration in the reaction mixture above zero. In some embodiments, each NTP in the reaction mixture of step (a) is present in a molar ratio equivalent to the calculated percentage consumption value for each NTP.

[0013] In some embodiments, the NTPs are balanced using the consumption percentage.In other embodiments, the NTPs are maintained at different ratios to change the product properties or improve the utilization of different NTPs.For example, the consumption percentage can be used to maintain desired conditions throughout the reaction.

[0014] The feed mixture can be fed to the ongoing IVT reaction mixture using a bolus or a continuous feed over time, e.g., every 10-250 minutes, optionally every 20-200 minutes. Continuous feeding includes feeding the feed mixture to the ongoing IVT reaction mixture at a continuous flow rate, e.g., 2-8 mL / min or 4-6 mL / min.

[0015] In some embodiments, the reaction mixture further comprises an RNA cap analog, thereby generating transcribed RNA that incorporates the RNA cap analog (e.g., at least 90% or at least 95% of the transcribed RNA comprises the RNA cap analog).The initial and / or ongoing IVT reaction mixture comprises an [RNA cap analog]:[purine] ratio of 1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1. [Brief description of the drawings]

[0016] [Figure 1] Example of empirical equilibration of nucleotides. In vitro transcription (IVT) reactions were performed with known initial nucleotide concentrations. The concentrations of CTP, GTP, UTP, and ATP (A) and total nucleotide concentration (B) were measured over time. The % consumption of each nucleotide was calculated by dividing the individual NTP consumption rate (e.g., identified in A) by the total NTP consumption rate (e.g., identified in B). [Diagram 2] Observation of NTP and cap analog concentrations. NTP and trinucleotide concentrations can be observed over time using ultra-performance liquid chromatography (UPLC-UV) with a UV photodetector set at 260 nm. This method can also be used to calculate individual NTP concentrations or total NTP concentrations. [Diagram 3] Example of empirical equilibration of nucleotides for two sample constructs, RNA#1 (A) and RNA#2 (B). Concentrations of CTP, GTP, UTP, and ATP and total nucleotide concentrations were measured over time. The % consumption of each nucleotide was calculated by dividing the individual NTP consumption rate by the total NTP consumption rate. [Figure 4] Example of two NTP master mixes generated from % consumption of RNA#1 to achieve different process requirements. Batch IVT in A shows that all four nucleotide reagents reach 0 mM at approximately the same time, thus maximizing reagent utilization. B batch IVT reaction in B shows that for 10 mM total [NTP], all four nucleotide reagents reach 2.5 mM at the same time. The latter scenario is particularly useful for developing fed-batch IVT reactions that require specific NTP ratios in the reaction to generate a desired product profile or to fully utilize specific reagents. [Diagram 5] Determination of Fed-Batch Feed Schedule. Scouting fed-batch IVT reactions were performed using the DNA of interest (RNA#1 and RNA#2 shown in A and B, respectively), an empirically balanced nucleotide master mix, and an initial feed schedule based on predicted NTP consumption rates. Throughout this experiment, total nucleotide concentrations were measured at time points immediately prior to and after feeding of the IVT reactions. [Figure 6] Determination of Fed-Batch Feed Schedule. Scouting fed-batch IVT reactions were performed using the DNA of interest (RNA#1 and RNA#2 shown in A and B, respectively), an empirically balanced nucleotide master mix, and an initial feed schedule based on predicted NTP consumption rates. These concentrations and times were used to calculate the consumption rates between each feed for reactions producing either RNA#1 or RNA#2. [Figure 7] Nucleotide consumption rates were plotted versus either [DNA] (A), [mRNA] (FIG. 7B), or reaction time (C) for the scouting fed-batch IVT of RNA#1. Curve fits (linear approximations shown) provide an empirical model of the nucleotide consumption rates throughout the run of a fed-batch IVT reaction. [Figure 8]IVT reaction modeling of RNA#1. Additional construct-specific parameters were used to generate a reaction model for RNA#1, including the ratio of CTP, GTP, UTP, and ATP in the master mix, the consumption rates of CTP, GTP, UTP, and ATP, the initial concentration of cap analog, the initial % tailed RNA product, and the % tailed RNA product over time, to estimate concentrations of NTP or trinucleotide reactants. [Figure 9] IVT reaction modeling of RNA#1. Additional construct-specific parameters including the ratio of CTP, GTP, UTP, and ATP in the master mix, the consumption rates of CTP, GTP, UTP, and ATP, the initial concentration of cap analog, the initial % tailed RNA product, and the % tailed RNA product over time were used to generate a reaction model for RNA#1 to estimate RNA product production. [Figure 10] IVT reaction modeling of RNA#1. Additional construct-specific parameters including the ratio of CTP, GTP, UTP, and ATP in the master mix, the consumption rates of CTP, GTP, UTP, and ATP, the initial concentration of cap analog, the initial % tailed RNA product, and the % tailed RNA product over time were used to generate a reaction model for RNA#1 to estimate the % tailed RNA product. [Figure 11] [NTP] measurements for specific fed-batch IVT reactions of RNA#1. A-B indicate the total and individual [NTP] measurements during the fed-batch IVT reaction of RNA#1. [Figure 12] [NTP] measurements for a particular fed-batch IVT reaction of RNA#2. A-B indicate the total and individual [NTP]s measured during the fed-batch IVT reaction of RNA#2. [Figure 13] Measurement of RNA product yield for certain fed-batch IVT reactions of RNA#1 and RNA#2. A-B show the measured concentrations of total RNA and tailed RNA for RNA#1 and RNA#2. [Figure 14]Measured RNA product yields for certain fed-batch IVT reactions of RNA#1 and RNA#2. A-B show the measured mass of total and tailed RNA versus initial IVT reaction volume for RNA#1 and RNA#2. [Figure 15] Measurement of % tail RNA for specific fed-batch IVT reactions of RNA#1 and RNA#2. [Figure 16] Graph A shows the measured % of capped RNA product for RNA#1 and RNA#2. Graph B shows the measured % of capped RNA product over time for RNA#1. [Figure 17] Total nucleotide concentrations measured during exemplary bolus (A) and continuous (B) fed-batch IVT reactions for RNA#3. [Figure 18] RNA product yield measurements for bolus and continuous fed-batch IVT reactions of RNA#3. Measured concentrations of tailed RNA are shown for the bolus (A) and continuous (B) processes. [Figure 19] Measured RNA product yield for bolus and continuous fed-batch IVT reactions of RNA#3. Shown is the measured mass of tailed RNA versus initial IVT reaction volume for the bolus (A) and continuous (B) processes. [Figure 20] Measured % of tailed RNA product in bolus (A) and continuous (B) fed-batch IVT reactions for RNA#3. [Figure 21] Measured % capped RNA product in bolus (A) and continuous (B) fed-batch IVT reactions for RNA#3. [Figure 22] Measurement of average A / GTP to trinucleotide ratios across continuous fed-batch reactions. [Figure 23] [ATP] measurements through fed-batch IVT reactions compared to batch IVT reactions. [Figure 24] Total RNA yield measurements over a 2 hour reaction time for fed-batch IVT reactions compared to batch IVT reactions (mg / mL initial IVT). [Diagram 25]Shown is the % capped RNA product after fed-batch IVT reactions compared to batch IVT reactions after 2 hours reaction time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Provided herein is an empirically balanced fed-batch in vitro transcription (IVT) platform developed to improve the overall product yield of a single batch and maintain high product quality while increasing reactant utilization. Unlike existing IVT methods that have low utilization of reaction components, high percentage of unused NTPs, and require high concentrations of RNA cap analogs or trinucleotide capping reagents, the empirically balanced fed-batch IVT method of the present disclosure maximizes the use of expensive reagents present in the reaction, increasing product yield without compromising product reliability. This improved fed-batch IVT process, in some embodiments, begins with an empirical balancing reaction of initial nucleotides. This is used to calculate the percentage consumption (%) value for each NTP type in the initial reaction specific to the RNA being transcribed. These empirically determined values ​​are then used to balance the nucleotide ratios in subsequent fed-batch IVT reactions for efficient, high-yield RNA production.

[0018] Consumption percentage as used herein generally refers to the relative consumption rate of each NTP compared to the consumption rate of all NTPs for a given DNA encoding RNA of interest.In some embodiments, consumption rate percentage is determined by dividing each NTP consumption rate by total NTP consumption rate.In some embodiments, each NTP consumption rate is a measure of each NTP concentration over time (e.g., during the empirical equilibration reaction of nucleotide), and total NTP consumption rate is a measure of total NTP concentration over time (e.g., during the empirical equilibration reaction of nucleotide).

[0019] As used herein, empirical equilibration of NTPs generally refers to a process used to determine the percentage consumption of each NTP with respect to DNA encoding an RNA of interest. The process involves performing a batch IVT reaction with known initial concentrations of CTP, GTP, UTP, and ATP, and measuring the rate at which the concentration of at least one of these NTPs decreases during the course of the reaction, for example, over a period of 20-40 minutes, until the concentration of each nucleotide falls below a threshold value, for example, 1-5 mM or 20-50% of the starting concentration. The collected measured concentrations and times can then be used to calculate the percentage consumption of CTP, GTP, UTP, and ATP consumption during the reaction.

[0020] As used herein, batch IVT generally refers to an in vitro transcription reaction in which all components of the reaction, e.g., NTPs, polymerase, salts, and / or DNA, are added to the reaction mixture only once, e.g., at the beginning of the reaction. In some embodiments, the reaction mixture is held at specific conditions, e.g., temperature, for a period of time, after which the transcribed RNA product is collected.

[0021] As used herein, fed-batch IVT generally refers to an in vitro transcription reaction in which an active reaction mixture is replenished with reaction components, e.g., NTPs, polymerase, salts, and / or DNA, to prevent depletion of limiting reagents or to counteract degradation of labile components as the reaction proceeds over time. In some embodiments, the mixture added to the reaction is referred to as a feedstock. In some embodiments, reaction components or feedstocks are replenished to the active reaction mixture using a bolus feed. In some embodiments, a bolus feed involves the addition of discrete volumes of reaction components or feedstocks to the active reaction mixture at specific time intervals, e.g., the addition of 5 mL of feedstock to the reaction mixture every 20 minutes. In some embodiments, reaction components or feedstocks are replenished to the active reaction mixture using a continuous feed. In some embodiments, continuous feeding includes the addition of a reactant or feedstock to an active reaction mixture by a continuous flow rate of the reactant feedstock over a specified period of time, e.g., addition of the feedstock at a continuous flow rate of 2-8 mL / min, 2-6 mL / min, or 4-6 mL / min, or at a continuous flow rate of 0.0030-0.007 mL / min per mL of initial volume, 0.0040-0.0060 mL / min per mL of initial volume, 0.0050-0.0080 mL / min per mL of initial volume, or 0.0060-0.0090 mL / min per mL of initial volume.

[0022] As used herein, Scouting Fed-Batch IVT generally refers to an initial Fed-Batch IVT reaction using a bolus feed that is used to model the reaction rate(s) over time and to set appropriate feed volumes and times in downstream Fed-Batch IVT reactions.

[0023] As used herein, the percentage of tailed RNA generally refers to the relative abundance of transcription RNA products that contain a 3' polyA tail. In some embodiments, the 3' polyA tail is 100It is a poly-A tail (i.e., consists of 100 alanine residues). In some embodiments, the percentage of tailed RNA (the percentage (%) of transcription RNA products that contain a 3' poly-A tail) is greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. In some embodiments, the percentage (%) of tailed RNA is greater than 90%, 95%, 97%, or 99%. In some embodiments, the percentage of tailed RNA (percentage of transcribed RNA products that contain a 3' polyA tail) is between 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 25-75%, 30-50%, 40-60%, 50-70%, 45-60%, 55-70%, 60-80%, 60-100%, 75-100%, 50-95%, 75-95%, 80-100%, 80-90%, 90-95%, 95-100%, 90-99%, or 95-99%.

[0024] As used herein, percent capped RNA generally refers to the relative abundance of transcribed RNA products that contain a cap analog incorporated at their 5' ends. In some embodiments, the cap analog is an RNA cap analog. In some embodiments, the RNA cap analog is a dinucleotide, trinucleotide, or tetranucleotide. In some embodiments, the percent capped RNA (percentage of transcribed RNA products that contain a 5' cap analog) is greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. In some embodiments, the percent capped RNA is greater than 90%, 95%, 97%, or 99%. In some embodiments, the percentage of RNA that is capped is 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 25-75%, 30-50%, 40-60%, 50-70%, 45-60%, 55-70%, 60-80%, 60-100%, 75-100%, 50-95%, 75-95%, 80-100%, 80-90%, 90-95%, 95-100%, 90-99%, or 95-99%.

[0025] In some embodiments, an RNA cap analog is added to the IVT reaction mixture that preferentially initiates the 5' end of the RNA sequence during transcription. This allows for a "co-transcriptional capping" IVT reaction in which the IVT mixture contains an RNA cap. Examples of RNA cap analogs that allow for a "co-transcriptional capping" IVT reaction include 7-methylguanosine (m7G) and 3'-O-me-7-meGpppG.

[0026] As used herein, an initial IVT reaction mixture for use in fed-batch applications generally refers to an IVT reaction mixture at the beginning of a fed-batch IVT reaction, before additional reaction components or feedstocks are replenished. In some embodiments, the initial IVT reaction mixture includes NTPs (e.g., naturally occurring and / or modified NTPs, such as ATP, UTP, GTP, and CTP), buffers (e.g., Tris and / or Good's buffer), cofactors (e.g., magnesium), RNA cap analog(s) (e.g., trinucleotide cap analogs), RNA polymerase (e.g., T7 RNA polymerase), detergents (e.g., Triton X-100), DNA encoding an RNA of interest, reducing agents (e.g., dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP)), small molecule additives (e.g., spermidine), and / or enzyme additives (e.g., inorganic phosphatase (PPiase)).

[0027] As used herein, an ongoing IVT reaction mixture for use in fed-batch applications generally refers to an IVT reaction mixture after the initial reaction conditions. In some embodiments, the concentrations of components in the ongoing IVT reaction mixture, such as NTPs and RNA cap analogs, change over time. In some embodiments, the ongoing IVT reaction mixture includes NTPs (e.g., naturally occurring and / or modified NTPs, such as ATP, UTP, GTP, and CTP), buffers (e.g., Tris and / or Good's buffer), cofactors (e.g., magnesium), RNA cap analog(s) (e.g., trinucleotide cap analogs), RNA polymerase (e.g., T7 RNA polymerase), detergents (e.g., Triton X-100), DNA encoding the RNA of interest, reducing agents (e.g., DTT, TCEP), small molecule additives (e.g., spermidine), enzyme additives (e.g., inorganic phosphatase or PPiase), and / or the transcribed RNA of interest.

[0028] As used herein, the first coding position of the target RNA generally refers to the first nucleotide transcribed after a promoter sequence (e.g., T7 RNA polymerase promoter).In some embodiments, the first coding position is any NTP, such as ATP, UTP, GTP, or CTP.

[0029] As used herein, the RNA of interest generally refers to an RNA molecule encoded by DNA in an IVT reaction. In some embodiments, the RNA of interest is a transcribed RNA of interest, and the transcribed RNA of interest is produced by an IVT reaction, for example, a fed-batch IVT reaction. In some embodiments, the RNA of interest is an mRNA, optionally including a 5' cap, a 5' untranslated region (5'UTR), an open reading frame (ORF) encoding a protein of interest, a 3' untranslated region (3'UTR), and / or a polyA tail. In some embodiments, the 5' cap analog is a 5' trinucleotide cap.

[0030] Empirical equilibration of nucleotides Provided herein, in some embodiments, is a method for determining the percentage (%) of nucleoside triphosphate (NTP) consumption of an in vitro transcription (IVT) reaction. The percentage (%) of NTP consumption (also more simply referred to as "% consumed") is the value obtained per NTP of an initial nucleotide empirical equilibration reaction and is calculated using the following formula: % Consumption = (individual NTP consumption rate) / (total NTP consumption rate), where the individual NTP consumption rate is a measure of the individual NTP concentration over time (during the empirical equilibration reaction of the nucleotides), and the total NTP consumption rate is a measure of the total NTP concentration over time (during the empirical equilibration reaction of the nucleotides).

[0031] Thus, individual ATP consumption rates are calculated by measuring the concentration of consumed ATP at various times during the empirical equilibration reaction of the initial nucleotides until the concentration of the ATP, or the concentration of another NTP in the reaction, falls below a threshold value, e.g., 5 mM to 20 mM, and individual UTP consumption rates are calculated by measuring the concentration of consumed UTP at various times during the empirical equilibration reaction of the initial nucleotides until the concentration of the UTP, or the concentration of another NTP in the reaction, falls below a threshold value, e.g., 5 mM to 20 mM. The individual GTP consumption rates are calculated by measuring the concentration of consumed GTP at various time points during the empirical equilibration reaction of the initial nucleotide until the concentration of the GTP, or the concentration of another NTP in the reaction, falls below a threshold value, for example, 5 mM to 20 mM, and the individual CTP consumption rates are calculated by measuring the concentration of consumed CTP at various time points during the empirical equilibration reaction of the initial nucleotide until the concentration of the CTP, or the concentration of another NTP in the reaction, falls below a threshold value, for example, 5 mM to 20 mM.

[0032] The total NTP consumption rate is calculated by measuring the concentrations of all (eg, all four) NTPs consumed at various times during an empirical equilibration reaction of the nascent nucleotides.

[0033] Then, the % consumption value for ATP is calculated by dividing each ATP consumption rate by the total NTP consumption rate.Similarly, the percentage consumption value for UTP is calculated by dividing each UTP consumption rate by the total NTP consumption rate, the percentage consumption value for GTP is calculated by dividing each GTP consumption rate by the total NTP consumption rate, and the percentage consumption value for CTP is calculated by dividing each CTP consumption rate by the total NTP consumption rate.

[0034] The initial nucleotide empirical equilibration reaction, in some embodiments, includes a mixture of DNA (e.g., a DNA plasmid encoding an RNA of interest), an RNA polymerase (e.g., T7 polymerase), and NTPs. In the initial nucleotide empirical equilibration reaction, the starting concentrations of NTPs (e.g., each of ATP, UTP, GTP, and CTP) are known (predetermined). In some embodiments, the initial nucleotide empirical equilibration reaction also includes a buffer (e.g., Tris-HCl), magnesium (e.g., magnesium acetate), pyrophosphatase, and / or dithiothreitol (DTT). In some embodiments, the initial nucleotide empirical equilibration reaction also includes an RNA cap analog, such as a trinucleotide cap analog (e.g., GAG) as discussed elsewhere herein.

[0035] In some embodiments, the concentration of each NTP in the initial nucleotide empirical equilibration reaction is kept above zero (0) millimolar (mM). That is, the NTP concentrations (individual and total) are collected during the initial nucleotide empirical equilibration reaction only until one of the NTP concentrations falls below a certain threshold (the NTP consumption rate is calculated). In some embodiments, the threshold is 1 mM to 50 mM, 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 1 mM to 10 mM, 2 mM to 50 mM, 2 mM to 40 mM, 2 mM to 30 mM, 2 mM to 20 mM, 2 mM to 10 mM, 3 mM to 50 mM, 3 mM to 40 mM, 3 mM to 30 mM, 3 mM to 20 mM, 3 mM to 10 mM, 4 mM to 50 mM, 4 mM to 40 mM, 4 mM to 30 mM, 4 mM to 20 mM, 4 mM to 10 mM, 5 mM to 50 mM, 5 mM to 40 mM, 5 mM to 30 mM, 5 mM to 20 mM, or 5 mM to 10 mM. In some embodiments, the threshold is 10 mM to 20 mM. In some embodiments, the threshold is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. In some embodiments, the threshold is a lower limit of 2 mM to 10 mM, or higher than a lower limit of 2 mM to 9 mM, or higher than a lower limit of 2 mM to 8 mM, or higher than a lower limit of 2 mM to 7 mM, or higher than a lower limit of 2 mM to 6 mM, or higher than a lower limit of 2 mM to 6 mM.

[0036] In some embodiments, the threshold is within 5% to 200% of the initial NTP concentration. For example, the threshold can be within 5% to 175%, 5% to 150%, 5% to 125%, 5% to 100%, 5% to 75%, 5% to 50%, or 5% to 25% of the initial NTP concentration. In some embodiments, the threshold is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the initial NTP concentration. The initial NTP concentration is the total NTP concentration in the IVT reaction before transcription begins (after all IVT reaction components are mixed and before the polymerase is active).

[0037] The initial NTP concentration of the initial nucleotide empirical equilibration reaction is therefore known in some embodiments. In some embodiments, the known initial NTP concentration is an equimolar NTP concentration. That is, each NTP (e.g., ATP, UTP, GTP, and UTP) is present in the reaction in equimolar ratio (1:1:1:1). In other embodiments, the known initial NTP concentration is not an equimolar NTP concentration. For example, one or more NTPs (e.g., ATP and / or UTP) may be present in excess of other NTPs.

[0038] The timing of the empirical equilibration reaction of the initial nucleotides may vary. Advantageously, however, the reaction time may be as short as 10-60 minutes. In some embodiments, the empirical equilibration reaction time of the initial nucleotides is 10-50 minutes, 10-40 minutes, 10-30 minutes, 10-20 minutes, 20-60 minutes, 20-50 minutes, 20-40 minutes, 20-30 minutes, 30-60 minutes, 30-50 minutes, or 30-40 minutes. In some embodiments, the empirical equilibration reaction time of the initial nucleotides is 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes. Longer reaction times may be used depending on the time point at which the concentration of any one NTP falls below a threshold value.

[0039] Empirical equilibrated fed-batch in vitro transcription reactions The initial nucleotide empirical equilibration reaction is specific to a specific DNA encoding an RNA of interest, and the percentage consumption (%) value calculated from the initial reaction can be used in a subsequent fed-batch IVT reaction for the synthesis of a specific RNA of interest.Thus, in some embodiments, a fed-batch IVT method for an RNA of interest includes (a) performing an IVT reaction in a reaction mixture containing deoxyribonucleic acid (DNA) encoding an RNA of interest, RNA polymerase, and nucleoside triphosphates (NTPs), and (b) feeding the IVT reaction mixture with a feed mixture containing NTPs over time, each NTP being present in a molar ratio based on a percentage consumption value calculated for each NTP, the percentage consumption value being specific to the RNA of interest, and the feed mixture being provided in an amount that maintains the total NTP concentration in the reaction mixture above zero.

[0040] In some embodiments, the feed mixture is provided in an amount that maintains an [ATP]:[UTP] ratio of 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1. In some embodiments, the feed mixture is provided in an amount that maintains an [ATP]:[UTP] ratio of 2:1. In some embodiments, the feed mixture is provided in an amount that maintains an [GTP]:[CTP] ratio of 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1. In some embodiments, the feed mixture is provided in an amount that maintains an [GTP]:[CTP] ratio of 4:1. In some embodiments, the feed mixture is provided in an amount that maintains an [ATP]:[UTP] ratio of 2:1 and an [GTP]:[CTP] ratio of 4:1.

[0041] The empirically balanced fed-batch IVT reaction mixture, in some embodiments, includes components selected from the following: deoxyribonucleic acid (DNA), ribonucleic acid (RNA) polymerase, nucleoside triphosphate (NTP), RNA cap analog, buffer, magnesium, pyrophosphatase, and reducing agent (e.g., dithiothreitol). The feedstock mixture, on the other hand, includes NTP, RNA polymerase, buffer, magnesium, pyrophosphatase, and / or reducing agent, but generally does not include DNA and RNA cap analog. The exact conditions used in the IVT reaction will vary, for example, depending on the amount of RNA required for a particular use. Similarly, the total transcription reaction time can vary, but in some embodiments, the total transcription reaction time is longer than a conventional IVT reaction time. In some embodiments, the total transcription reaction time is between 100 minutes and 1000 minutes. For example, the total transcription reaction time is 100-800, 100-600, 100-400, 150-1000, 150-800, 150-600, 150-400, 200-1000, 200-800, 200-600, 200-400, 200-800, 200-600, 200-400, 300-1000, 300-800, 300-400, 300-500, 300-600, 300-800, 300-1000, 300-800, 300-400, 300-1000, 300-200, 300-300, 300-400, 300-500, 300-600, 300-800, 300-400, 300-500, 300-600, 300-800, 300-1000, 300-1000, 300-200, 300-300, 300-400, 300-500, 300-600, 300-400, 300-500, 300-600, 300-800, 300-400, 300-1000, 300-1000, 300-200, 300-300, 300-400, 300-500, 300-600, 300-400, 300-50 The time may be 00-600, 300-400, 300-800, 300-600, 300-400, 400-1000, 400-800, 400-600, 400-400, 400-800, 400-600, 500-1000, 500-800, 500-600, 500-500, 500-800, or 500-600 minutes. In some embodiments, the total transcription reaction time is at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 minutes.

[0042] DNA encoding the RNA of interest The DNA may be single-stranded or double-stranded. In some embodiments, the DNA is present on a plasmid or other vector. The DNA may include a polynucleotide encoding a polypeptide of interest. In some embodiments, the DNA may include an RNA polymerase promoter (e.g., a T7 RNA polymerase promoter) located 5' from the polynucleotide encoding the polypeptide of interest and operably linked to the polynucleotide. The DNA may also include a nucleotide sequence encoding a polyadenylation (poly A) tail located at the 3' end of the polynucleotide.

[0043] The length of the DNA, and therefore the length of the RNA of interest, may vary. For example, the DNA (and / or the RNA of interest) may have a length of 200 nucleotides to 10,000 nucleotides. In some embodiments, the DNA (and / or the RNA of interest) has a length of 200-500, 200-1000, 200-1500, 200-2000, 200-2500, 200-3000, 200-3500, 200-4000, 200-4500, 200-5000, 200-5500, 200-6000, 200-6500, 200-7000, 200-7500, 200-8000, 200-8500, 200-9000, or 200-9500 nucleotides. In some embodiments, the DNA (and / or RNA of interest) has a length of at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, or at least 10,000 nucleotides.

[0044] In some embodiments, the reaction mixture is not supplemented with DNA during an empirically balanced fed-batch IVT reaction, i.e., in some embodiments, the only DNA present throughout the IVT reaction is that which was in the reaction mixture before transcription began (no additional DNA is added to the IVT reaction).

[0045] In some embodiments, the concentration of DNA in the initial or ongoing IVT reaction mixture is about 0.01-0.10 mg / mL, 0.01-0.09 mg / mL, 0.01-0.075 mg / mL, 0.025-0.075 mg / mL, 0.01-0.05 mg / mL, 0.02-0.08 mg / mL, 0.02-0.06 mg / mL, 0.03-0.055 mg / mL, 0.04-0.05 mg / mL, or 0.05 mg / mL. In some embodiments, the concentration of DNA is maintained at greater than 0.01 mg / mL throughout the IVT reaction. In some embodiments, the concentration of DNA is maintained at a concentration of about 0.01-0.10 mg / mL, 0.01-0.09 mg / mL, 0.01-0.075 mg / mL, 0.025-0.075 mg / mL, 0.01-0.05 mg / mL, 0.02-0.08 mg / mL, 0.02-0.06 mg / mL, 0.03-0.055 mg / mL, or 0.04-0.05 mg / mL throughout the IVT reaction.

[0046] RNA product In some embodiments, the transcribed RNA of interest provided herein is messenger RNA (mRNA). In some embodiments, the NTP present at the first position of the RNA of interest is ATP. In some embodiments, the NTP present at the first position of the RNA of interest is GTP. In some embodiments, the NTP present at the first position of the RNA of interest is UTP. In some embodiments, the NTP present at the first position of the RNA of interest is CTP.

[0047] In some embodiments, the method further comprises isolating (e.g., purifying) RNA (e.g., mRNA) from the empirically equilibrated fed-batch IVT reaction mixture. In some embodiments, the method further comprises formulating the isolated RNA into nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, e.g., cationic lipid nanoparticles. The lipid nanoparticles can include, for example, a molar ratio of 20-60% ionic amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. See, e.g., WO2017 / 070624, published April 27, 2017, which is incorporated herein by reference. Other nanoparticles may be used.

[0048] In some embodiments, the yield of transcribed RNA of interest is higher than the yield of RNA transcribed using a batch IVT reaction or method or a conventional fed-batch method (a fed-batch method that is not empirically balanced for NTP consumption). See Kerr et al., Biotechnol. Prog. 15:174-184 (1999). For example, the yield of transcribed RNA of interest can be 20% to 200% higher than the yield of RNA transcribed using a batch IVT reaction or method. In some embodiments, the yield of transcribed RNA of interest is between 20% and 175%, between 20% and 150%, between 20% and 125%, between 20% and 100%, between 20% and 75%, between 20% and 50%, between 30% and 200%, between 30% and 175%, between 30% and 150%, between 30% and 125%, between 30% and 100%, between 30% and 75%, between 30% and 50%, between 40% and 200%, between 40% and 175%, between 40% and 150%, between 40% and 125%, between 40% and 100%, between 40% and 75%, or between 40% and 50% higher than the yield of RNA transcribed using a batch IVT reaction or method. In some embodiments, the yield of transcribed RNA of interest is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than the yield of RNA transcribed using a batch IVT reaction or method.

[0049] In some embodiments, the yield of the transcribed RNA of interest is greater than 5 mg / mL initial reaction volume. In some embodiments, the yield of the transcribed RNA of interest is greater than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 mg / mL initial reaction volume. In some embodiments, the yield of the transcribed RNA of interest is 5-10 mg / mL initial reaction volume, 5-15 mg / mL initial reaction volume, 10-20 mg / mL initial reaction volume, 15-25 mg / mL initial reaction volume, 20-30 mg / mL initial reaction volume, 25-35 mg / mL initial reaction volume, or 30-40 mg / mL initial reaction volume.

[0050] In some embodiments, the % RNA with the desired tail (percentage (%) of RNA transcripts containing a polyA tail) is greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% at reaction times (in minutes) of 150, 180, 210, 240, 270, 300, 330, and / or 360 minutes in a fed-batch reaction.

[0051] RNA polymerase The examples of RNA polymerase provided herein that can be used include, but are not limited to, T7RNA polymerase, T3RNA polymerase, and SP6RNA polymerase, and their homologs, orthologs, and variants.In some embodiments, the RNA polymerase is a T7 polymerase variant. In some embodiments, a T7 RNA polymerase is modified to include an amino acid substitution of at least one high helix-prone amino acid at at least one position selected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45 (e.g., E45R / L), M46 (e.g., M46A), G47 (e.g., G47A), A255 (e.g., A255K / Q / Y / I), R257 (e.g., R257A), A258 (e.g., A258R / E / L), G259 (e.g., G259A), A260 (e.g., A260R / E / L), L261 (e.g., L261A), and A262 (e.g., A262R / E / L) relative to a wild-type T7 RNA polymerase. The T7 RNA polymerase may further, in some embodiments, include one or more additional amino acid substitutions (in addition to at least one highly helical-prone amino acid substitution). Thus, the present disclosure encompasses further modification of existing (e.g., currently available and / or commercially available) T7 RNA polymerase variants with one or more highly helical-prone amino acid substitutions provided herein.

[0052] In some embodiments, the RNA polymerase variant (e.g., a T7 RNA polymerase variant) comprises an additional amino acid at its C-terminus. In some embodiments, the additional amino acid is glycine (G). In some embodiments, the additional amino acid is alanine (A). In some embodiments, the RNA polymerase variant (e.g., a T7 RNA polymerase variant) comprises at least two (e.g., two, three, four, five, or more) additional amino acids at its C-terminus. In some embodiments, the RNA polymerase variant (e.g., a T7 RNA polymerase variant) comprises at least two (e.g., two, three, four, five, or more) additional amino acids at its C-terminus. In some embodiments, the RNA polymerase variant (e.g., a T7 RNA polymerase variant) comprises at least one additional amino acid at its C-terminus, e.g., E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45 (e.g., E45R / L), M46 (e.g., M46A), G47 (e.g., G47A), A255 (e.g., A255K / Q / Y / I), R25 (e.g., ... 7 (e.g., R257A), A258 (e.g., A258R / E / L), G259 (e.g., G259A), A260 (e.g., A260R / E / L), L261 (e.g., L261A) and A262 (e.g., A262R / E / L), and further amino acids at its C-terminus.

[0053] In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 modified to include at least one amino acid substitution of a high helix-prone amino acid at a position selected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45 (e.g., E45R / L), M46 (e.g., M46A), and G47 (e.g., G47A). In some embodiments, the at least one amino acid substitution comprises G47A.

[0054] In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an additional C-terminal amino acid relative to the wild-type RNA polymerase. In some embodiments, the additional C-terminal amino acid is selected from glycine, alanine, threonine, proline, glutamine, serine. In some embodiments, the additional C-terminal amino acid (e.g., at position 884 relative to the wild-type RNA polymerase that comprises the amino acid sequence of SEQ ID NO: 1) is glycine.

[0055] In some embodiments, the T7 RNA polymerase comprises an additional glycine at the C-terminus relative to wild-type T7 RNA polymerase. In some embodiments, the T7 RNA polymerase comprises a G47A substitution relative to wild-type T7 RNA polymerase. In some embodiments, the T7 RNA polymerase comprises a G47A substitution and an additional glycine at the C-terminus relative to wild-type T7 RNA polymerase.

[0056] In some embodiments, the RNA polymerase variant comprises an RNA polymerase comprising (a) an amino acid substitution at a position selected from 350, 351, 387, 394, 425, 427, 437, 441, 632, 811, and 880, and (b) an additional amino acid substitution and / or amino acid modification at the C-terminus, relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0057] In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 350, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), a lysine (K) at position 350 (E350K), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an asparagine (N) at position 350 (E350N), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an alanine (A) at position 350 (E350A), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), a tryptophan at position 350 (E350W), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0058] In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 351, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 351 (D351V), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0059] In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 387, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), a serine at position 387 (K387S), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), a histidine (H) at position 387 (K387H), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an asparagine at position 387 (K387N), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0060] In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 394, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0061] In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 425, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0062] In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 427, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0063] In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 437, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), a threonine at position 437 (N437T), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an isoleucine at position 437 (N437I), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), a tyrosine at position 437 (N437Y), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), a phenylalanine at position 437 (N437F), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0064] In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 441, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an arginine at position 441 (K441R), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0065] In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 632, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0066] In some embodiments, the RNA polymerase variant comprises an RNA polymerase that comprises an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 811, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, wherein the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO:1.

[0067] In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), an amino acid substitution at position 880, and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RNA polymerase variants include RNA polymerases that include an amino acid substitution at position 47 (e.g., G47A), a tyrosine at position 880 (F880Y), and / or an additional amino acid (e.g., G) at the C-terminus (position 884) relative to a wild-type RNA polymerase, where the wild-type RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1.

[0068] In some embodiments, the RNA polymerase variant comprises an RNA polymerase comprising an amino acid substitution at a position selected from the group consisting of E350, D351, K387, N437, K441, D506, R632, D653, S628, P657, F880, and G884 relative to an RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4.

[0069] In some embodiments, the RNA polymerase comprises an amino acid substitution at E350. In some embodiments, the RNA polymerase comprises an amino acid substitution at D351. In some embodiments, the RNA polymerase comprises an amino acid substitution at K387. In some embodiments, the RNA polymerase comprises an amino acid substitution at N437. In some embodiments, the RNA polymerase comprises an amino acid substitution at K441. In some embodiments, the RNA polymerase comprises an amino acid substitution at D506. In some embodiments, the RNA polymerase comprises an amino acid substitution at R632. In some embodiments, the RNA polymerase comprises an amino acid substitution at D653. In some embodiments, the RNA polymerase comprises an amino acid substitution at S628. In some embodiments, the RNA polymerase comprises an amino acid substitution at P657. In some embodiments, the RNA polymerase comprises an amino acid substitution at F880. In some embodiments, the RNA polymerase comprises an amino acid substitution at G884.

[0070] In some embodiments, the RNA polymerase comprises at least two, at least three, at least four, or at least five amino acid substitutions at positions selected from the group consisting of E350, D351, K387, N437, K441, D506, R632, D653, S628, P657, F880, and G884.

[0071] In some embodiments, the RNA polymerase comprises an amino acid substitution at a position selected from the group consisting of E350 and D351, E350 and K387, E350 and N437, E350 and K441, E350 and D506, E350 and R632, E350 and D653, E350 and S628, E350 and P657, E350 and F880, E350 and G884, D351 and K387, D351 and N437, D 351 and K441, D351 and D506, D351 and R632, D351 and D653, D351 and S628, D351 and P657, D351 and F880, D351 and G884, K387 and N437, K387 and K441, K387 and D506, K387 and R632, K387 and D653, K387 and S628, K387 and P657, K387 and F880, and K387 and G884, N437 and K 441, N437 and D506, N437 and R632, N437 and D653, N437 and S628, N437 and P657, N437 and F880, N437 and G884, K441 and D506, K441 and R632, K441 and D653, K441 and S628, K441 and P657, K441 and F880, K441 and G884, D506 and R632, D506 and D653, D506 and S628, D506 and and P657, D506 and F880, D506 and G884, R632 and D653, R632 and S628, R632 and P657, R632 and F880, R632 and G884, D653 and S628, D653 and P657, D653 and F880, D653 and G884, S628 and P657, S628 and F880, S628 and G884, P657 and F880, P657 and G884, and F880 and G884.

[0072] In some embodiments, the RNA polymerase comprises an acid substitution at a position selected from the group consisting of K387, D653, and G884, E350, D351, and K387, and D653, P657, and R632. In some embodiments, the amino acid substitution at E350 is selected from the group consisting of E350A, E350K, E350N, and E350W, and optionally, the amino acid substitution at E350 is E350N. In some embodiments, the amino acid substitution at D351 is D351V. In some embodiments, the amino acid substitution at K387 is selected from the group consisting of K387H, K387N, and K387S, and optionally, the amino acid substitution at K387 is K387N. In some embodiments, the amino acid substitution at N437 is selected from the group consisting of N437F, N437I, N437T, and N437Y, optionally, the amino acid substitution at N437 is N437F. In some embodiments, the amino acid substitution at K441 is K441R. In some embodiments, the amino acid substitution at D506 is selected from the group consisting of D506F, D506L, D506R, D506W, and D506Y. In some embodiments, the amino acid substitution at R632 is R632K or R632T.

[0073] In some embodiments, the amino acid substitution at D653 is selected from the group consisting of D653A, D653F, D653G, D653H, D653I, D653K, D653L, D653M, D653N, D653P, D653Q, D653R, D653S, D653T, D653V, D653W, and D653Y, and optionally, the amino acid substitution at D653 is D653W. In some embodiments, the amino acid substitution at S628 is S628W. In some embodiments, the amino acid substitution at P657 is selected from the group consisting of P657A, P657R, and P657W. In some embodiments, the amino acid substitution at F880 is F880Y. In some embodiments, the amino acid substitution at G884 is selected from the group consisting of G884A, G884S, G884T, and G884P.

[0074] In some embodiments, the RNA polymerase comprises an amino acid substitution at two positions selected from the group consisting of E350, D351, K387, and D653 relative to an RNA polymerase comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 4. In some embodiments, the RNA polymerase comprises an amino acid substitution at E350 and D351. In some embodiments, the RNA polymerase comprises an amino acid substitution at E350 and K387. In some embodiments, the RNA polymerase comprises an amino acid substitution at K387 and D653. In some embodiments, the amino acid substitution at E350 is E350W, E350A, E350K, or E350N. In some embodiments, the amino acid substitution at D351 is D351V. In some embodiments, the amino acid substitution at K387 is K387N, K387S, or K387H. In some embodiments, the amino acid substitution at position D653 is D653T or D653K.

[0075] In some embodiments, the RNA polymerase comprises amino acid substitutions at positions E350 and K387 relative to an RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 1-4, optionally, the substitutions are E350W and K387N.

[0076] In some embodiments, the RNA polymerase comprises amino acid substitutions at positions E350 and D351 relative to an RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 1-4, optionally, the substitutions are E350W and D351V.

[0077] In some embodiments, the RNA polymerase comprises amino acid substitutions at positions K387 and D653 relative to an RNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 4, optionally, the substitutions are K387N and D653T.

[0078] In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the T7 RNA polymerase comprises the amino acid sequence of SEQ ID NO:12.

[0079] T7 RNA polymerase sequence Wild-type T7 RNA polymerase [ka]

[0080] C-terminal G T7 RNA polymerase [ka]

[0081] G47A T7 RNA polymerase [ka]

[0082] G47A; C-terminal G T7 RNA polymerase [ka]

[0083] G47A;E350K;C-terminal G T7 RNA polymerase [ka]

[0084] G47A;E350N;C-terminal G T7 RNA polymerase [ka]

[0085] G47A;E350A;C-terminal G T7 RNA polymerase [ka]

[0086] G47A;E350W;C-terminal G T7 RNA polymerase [ka]

[0087] G47A;D351V;C-terminal G T7 RNA polymerase [ka]

[0088] G47A;K487S;C-terminal G T7 RNA polymerase [ka]

[0089] G47A;K387H;C-terminal G T7 RNA polymerase [ka]

[0090] G47A;K387N;C-terminal G T7 RNA polymerase [ka]

[0091] G47A;E350W;D351V;C-terminal G T7 RNA polymerase [ka]

[0092] E350W;D351V;T7 RNA polymerase [ka]

[0093] In some embodiments, the RNA polymerase (e.g., a T7 RNA polymerase variant) is present in the empirically balanced fed-batch IVT reaction mixture at a concentration of 0.01 mg / ml to 1 mg / ml. For example, the RNA polymerase can be present in the reaction mixture at a concentration of 0.01 mg / mL, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1.0 mg / ml.

[0094] Nucleoside triphosphates The NTP of the present disclosure can be naturally occurring NTP, synthetic NTP, and / or modified NTP. A reaction mixture can include naturally occurring NTP, synthetic NTP, modified NTP, or any combination thereof. Thus, the NTP of reaction mixture can include unmodified and / or modified adenosine triphosphate (ATP), modified and / or unmodified uridine triphosphate (UTP), modified and / or unmodified guanosine triphosphate (GTP), and / or modified and / or unmodified cytidine triphosphate (CTP). In some embodiments, the NTP includes modified nucleobase. Non-limiting examples of modified nucleobases that can be used provided herein include pseudouridine (ψ), 1-methylpseudouridine (m1ψ), 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine (mo5U) and 2'-O-methyluridine. In some embodiments, the mixture of NTPs (and thus the RNA transcripts thereof) comprises a combination of at least two (e.g., two, three, four or more) of the aforementioned modified nucleobases. In some embodiments, the mixture of NTPs comprises 1-methylpseudouridine (m1ψ). In some embodiments, the mixture of NTPs comprises 1-ethylpseudouridine.

[0095] In some embodiments, each NTP (e.g., ATP, UTP, GTP, and CTP) in the fed-batch IVT reaction mixture is present in a molar ratio equal to the calculated percentage (%) consumption value for each NTP (e.g., calculated during the initial nucleotide empirical equilibration reaction). For example, if the calculated percentage consumption values ​​for ATP, UTP, GTP, and CTP for a particular RNA of interest during the initial nucleotide empirical equilibration reaction are 35%, 20%, 25%, and 20%, respectively, then in an IVT reaction mixture containing a total NTP concentration of 20 mM (to transcribe the same RNA of interest), the corresponding molar ratios for ATP, UTP, GTP, and CTP are 7 mM, 4 mM, 5 mM, and 4 mM, respectively.

[0096] In some embodiments, individual (and therefore total) NTP concentrations in a fed-batch IVT reaction mixture are maintained above zero (0) millimolar (mM) throughout the reaction. For example, the NTP concentration may be maintained at 1 mM to 50 mM, 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 1 mM to 10 mM, 2 mM to 50 mM, 2 mM to 40 mM, 2 mM to 30 mM, 2 mM to 20 mM, 2 mM to 10 mM, 3 mM to 50 mM, 3 mM to 40 mM, 3 mM to 30 mM, 3 mM to 20 mM, 3 mM to 10 mM, 4 mM to 50 mM, 4 mM to 40 mM, 4 mM to 30 mM, 4 mM to 20 mM, 4 mM to 10 mM, 5 mM to 50 mM, 5 mM to 40 mM, 5 mM to 30 mM, 5 mM to 20 mM, or 5 mM to 10 mM. In some embodiments, the NTP concentration is maintained at 10 mM to 20 mM. In some embodiments, the NTP concentration is maintained at (or at least at) 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. In some embodiments, the NTP concentration is maintained above a lower limit of 2 mM to 10 mM, above a lower limit of 2 mM to 9 mM, above a lower limit of 2 mM to 8 mM, above a lower limit of 2 mM to 7 mM, above a lower limit of 2 mM to 6 mM, or above a lower limit of 2 mM to 6 mM.

[0097] In some embodiments, individual (and therefore total) NTP concentrations are maintained within 5%-200% of the initial NTP concentration throughout a fed-batch IVT reaction. For example, the NTP concentrations can be maintained within 5%-175%, 5%-150%, 5%-125%, 5%-100%, 5%-75%, 5%-50%, or 5%-25% of the initial NTP concentration. In some embodiments, the NTP concentration is maintained within 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the initial NTP concentration.The initial NTP concentration is the total NTP concentration in the IVT reaction before transcription starts.As used herein, [NTP] (i.e., brackets around NTP) generally refers to the concentration of the NTP contained in the brackets. For example, [ATP] generally refers to the concentration of ATP, [GTP] generally refers to the concentration of GTP, [CTP] generally refers to the concentration of CTP, and [UTP] generally refers to the concentration of UTP.

[0098] The concentrations of NTPs and cap analogs present in the IVT reaction mixture can vary. In some embodiments, the NTPs and RNA cap analogs ("cap analogs") of each purine are present in the reaction at equimolar (1:1) concentrations. In some embodiments, the molar ratio of cap analogs (e.g., trinucleotide caps) to NTPs of each purine in the reaction is greater than 1:1. For example, the molar ratio of cap analogs to NTPs in the reaction can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 50:1, or 100:1. In some embodiments, the molar ratio of cap analogs (e.g., trinucleotide caps) to NTPs in the reaction is less than 1:1. For example, the molar ratio of cap analog (e.g., trinucleotide cap) to each NTP in the reaction can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:50, or 1:100.

[0099] The relative concentrations of individual NTPs in an IVT reaction may also vary. For example, ATP may be used over GTP, CTP, and UTP. As another example, GTP may be used over ATP, CTP, and UTP, CTP may be used over ATP, GTP, and UTP, and UTP may be used over ATP, GTP, and CTP.

[0100] In some embodiments, the relative concentration of NTPs in the initial IVT reaction mixture comprises an [ATP]:[UTP] ratio of 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1. In some embodiments, the relative concentration of NTPs in the initial IVT reaction mixture comprises an [ATP]:[UTP] ratio of 2:1. In some embodiments, the relative concentration of NTPs in the initial IVT reaction mixture comprises an [GTP]:[CTP] ratio of 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1. In some embodiments, the relative concentration of NTPs in the initial IVT reaction mixture comprises a [GTP]:[CTP] ratio of 4:1. In some embodiments, the relative concentration of NTPs in the initial IVT reaction mixture comprises an [ATP]:[UTP] ratio of 2:1 and a [GTP]:[CTP] ratio of 4:1.

[0101] In some embodiments, the total NTP concentration in the ongoing IVT reaction mixture is maintained above a lower limit of 0.5 mM. In some embodiments, the total NTP concentration in the ongoing IVT reaction mixture is maintained at 0.5-20 mM, 1-20 mM, 0.5-5 mM, 2-8 mM, 2-5 mM, 5-10 mM, 5-30 mM, 5-20 mM, 10-20 mM, or 5-15 mM. In some embodiments, the total NTP concentration in the ongoing IVT reaction mixture is maintained at about 10-20 mM, about 8-16 mM, about 6-14 mM, or about 10-15 mM.

[0102] RNA Cap Analogs In some embodiments, the empirically balanced fed-batch IVT reaction is a "co-transcriptional capping" IVT reaction in which the IVT reaction mixture includes an RNA cap. That is, the mRNA is produced in a "one-pot" reaction and no separate capping reaction is required. Thus, in some embodiments, the IVT reaction mixture of the present disclosure includes an RNA cap analog. RNA cap analogs generally increase mRNA stability and translation efficiency. Conventional cap analogs include GpppG, m7GpppG, and m2,2,7GpppG. In some embodiments, the RNA cap analog of the present disclosure is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

[0103] In some embodiments, the cap analog is a trinucleotide cap, hi some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.

[0104] In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: 7 GppApA, m 7 GpppApC, m 7 GppApG, m 7 GppApU,m 7 GppCpA, m 7 GppCpC, m 7 GppCpG, m 7 GppCpU,m 7 GppGpA, m 7 GppGpC, m 7 GppGpG, m 7 GppGpU,m 7 GpppUpA,m 7 GpppUpC,m 7 GpppUpG, and m 7 In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences:7 G 3’OMe pppApA, m 7 G 3’OMe pppApC, m 7 G 3’OMe pppApG, m 7 G 3’OMe pppApU,m 7 G 3’OMe pppCpA, m 7 G 3’OMe pppCpC, m 7 G 3’OMe pppCpG,m 7 G 3’OMe pppCpU,m 7 G 3’OMe pppGpA,m 7 G 3’OMe pppGpC, m 7 G 3’OMe pppGpG, m 7 G 3’OMe pppGpU,m 7 G 3’OMe pppUpA,m 7 G 3’OMe pppUpC,m 7 G 3’OMe pppUpG, and m 7 G 3’OMe In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: 7 G 3’OMe pppA 2’OMe pA, m 7 G 3’OMe pppA 2’OMe pC, m 7 G 3’OMe pppA 2’OMe pG,m 7 G 3’OMe pppA 2’OMe pU,m 7 G 3’OMe pppC 2’OMe pA, m 7 G 3’OMe pppC 2’OMe pC, m 7 G 3’OMe pppC 2’OMe pG,m 7 G 3’OMe pppC 2’OMe pU,m 7G 3’OMe pppG 2’OMe pA, m 7 G 3’OMe pppG 2’OMe pC, m 7 G 3’OMe pppG 2’OMe pG,m 7 G 3’OMe pppG 2’OMe pU,m 7 G 3’OMe pppU 2’OMe pA, m 7 G 3’OMe pppU 2’OMe pC, m 7 G 3’OMe pppU 2’OMe pG, and m 7 G 3’OMe pppU 2’OMe In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: 7 GpppA 2’OMe pA, m 7 GpppA 2’OMe pC, m 7 GpppA 2’OMe pG,m 7 GpppA 2’OMe pU,m 7 GpppC 2’OMe pA, m 7 GpppC 2’OMe pC, m 7 GpppC 2’OMe pG,m 7 GpppC 2’OMe pU,m 7 GpppG 2’OMe pA, m 7 GpppG 2’OMe pC, m 7 GpppG 2’OMe pG,m 7 GpppG 2’OMe pU,m 7 GpppU 2’OMe pA, m 7 GpppU 2’OMe pC, m 7 GpppU 2’OMe pG, and m 7 GpppU 2’OMe pU.

[0105] In some embodiments, the trinucleotide cap comprises a sequence selected from the following sequences: GAG, GCG, GUG, and GGG. In some embodiments, the trinucleotide cap comprises the sequence GAG. In some embodiments, the trinucleotide cap comprises the sequence 7 GpppA 2’Ome In some embodiments, the trinucleotide cap comprises m 7 GpppmA 2’Ome In some embodiments, the trinucleotide cap comprises m 7 Gpppm 6 A 2’Ome In some embodiments, the trinucleotide cap comprises m 7 Gpppe 6 A 2’Ome Contains pG.

[0106] In some embodiments, the trinucleotide cap comprises the following structure: [ka]

[0107] In other embodiments, the trinucleotide cap comprises the following structure: [ka]

[0108] In yet other embodiments, the trinucleotide cap comprises the following structure: [ka]

[0109] In yet other embodiments, the trinucleotide cap comprises the following structure: [ka]

[0110] In some embodiments, the cap analog is a tetranucleotide cap analog, hi some embodiments, the cap analog is a tetranucleotide cap analog that comprises a GGAG sequence.

[0111] In some embodiments, the tetranucleotide cap comprises the following structure: [ka]

[0112] In other embodiments, the tetranucleotide cap comprises the following structure: [ka]

[0113] In yet other embodiments, the tetranucleotide cap comprises the following structure: [ka]

[0114] In yet other embodiments, the tetranucleotide cap comprises the following structure: [ka]

[0115] In some embodiments, R is an alkyl (e.g., a C1-C6 alkyl). In some embodiments, R is a methyl group (e.g., a C1 alkyl). In some embodiments, R is an ethyl group (e.g., a C2 alkyl). In some embodiments, R is hydrogen.

[0116] In some embodiments, between 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, or 90% and 100% of the RNA produced by the methods of the disclosure is capped (RNA cap analogs, e.g., m 7 GpppA 2’Ome For example, greater than 70%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% of the RNA produced by the methods of the disclosure is capped (including RNA cap analogs, e.g., m 7 GpppA 2’Ome In some embodiments, 100% of the RNA produced by the methods of the disclosure is capped (including RNA cap analogs, such as m 7 GpppA 2’Ome (including pG).

[0117] In some embodiments, the % capping of the RNA of interest (percentage (%) of RNA transcripts that contain an RNA cap analog) is greater than 80%, greater than 90%, or greater than 95% at 150, 180, 210, 240, 270, 300, 330, and / or 360 minutes in a fed-batch reaction.

[0118] In some embodiments, the reaction mixture is not replenished with an RNA cap analog during transcription of the RNA of interest, i.e., throughout the entire IVT reaction, the only RNA cap analog present is the one that was in the reaction mixture before transcription began (no additional RNA cap analog is added to the IVT reaction).

[0119] In some embodiments, the IVT reaction mixture comprises a starting RNA cap analog concentration (the concentration of RNA cap analog prior to the start of transcription) that is at least 10% (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) higher than the concentration of the NTP (e.g., ATP, UTP, GTP, or CTP (modified or unmodified)) with the highest percent consumed value. In some embodiments, the IVT reaction mixture comprises a starting RNA cap analog concentration that is higher than the concentration of the NTP (e.g., ATP, UTP, GTP, or CTP (modified or unmodified)) with the highest percent consumed value.

[0120] In some embodiments, the IVT reaction mixture comprises a starting RNA cap analog concentration (the concentration of RNA cap analog prior to the start of transcription) that is at least 10% (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) higher than the concentration of the purine (modified or unmodified) with the highest percent consumed value. In some embodiments, the IVT reaction mixture comprises a starting RNA cap analog concentration that is higher than the concentration of the purine (modified or unmodified) with the highest percent consumed value.

[0121] In some embodiments, the IVT reaction mixture comprises a starting RNA cap analog concentration (concentration of RNA cap analog prior to the start of transcription) that is at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, or at least 100%) higher than the concentration of the individual NTP (e.g., ATP, UTP, GTP, or CTP (modified or unmodified)) present at the first coding position of the RNA of interest. In some embodiments, the IVT reaction mixture comprises a starting RNA cap analog concentration (concentration of RNA cap analog prior to the start of transcription) that is 10%-100%, 10%-50%, 10%-40%, or 10%-30% higher than the concentration of the individual NTP (e.g., ATP, UTP, GTP, or CTP (modified or unmodified)) present at the first coding position of the RNA of interest.

[0122] In some embodiments, the IVT reaction mixtures, e.g., initial and ongoing IVT reaction mixtures, include an [RNA cap analog]:[purine] ratio of 1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1. In some embodiments, the concentration of RNA cap analog to purine nucleotides (e.g., ATP and GTP) is 1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1.

[0123] Additional IVT reaction components In some embodiments, the IVT reaction mixture comprises a buffer, such as Tris, phosphate, or Good's buffer. The concentration of the buffer used in the empirically balanced fed-batch IVT reaction mixture can be, for example, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, or at least 110 mM phosphate. In some embodiments, the concentration of phosphate is 20-60 mM or 10-100 mM. In some embodiments, the buffer comprises Tris-HCl. For example, the buffer can comprise 10-100 mM, 10-80 mM, 10-60 mM, 20-100 mM, 20-18 mM, or 20-60 mM Tris-HCl. In some embodiments, the buffer comprises 40 mM Tris-HCl.

[0124] In some embodiments, the fed-batch IVT reaction mixture includes a reductant or reducing agent, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). The concentration of DTT used in the IVT reaction mixture can be, for example, at least 1 mM, at least 5 mM, or at least 50 mM. In some embodiments, the concentration of DTT used in the IVT reaction mixture is 1-50 mM or 5-50 mM. In some embodiments, the concentration of DTT used in the IVT reaction mixture is 5 mM. The concentration of TCEP used in the IVT reaction mixture can be, for example, at least 1 mM, at least 5 mM, or at least 50 mM. In some embodiments, the concentration of TCEP used in the IVT reaction mixture is 1-50 mM or 5-50 mM. In some embodiments, the concentration of TCEP used in the IVT reaction mixture is 2 mM.

[0125] In some embodiments, the empirically equilibrated fed-batch IVT reaction mixture comprises magnesium. In some embodiments, the magnesium ion (Mg 2+ For example, the molar ratio of NTP to magnesium ion can be 1:1 to 1:5. For example, the molar ratio of NTP to magnesium ion can be 1:1, 1:2, 1:3, 1:4, or 1:5.

[0126] Supply Schedule A fed-batch IVT reaction typically includes a periodic feeding schedule. A periodic feeding schedule may be used to maintain the NTP concentration in the IVT reaction at a desired level, e.g., above a threshold value, e.g., above 5%, 10%, 20%, 30%, or 50% of the initial NTP concentration. In some embodiments, the timing and / or amount used in the feeding schedule is determined using a scouting fed-batch IVT reaction. In some embodiments, if the IVT reaction is overfed (i.e., too much of any given NTP is added during the reaction), the ratio of cap analog to purine may fall below 1:1, in which case overfeeding may result in a lower % capped RNA product. In other embodiments, if the IVT reaction is underfed (i.e., not enough of any given NTP is added during the reaction), one or more nucleotides may be depleted below a threshold value, e.g., to 0 mM, and the overall yield of transcribed RNA may be lower. In some embodiments, the feeding schedule may include a bolus feed of the feedstock. In some embodiments, the feeding schedule may include a continuous feed of the feedstock.

[0127] In some embodiments, bolus delivery of feedstock to an ongoing IVT reaction comprises adding a discrete volume or amount of feedstock once every 10-250 minutes, 20-200 minutes, 10-175 minutes, 10-100 minutes, 10-20 minutes, 10-30 minutes, 30-60 minutes, 30-100 minutes, 30-150 minutes, 50-100 minutes, 50-150 minutes, 100-300 minutes, 100-250 minutes, or 100-150 minutes. In some embodiments, a bolus supply of feedstock to an ongoing IVT reaction comprises adding a separate volume or amount of feedstock once every 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 minutes. In some embodiments, the separate volumes included in the bolus supply are 1-20 mL, 5-20 mL, 10-50 mL, 25-100 mL, 50-500 mL, 250-1000 mL, or more. In some embodiments, the individual volumes included in the bolus delivery are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 750, or 1000 mL. In some embodiments, the individual amounts of feedstock are any amount sufficient to maintain the NTP concentration above the thresholds, for example, within the range of 5%-200%, 5%-100%, 5%-75%, 20%-100%, 20%-75%, or 25%-50% of the initial NTP concentration. In some embodiments, the individual amounts of feedstock are any amount sufficient to maintain the [ATP]:[UTP] ratio at 1:1-5:1, 1:1-4:1, 1:1-3:1, or 1:1-2:1. In some embodiments, the individual amounts of feedstock are any amount sufficient to maintain the [ATP]:[UTP] ratio at 2:1. In some embodiments, the individual amounts of the feedstocks are any amount sufficient to maintain a [GTP]:[CTP] ratio of 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1. In some embodiments, the individual amounts of the feedstocks are any amount sufficient to maintain a [GTP]:[CTP] ratio of 4:1.In some embodiments, the individual amounts of feedstocks are any amount sufficient to maintain an [ATP]:[UTP] ratio of 2:1 and an [GTP]:[CTP] ratio of 4:1.

[0128] In some embodiments, the IVT reaction is replenished with 1, 2, or 3 bolus feeds of feedstock throughout the reaction. In some embodiments, the IVT reaction is replenished with 4, 5, 6, 7, 8, 9, 10, or more bolus feeds of feedstock throughout the reaction. In some embodiments, the IVT reaction is replenished with 1-5, 2-5, 2-10, 3-10, 3-7, 4-8, 5-10, 5-15, or more bolus feeds of feedstock throughout the reaction.

[0129] In some embodiments, continuous supply of feedstock to an ongoing IVT reaction comprises continuous or sustained addition of feedstock over time, e.g., throughout the entire IVT reaction. In some embodiments, the continuous supply schedule is determined based on a bolus supply schedule of the same RNA of interest, e.g., by dividing the total volume or amount added over a period of time during the reaction by the total time that volume or amount was added. In some embodiments, continuous supply comprises sustained addition of feedstock to an ongoing IVT reaction at a continuous flow rate of 1-50 mL / min, 2-25 mL / min, 2-10 mL / min, 2-8 mL / min, 4-6 mL / min, 3-6 mL / min, or any operable flow rate. In some embodiments, the continuous feed comprises the continuous addition of a feedstock to an ongoing IVT reaction at a continuous flow rate of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mL / min. In some embodiments, the continuous feed comprises the continuous addition of a feedstock to an ongoing IVT reaction at a continuous flow rate of 0.0030-0.007 mL / min per mL of initial volume, 0.0040-0.0060 mL / min per mL of initial volume, 0.0050-0.0080 mL / min per mL of initial volume, or 0.0060-0.0090 mL / min per mL of initial volume. In some embodiments, the concentration of NTPs in the feedstock used for continuous feeding is sufficient to maintain the NTP concentration above the thresholds described above, for example, within the range of 5%-200%, 5%-100%, 5%-75%, 20%-100%, 20%-75%, or 25%-50% of the initial NTP concentration. In some embodiments, the continuous feeding is performed using a peristaltic pump to accurately deliver a constant flow rate (volume) over time.

[0130] In some embodiments, the concentration of NTP in the feedstock used for continuous or bolus delivery is sufficient to maintain the NTP concentration above a threshold value. In some embodiments, the individual amount of the feedstock is any amount sufficient to maintain the NTP concentration above a threshold value. In some embodiments, the threshold is 1 mM to 50 mM, 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 1 mM to 10 mM, 2 mM to 50 mM, 2 mM to 40 mM, 2 mM to 30 mM, 2 mM to 20 mM, 2 mM to 10 mM, 3 mM to 50 mM, 3 mM to 40 mM, 3 mM to 30 mM, 3 mM to 20 mM, 3 mM to 10 mM, 4 mM to 50 mM, 4 mM to 40 mM, 4 mM to 30 mM, 4 mM to 20 mM, 4 mM to 10 mM, 5 mM to 50 mM, 5 mM to 40 mM, 5 mM to 30 mM, 5 mM to 20 mM, or 5 mM to 10 mM. In some embodiments, the threshold is 10 mM to 20 mM. In some embodiments, the threshold is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. In some embodiments, the threshold is a lower limit of 2 mM to 10 mM, or higher than a lower limit of 2 mM to 9 mM, or higher than a lower limit of 2 mM to 8 mM, or higher than a lower limit of 2 mM to 7 mM, or higher than a lower limit of 2 mM to 6 mM, or higher than a lower limit of 2 mM to 6 mM. In some embodiments, the threshold is within 5% to 200% of the initial NTP concentration. For example, the threshold value may be within 5% to 175%, 5% to 150%, 5% to 125%, 5% to 100%, 5% to 75%, 5% to 50%, or 5% to 25% of the initial NTP concentration. In some embodiments, the threshold value is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the initial NTP concentration.

[0131] The results of fed-batch IVT consistently show a decrease in reaction rate (both NTP consumption and RNA production) throughout the reaction. As a result, the feed rate also needs to be changed throughout the reaction. If a single continuous feed rate (described above) is used to feed an IVT where the reaction rate decreases over the entire period, the reaction will be underfed at the beginning of the fastest reaction and overfed at the end of the slowest reaction. To prevent the risk of under- and over-feeding, the reaction can be split using two or more continuous feed rates at certain time intervals of the reaction. The number of different feed rates and switch times required may vary depending on the observed decrease in reaction rate throughout fed-batch IVT.

[0132] Scouting Fed-batch IVT Reactions To determine an appropriate feeding schedule, a scouting fed-batch IVT reaction can be performed. In this reaction, a bolus feed is used to add feedstock to the active IVT reaction at specific time intervals and specific volumes or amounts. In some embodiments, these initial feeding conditions are selected based on the results of previous fed-batch IVT reactions with other DNAs encoding different RNA sequences and are not intended to produce high quality products or high yields. In some embodiments, the scouting IVT reaction includes sampling time points from the active fed-batch IVT immediately before each bolus feed. The NTP concentrations measured in each of these samples can then be used to calculate the NTP consumption rate in the IVT reaction for the unique DNA transcribed over time. The feeding schedule can then be adjusted to match the observed NTP consumption and maintain the target NTP concentration throughout the reaction. In some embodiments, these individual feeding times and volumes constitute the bolus feeding schedule for the fed-batch IVT reaction.

[0133] Further embodiments 1. A method for determining the percentage (%) of nucleoside triphosphate (NTP) consumption of an in vitro transcription (IVT) reaction, comprising: (a) performing an IVT reaction in a reaction mixture containing a known initial concentration of NTPs, deoxyribonucleic acid (DNA) encoding a ribonucleic acid (RNA) of interest, and an RNA polymerase; (b) measuring individual NTP concentrations at discrete intervals over a period of time; and (c) calculating a percent (%) value for each NTP consumed in said reaction mixture; The method.

[0134] 2. The method of paragraph 1, wherein the NTPs include adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP).

[0135] 3. The method of paragraph 1, wherein step (b) comprises (i) dividing each individual NTP consumption rate by the total NTP consumption rate.

[0136] 4. The method of paragraph 3, wherein said individual NTP consumption rates are calculated by measuring individual NTP concentrations at discrete intervals over a period of time, and said total NTP consumption rate is calculated by measuring the total NTP concentration at discrete intervals over a period of time.

[0137] 5. The method of paragraph 4, wherein measurements of the individual NTP concentrations and total NTP concentration are collected until the concentration of at least one of the NTPs falls below a threshold concentration.

[0138] 6. The method of paragraph 5, wherein the threshold concentration is greater than zero (0) millimolar (mM), optionally, the threshold concentration is between 5 mM and 20 mM, and optionally, the threshold concentration is within 5% to 75% of the initial NTP concentration.

[0139] 7. The method of any one of paragraphs 1-6, wherein the known initial NTP concentrations comprise equimolar concentrations of each of [ATP], [CTP], [UTP], and [GTP].

[0140] 8. The method of any one of paragraphs 1 to 6, wherein the known initial NTP concentrations comprise an [ATP]:[UTP] ratio of 1:1 to 4:1, optionally 1:1 to 2:1, and / or an [GTP]:[CTP] ratio of 1:1 to 4:1.

[0141] 9. The method of any one of paragraphs 1-8, wherein the known initial NTP concentrations comprise an [ATP]:[UTP]:[CTP]:[GTP] ratio of 2:1:1:4.

[0142] 10. The method of any of the above paragraphs, wherein the NTP is a chemically modified NTP, a naturally occurring NTP, or a synthetic NTP.

[0143] 11. The method according to any one of paragraphs 1 to 10, wherein the IVT reaction is carried out within a fixed time period of 20 to 40 minutes.

[0144] 12. A method for fed-batch in vitro transcription (IVT) of a ribonucleic acid (RNA) of interest, comprising: (a) performing an IVT reaction with an initial reaction mixture that includes deoxyribonucleic acid (DNA) encoding an RNA of interest, an RNA polymerase, and nucleoside triphosphates (NTPs); (b) supplying, over time, to the ongoing IVT reaction mixture a feed mixture comprising NTPs, wherein each NTP is present in the feed mixture in a molar ratio based on a percentage consumption value calculated separately for each NTP, the percentage consumption value being specific to the RNA of interest, and wherein the feed mixture is provided in an amount that maintains a total NTP concentration in the reaction mixture greater than zero mM; Thereby, the desired transcription RNA is generated. The method.

[0145] 13. The method of paragraph 12, wherein the NTPs include adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP).

[0146] 14. The method of paragraph 12 or 13, wherein the initial reaction mixture of (a) comprises an [ATP]:[UTP] ratio of 1:1 to 4:1, optionally 1:1 to 2:1, and / or an [GTP]:[CTP] ratio of 1:1 to 4:1.

[0147] 15. The method of any one of paragraphs 12 to 14, wherein the initial reaction mixture of (a) comprises an [ATP]:[UTP]:[CTP]:[GTP] ratio of 2:1:1:4.

[0148] 16. The method of any one of paragraphs 12 to 15, wherein each NTP in the initial reaction mixture of (a) is present in a molar equivalent amount different from the calculated percentage (%) consumed value for each NTP.

[0149] 17. The method of any one of paragraphs 12 to 16, wherein each NTP in the initial reaction mixture of (a) is present in an equimolar concentration with respect to each NTP.

[0150] 18. The method of any one of paragraphs 12 to 17, wherein each NTP in the initial reaction mixture of (a) is present at a concentration of 1 to 10 mM, 1 to 6 mM, 2 to 6 mM, or 3 to 6 mM.

[0151] 19. The method of any one of paragraphs 12-18, wherein the concentration of each NTP in the ongoing IVT reaction mixture is maintained within the range of 5%-200%, 5%-100%, 5%-75%, 20%-100%, 20%-75%, or 25%-50% of the corresponding initial NTP concentration.

[0152] 20. The method of any one of paragraphs 12 to 19, wherein the reaction mixture is maintained at an [ATP]:[UTP] ratio of 1:1 to 4:1, optionally 1:1 to 2:1, and / or an [GTP]:[CTP] ratio of 1:1 to 4:1.

[0153] 21. The method of any one of paragraphs 12 to 20, wherein the reaction mixture is maintained at an [ATP]:[UTP]:[CTP]:[GTP] ratio of 2:1:1:4.

[0154] 22. A method for fed-batch in vitro transcription (IVT) of a ribonucleic acid (RNA) of interest, comprising: (a) performing an IVT reaction with an initial reaction mixture comprising deoxyribonucleic acid (DNA) encoding an RNA of interest, an RNA polymerase, and nucleoside triphosphates (NTPs), the NTPs comprising an [ATP]:[UTP] ratio of 2:1 and an [GTP]:[CTP] ratio of 4:1; and (b) supplying to the ongoing IVT reaction mixture over time a feed mixture comprising NTPs, each NTP being present in the feed mixture in a molar ratio based on a percentage consumption value calculated separately for each NTP, the percentage consumption value being specific to the RNA of interest, and the feed mixture being supplied in an amount that maintains an [ATP]:[UTP] ratio of 2:1 and an [GTP]:[CTP] ratio of 4:1; Thereby, the desired transcription RNA is generated. The method.

[0155] 23. The method of any one of paragraphs 12 to 22, wherein the NTP is a chemically modified NTP, a naturally occurring NTP, or a synthetic NTP.

[0156] 24. The method of any one of paragraphs 12-23, wherein the feed mixture is fed to the ongoing IVT reaction mixture using a bolus feed over time.

[0157] 25. The method of paragraph 24, wherein the feed mixture is fed to the ongoing IVT reaction mixture every 10 to 250 minutes, optionally every 20 to 200 minutes.

[0158] 26. The method of any one of paragraphs 12 to 23, wherein the feed mixture is fed to the ongoing IVT reaction mixture using a continuous feed over time.

[0159] 27. The feed mixture is added to the ongoing IVT reaction mixture. (i) 2 to 8 mL / min, optionally 4 to 6 mL / min, or (ii) delivered at a continuous flow rate of 0.0030-0.007 mL / min per mL of initial volume, optionally 0.0040-0.0060 mL / min per mL of initial volume; 27. A method according to paragraph 26.

[0160] 28. The method of any one of paragraphs 12 to 27, wherein each NTP in the initial reaction mixture of (a) is present in a molar ratio equivalent to the calculated percentage (%) consumed value for each NTP.

[0161] 29. The method of any one of paragraphs 12-28, wherein the total NTP concentration in the ongoing IVT reaction mixture is maintained above a lower limit of 0.5 mM, and optionally between 10 mM and 20 mM.

[0162] 30. The method of any one of paragraphs 1 to 29, wherein the initial and / or ongoing IVT reaction mixture further comprises an RNA cap analog.

[0163] 31. The method of paragraph 30, wherein the RNA cap analog is a chemically modified RNA cap analog, a naturally occurring RNA cap analog, or a synthetic RNA cap analog.

[0164] 32. The method of paragraph 30 or 31, wherein the RNA cap analog is (i) a trinucleotide RNA cap analog, optionally selected from trinucleotide cap analogs including a cap1, cap2, cap3, or cap4 structure, or (ii) a tetranucleotide RNA cap analog, optionally selected from trinucleotide cap analogs including a cap5, cap6, cap7, or cap8 structure.

[0165] 33. The method of any one of paragraphs 30-32, wherein the initial and ongoing IVT reaction mixtures comprise an [RNA cap analog]:[purine] ratio of 1:1 to 20:1, 1:1 to 15:1, 1:1 to 10:1, 1:1 to 5:1, 1:1 to 3:1, or 1:1 to 2:1.

[0166] 34. The method of any one of paragraphs 12-33, wherein the yield of the transcribed RNA of interest is greater than the yield of RNA transcribed using a batch IVT reaction.

[0167] 35. The method of paragraph 34, wherein the yield of the transcribed RNA of interest is at least 100% greater than the yield of RNA transcribed using a batch IVT reaction.

[0168] 36. The method of any one of paragraphs 12 to 35, wherein the yield of the transcribed RNA of interest is greater than 5, 10, 15, 20, 25, or 30 mg / mL initial reaction volume.

[0169] 37. The method of any one of paragraphs 12 to 36, wherein the initial and ongoing IVT reaction mixtures further comprise a buffering agent and / or magnesium.

[0170] 38. The method of paragraph 37, wherein the buffer is Tris-HCl, optionally the buffer is 20-60 mM Tris-HCl, optionally the buffer is 40 mM Tris-HCl.

[0171] 39. The method of any one of paragraphs 30 to 3387, wherein at least 90%, optionally at least 95%, of the transcribed RNA of interest comprises the RNA cap analog.

[0172] 40. The method of any one of paragraphs 30 to 39, wherein the ratio of the cap analog to ATP or the ratio of the cap analog to GTP is greater than 0.6, and at least 90% of the transcribed RNA of interest contains a cap analog.

[0173] 41. The method of paragraph 39 or 40, wherein the transcribed RNA of interest has a length of at least 2000 nucleotides.

[0174] 42. The method of any one of paragraphs 30 to 41, wherein at least 90% of the transcribed RNA of interest contains the RNA cap analog by 180 minutes and / or 360 minutes of the IVT reaction.

[0175] 43. The method of any one of paragraphs 12-42, wherein the initial and / or ongoing IVT reaction mixture is not supplemented with an RNA cap analog during the IVT reaction.

[0176] 44. The method of any one of paragraphs 12 to 43, wherein the DNA concentration in the initial reaction mixture is 0.025 to 0.075 mg / mL, optionally 0.05 mg / mL.

[0177] 45. The method of any one of paragraphs 12 to 44, wherein the DNA concentration is maintained at a concentration greater than 0.01 mg / mL, optionally between 0.01 and 0.05 mg / mL, during the IVT reaction.

[0178] 46. ​​The method of any one of paragraphs 12 to 45, wherein the molar ratio of the transcribed RNA of interest to the DNA in the IVT reaction is at least 2-fold or at least 3-fold greater than the molar ratio of the transcribed RNA to DNA in a non-fed-batch control method.

[0179] 47. The method of any one of paragraphs 23 to 46, wherein the UTP is a modified UTP selected from 1-methylpseudouridine and 1-ethylpseudouridine.

[0180] 48. The method of any one of paragraphs 12 to 47, wherein the transcribed RNA of interest is messenger RNA (mRNA).

[0181] 49. The method of any one of paragraphs 12 to 48, wherein the transcribed RNA of interest has a length of more than 100 nucleotides.

[0182] 50. The method of any one of paragraphs 12 to 49, wherein the total IVT reaction time is 150 to 1000 minutes.

[0183] 51. The method of any one of paragraphs 12 to 50, wherein at least 50% or at least 70% of the transcribed RNA of interest contains a polyA tail by 420 minutes of the IVT reaction.

[0184] 52. The polyA tail is A 100 52. The method of paragraph 51, which is a polyA tail.

[0185] 53. The method of any one of paragraphs 30 to 52, wherein the initial reaction mixture comprises a concentration of RNA cap analog that is at least 10% or at least 20% higher than the concentration of the NTP present at the first coding position of the RNA of interest.

[0186] 54. The method of paragraph 53, wherein the NTP present in the first coding position of the RNA of interest is ATP or GTP.

[0187] 55. The method of any one of paragraphs 1 to 54, wherein the RNA polymerase is T7 RNA polymerase.

[0188] 56. The method of paragraph 55, wherein the T7 RNA polymerase comprises an additional glycine at the C-terminus relative to wild-type T7 RNA polymerase.

[0189] 57. The method of paragraph 55, wherein the T7 RNA polymerase comprises a G47A substitution relative to wild-type T7 RNA polymerase.

[0190] 58. The method of paragraph 55, wherein the T7 RNA polymerase comprises a G47A substitution and an additional glycine at the C-terminus relative to wild-type T7 RNA polymerase.

[0191] 59. The method of any one of paragraphs 30 to 58, wherein the RNA cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

[0192] 60. The RNA cap analog comprises the trinucleotide sequence GAG, optionally GpppA2' Ome 60. The method of paragraph 59, comprising pG.

[0193] 61. The method of paragraph 60, wherein the RNA cap analog comprises the tetranucleotide sequence GGAG.

[0194] 62. The method of any one of paragraphs 12 to 61, further comprising isolating the transcribed RNA of interest.

[0195] 63. The RNA of interest isolated by the method according to paragraph 62.

[0196] 64. Ribonucleic acid (RNA) produced by a method according to any one of paragraphs 12 to 63.

[0197] 65. The RNA of paragraph 63 or 64, formulated in a cationic lipid nanoparticle, optionally wherein the cationic lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. EXAMPLES

[0198] The present disclosure is further illustrated by the following examples, which are provided to aid in the understanding of the present disclosure and should not be construed as limiting thereof.

[0199] The empirical balanced fed-batch in vitro transcription (IVT) reaction of the present disclosure is based, in part, on the following: Empirical equilibration of nucleotides Identifying the percentage of each nucleotide consumed for a given DNA Formulate master mixes of nucleotides for use in IVT reactions and feedstocks using their consumption percentages Determining Feed Schedule and Feedstock Composition · Adjusting supply schedules to accommodate changes in reaction rates over time Supply a ratio of cap analog to maximum concentration of purine NTP greater than 1 Feedstock blending maximizes yield and product quality while minimizing cost of goods

[0200] Example 1. Empirical Equilibration of Nucleotides In this example, the fed-batch IVT reaction uses four nucleotides: CTP, GTP, UTP, and ATP. We have shown that the relative molar ratio of each nucleotide can be altered to maximize reactant utilization or to alter the properties of the RNA product. Here we present a platform for equilibrating the ratios of these four nucleotides according to their consumption rates so that the concentration of each is maintained throughout fed-batch IVT.

[0201] Once the DNA encoding the new RNA product is obtained, calculate the percentage consumption by following these steps: (1) Perform a batch IVT reaction with known initial concentrations of CTP, GTP, UTP, and ATP and measure the concentration of each nucleotide in the reaction. Ensure that all nucleotides are above 0 mM for all measurement time points, otherwise discard the data collected at that time point. For the data shown in Figures 1A-1B, all nucleotides had an initial concentration of 5 mM and their concentrations were measured for up to 30 min. (2) The measured concentrations and times collected are used to calculate the rates of consumption of CTP, GTP, UTP, and ATP during the reaction. In the case of multiple time points, this can be the slope of a linear fit to the data (see FIG. 1A). (3) Add the rates of CTP, GTP, UTP, and ATP to determine the total consumption rate of all nucleotides (Figure 1B). (4) The rate of each individual nucleotide is divided by the rate of all nucleotides to determine the percentage consumption of CTP, GTP, UTP, and ATP.

[0202] The relevant values ​​are given in Table 1. [Table 1]

[0203] The percentage consumption value can be used to formulate the reaction mixture for a batch IVT reaction, or the NTP master mixture and the feed mixture for a fed-batch IVT reaction. For example, the NTP master mixture can contain a molar ratio of NTPs equivalent to the percentage consumption value to ensure that all nucleotides are completely consumed simultaneously in a batch reaction. Alternatively, for fed-batch reactions, it may be preferable to instead formulate the NTP master mixture so that all nucleotides are at equal concentrations when the sum of all nucleotides is equal to 10 mM, or NTPs are maintained at some concentrations above 0 mM. Finally, it may be preferable to maintain a specific nucleotide ratio throughout the reaction to generate a desired product profile. The percentage consumption value allows the operator to achieve any of these process requirements.

[0204] The empirically determined percent consumption values ​​differ from the % abundance of each NTP observed in the desired RNA sequence. Furthermore, the determination of the percent consumption does not require prior knowledge of the sequence of the construct.

[0205] Ultra Performance Liquid Chromatography (UPLC®) may be used to monitor the concentrations of NTPs and RNA cap analogs, as shown in Figure 2. This information can then be used to calculate individual NTP concentrations and / or total NTP concentrations. As an example, the following conditions are provided: Column: ACQUITY UPLC Oligonucleotide BEH C18 Column, 130Å, 1.7μm, 2.1mmX150mm (Part Number 186005516) Column temperature: 40℃ Flow rate: 0.4mL / min Mobile phase A: 100 mM triethylammonium acetate (TEAA) Mobile phase B: 100 mM triethylammonium acetate (TEAA), 25% acetonitrile

[0206] Exemplary IVT master mix and feedstock formulations are shown in Tables 2 and 3 as follows, based on the data shown in Figures 1A-1B. [Table 2] [Table 3]

[0207] Example 2. Empirical Equilibration of NTP by Consumption Rate Empirical equilibration reactions of initial nucleotides were performed for DNA encoding two constructs, RNA#1 and RNA#2. Graphs showing the individual NTP concentrations per DNA are shown in Figures 3A and 3B. The following percentage consumption (%) values ​​were determined as described in Example 1: [Table 4]

[0208] Customizing NTP ratios in fed-batch IVT reactions The percentage consumption values ​​of CTP, GTP, UTP, and ATP can be used to control the NTP ratios remaining in the initial IVT reaction mixture, during the ongoing reaction, or after the completion of the IVT reaction. In one example, the process of making RNA#1 may require complete utilization of all four NTP reagents by the end of the reaction. In this case, a batch IVT reaction of RNA#1 was performed with the relative nucleotide ratios set equal to the empirical percentage consumption values. The graph shown in Figure 4A shows the NTP concentrations in this reaction. All four nucleotides approach 0 mM concentration at the same time, maximizing utilization of these components.

[0209] In other scenarios, the process may need to maintain specific NTP ratios during fed-batch or batch operation to generate a particular product profile or ensure full utilization of a particular reagent. For example, the process may require that [CTP], [GTP], [UTP], [ATP] be equal to the extent that the fed-batch IVT reaction is maintained, e.g., when total [NTP] is equal to 10 mM. In this case, the empirically determined percentage consumption values ​​were used to calculate the adjusted composition of the nucleotide master mix to meet this process requirement (Table 4). The graph shown in Figure 4B shows a batch IVT reaction with DNA encoding RNA#1, which used a unique nucleotide master mix for all NTPs to be equivalent at 10 mM total NTPs (Table 4). As shown in the figure, all four nucleotide reagents reached 2.5 mM simultaneously, i.e., total NTP concentration was equal to 10 mM. [Table 5]

[0210] Trinucleotide (GAG) Capping Efficiency Test Test IVT was performed to evaluate the impact of GAG:NTP ratios, where the NTP is either ATP or GTP. In this set of experiments (Figure 22), GAG:ATP or GAG:GTP ratios greater than 0.6 produced mRNA with greater than 90% cap. Use of RNA cap analogs with GAG:ATP or GAG:GTP ratios greater than 0.6 produced mRNA with optimal cap content. Thus, fed-batch IVT is designed to maintain a GAG:ATP or GAG:GTP ratio greater than 0.6.

[0211] Example 3. Setting a supply schedule An important aspect of the fed-batch IVT process is to identify the appropriate timing of additions for a given feedstock and IVT reaction mixture to maintain reagents in the desired range. To do this, a scouting fed-batch IVT reaction was performed with the DNA of interest using an empirical equilibrated nucleotide master mix (from Examples 1 and 2) and an initial feeding schedule based on the predicted NTP consumption rate for that DNA. This initial feeding schedule is modified depending on the results and analysis of this scouting fed-batch IVT reaction, as described below.

[0212] Throughout the scouting fed-batch IVT reactions, the total concentration of nucleotides was measured for each feed, immediately before and after the addition of the feedstock mixture to the IVT reaction (Figure 5A-B). These total concentration measurements and the feed times were then used to calculate the specific total nucleotide consumption rate between each feed of the reaction (Figure 6A-B). As shown in Figure 7A-C, these nucleotide consumption rates were plotted against either [DNA], [mRNA], or reaction time (in this example, [DNA] was used). Curve fitting of this plot (showing a linear model) provided an empirical model of the nucleotide consumption rate through the fed-batch IVT reaction. By incorporating these NTP consumption model parameters along with several additional parameters (below), the complete feeding schedule for a given feedstock and IVT reaction mixture used for RNA#1 was determined (see Table 5). Early [DNA] Minimum nucleotide concentration Initial nucleotide concentration Feedstock nucleotide concentration First feed volume fraction (feed volume / initial reaction volume) Either total reaction time or total nucleotides added [Table 6]

[0213] Example 4. Reaction modeling After completing the empirical equilibration experiments of nucleotides (Examples 1 and 2) and setting the feeding schedule for the given DNA, feedstock, and IVT reaction mixture (Example 3), a fed-batch IVT reaction model was created by combining several empirically determined reaction parameters with user-selected parameters of the fed-batch IVT reaction: Empirically determined reaction parameters % Consumption of CTP, GTP, UTP, and ATP (from Example 1) NTP Consumption Curve Fit Parameters (from Example 3) % of products with initial tail (observed in Example 3) Percentage of tailed product vs. time (as observed in Example 3) User-selected reaction parameters Ratio of CTP, GTP, UTP, and ATP in the nucleotide master mix for the initial IVT reaction Ratios of CTP, GTP, UTP, and ATP in the feedstock nucleotide master mix Initial [DNA] or reaction volume (if used for NTP consumption curve fitting) Initial trinucleotide concentration Minimum nucleotide concentration Initial nucleotide concentration Feedstock nucleotide concentration First feed volume fraction (feed volume / initial reaction volume) Either total reaction time or total nucleotides added

[0214] As shown in Figures 9-11, the reaction model was able to estimate total and individual nucleotide concentrations (Figure 9), yield of RNA product (Figure 10), and % RNA with tails (Figure 11). Additionally, the model provided guidance to the expected % capping of RNA products (by comparing [purine] and [trinucleotide]) and optimal reaction time for any DNA.

[0215] Example 5. Fed-batch IVT process testing of RNA#1 and RNA#2 Empirical equilibrated fed-batch IVT reactions were performed for RNA#1 and RNA#2. Feed schedules were set for both RNA#1 and RNA#2 based on kinetic analysis (Figures 6A-B and 7A-C). The following initial IVT conditions were used for each construct: Formulation of early IVT response: 40 mM Tris-HCl, pH 8.0 20 mM total nucleotides (empirically equilibrated to 10 mM NTPs) 30mM Magnesium Acetate RNA cap analog (trinucleotide) equal to 1.2*[ATP] 0.04mg / mL RNA polymerase 0.02% Triton X-100 (w / v) 0.05mg / mL plasmid 5mM DTT 1mM spermidine 1U / mL PPiase Test Details: ·Temperature: 37℃ Starting reaction volume: 500μL ·Tumble Mixture Feed material formulation: 40 mM Tris-HCl, pH 8.0 60mM total nucleotides (empirically equilibrated to 10mM NTPs) 60mM Magnesium Acetate 0.04mg / mL RNA polymerase 0.02% Triton X-100 5mM DTT 1mM spermidine 1U / mL PPiase Supply Schedule: ·Constant supply amount: 71.4μL Supply time: oRNA#1: 60, 85, 115, 150, 185, 220, 260 minutes (supplied 7 times) oRNA#2: 60, 100, 140, 180, 220, 270 minutes (supplied 6 times) Total reaction time: 360 minutes (6 hours) Final IVT reaction volume: oRNA#1 (supplied 7 times): 1000μL oRNA#2 (supplied 6 times): 928μL Analysis output: RNA product yield - Monitor NTP and RNA cap analog concentrations using ultra-high performance liquid chromatography Tailed RNA % Capped RNA %

[0216] Nucleotide concentration, yield, and product quality were assessed throughout fed-batch IVT reactions of RNA#1 and RNA#2, as shown in Figures 11-16. Figures 11A-B and Figures 12A-B show total [NTP] and individual [NTP] measurements during fed-batch IVT reactions for RNA#1 and RNA#2, respectively. Figures 13A and 13B show measured concentrations of total RNA and tailed RNA for RNA#1 and RNA#2, respectively. Figures 14A-B show measured mass of total RNA and tailed RNA versus initial IVT reaction volume for RNA#1 and RNA#2. Figure 15 shows % tailed RNA product for RNA#1 and RNA#2. Figure 16A shows % capped mRNA product for RNA#1 and RNA#2, and Figure 16B shows % capped RNA product over time for RNA#1. These experimental results can be compared to IVT model output using the same parameters to validate reaction results or improve the model. Construct-specific parameters can also be recorded and used to develop processes for future constructs.

[0217] Example 6. Continuous fed-batch at two flow rates for RNA#3 In Examples 3, 4, and 5, each fed-batch IVT reaction was performed using a bolus feed, in which the feed mixture was added in a fixed volume bolus addition at specific times during the reaction. This process resulted in fluctuations in nucleotide concentrations during the reaction at each feed (Figures 11A-B and 12A-B). To minimize these fluctuations and reduce the need for additional manufacturing controls required for different bolus feeds, the feeding schedule was transitioned to a continuous feed format.

[0218] An example of a continuous fed-batch IVT process is shown in Figures 17-22. Here, a bolus fed-batch reaction is compared to a continuous fed-batch reaction of DNA encoding RNA#3. Tables 6 and 7 show the process parameters for the bolus and continuous-feed IVT reaction modes used to test RNA#3. The bolus-fed version of this process required 18 process parameters, with an initial reaction volume of 1 L and each bolus feed volume of 14.28 mL. See Table 6. In contrast, the continuous-feed mode required five process parameters. See Table 7. This continuous-feed schedule included five feed parameters: start time, flow rate 1, switch time from flow rate 1 to flow rate 2, flow rate 2, and stop time (Table 7). These two flow rates were used to accommodate any changes in nucleotide consumption rate that occurred during the IVT reaction. [Table 7] [Table 8]

[0219] The graphs in Figures 17A-17B show that the total nucleotide concentrations throughout the IVT reactions for RNA#3 were much more consistent in the continuous fed-batch reactions than in the bolus fed-batch reactions. Figures 18A-18B and 19A-19B show comparable RNA product yields in the continuous and bolus fed-batch IVT reactions.

[0220] The overall product quality of RNA#3 was slightly improved in the continuous fed-batch IVT reaction compared to the bolus fed-batch. The graphs in Figures 20A-20B show that the percentage of RNA product with tails for RNA#3 was comparable but improved when using continuous fed-batch over bolus. The graphs in Figures 21A-21B show that capping was slightly better in the continuous fed-batch IVT reaction. This observation may be explained by the lower average A / GTP to trinucleotide ratios throughout the continuous fed-batch reaction. See Figure 22.

[0221] Example 7: Fed-batch IVT using tetranucleotide capped analogs In this example, a fed-batch IVT reaction was performed to synthesize RNA using a bolus feed with a tetranucleotide cap analog. An initial IVT mixture was created using nucleotides (Table 8), GGAG tetranucleotide cap analog, T7 RNA polymerase, pyrophosphatase, buffer, and DNA encoding RNA#4, present at equilibrated molar ratios to the RNA of interest. The initial molar ratio of tetranucleotide cap analog to ATP was 1.3:1. A feed master mix was composed of nucleotides, T7 RNA polymerase, pyrophosphatase, and buffer. The reaction was fed three times at 30 minute intervals using the feed master mix with a minimum of 8 mM NTPs as a target, and the reaction was allowed to proceed for 2 hours.

[0222] A batch IVT reaction using the same initial IVT mixture was run as a control experiment, in which no feed was added after the start of the reaction. [Table 9]

[0223] As shown in Figures 23-25, ATP concentration, total RNA yield, and percent capping were evaluated for both fed-batch IVT and control batch reactions. The fed-batch reaction provided RNA with high capping efficiency (about 90% of the RNA contained the cap analog). Surprisingly, the fed-batch IVT reaction significantly outperformed the control batch reaction with about 1.8-fold higher yield of RNA (about 5 mg / mL per initial volume for the control batch reaction compared to about 9 mg / mL per initial volume for the fed-batch reaction).

Claims

[Claim 1] 1. A method for determining the percentage (%) of nucleoside triphosphate (NTP) consumption of an in vitro transcription (IVT) reaction, comprising: (a) performing an IVT reaction in a reaction mixture containing a known initial NTP concentration, a deoxyribonucleic acid (DNA) encoding a ribonucleic acid (RNA) of interest, and an RNA polymerase; (b) measuring individual NTP concentrations at discrete intervals over a period of time; and (c) calculating a percent (%) value for each NTP of the reaction mixture.

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