Method and apparatus for preparing capped RNA by feed batch transcription

Abstract

The present invention relates to ribonucleic acids (RNAs) and methods for the synthesis and co-transcription of capped RNAs. The present invention relates, inter alia, to methods of enhancing mRNA co-transcription capping using cap analogs. The invention also relates to devices for performing such methods, as well as to devices capable of improving the synthesis of in vitro transcribed mRNA, and more particularly to the addition of 5 '-cap structures in in vitro transcribed mRNA.

Description

[0001] The present invention relates to ribonucleic acids (RNAs), in particular to methods of synthesis and co-transcriptional capping of RNAs, but not limited thereto. The present invention relates, inter alia, to methods of enhancing co-transcriptional capping of mRNA using cap analogs. The present invention also relates to devices for performing such methods, and devices enabling improved synthesis of in vitro transcribed mRNA, more specifically to the addition of 5' cap structures on in vitro transcribed mRNA.

[0002] All living organisms use DNA to store their genetic information. Most of these genes code for specific proteins, which control all cellular functions, including metabolic processes, cell signaling enabling the cell to respond to the environment, cellular structures and eventually the organism, and intercellular communication through cell-cell contact and hormones. The key to transforming genetic information from the DNA level into proteins is a molecule called messenger RNA (mRNA). Each gene is first transcribed into mRNA, which is then translated into proteins by ribosomes. From bacteria to fungi to plants to animals, the key elements of this process are very similar in all life kingdoms. After transcription of the mRNA, so-called post-transcriptional modifications occur, which further modify the primary structure of the mRNA. However, these post-transcriptional processes and / or their functional implications differ between the different life kingdoms.

[0003] Messenger RNA is a ribonucleic acid polymer, whose backbone consists of the pentose sugar ribose and phosphoric acid. The nucleobases adenine, guanine, cytosine and uridine are attached to these sugars via the 1 'C-atom. The resulting nucleosides are coupled to each other via phosphate molecules, which form esters with the hydroxyl group on the 3 'C-atom of one ribose and the hydroxyl group on the 5 'C-atom of another ribose. The 5'- and 3'-C atoms impart directionality to the mRNA molecule. During transcription, the mRNA is synthesized along the DNA template from 5' to 3'. In addition, translation by the ribosome also proceeds from 5' to 3'. One unit of the mRNA polymer consisting of a nucleoside and a phosphate is called a nucleotide.

[0004] As one of the structural modifications, for all eukaryotic mRNAs, an adenine nucleotide stretch is attached to the 3' end of the mRNA molecule. This poly-A tail plays a key role in controlling the lifetime and expression of the mRNA, as the poly-A is bound by proteins that protect the mRNA from degradation by RNA exonucleases. Poly-A tails also exist in prokaryotes, however, in this case, it destabilizes rather than stabilizes the mRNA.

[0005] Another important modification of mRNA is the addition of a so-called 5' cap structure, which is a chemical modification of the 5' end of the mRNA. The 5' cap is essential for the initiation of ribosome assembly at the mRNA and for the expression of proteins from the mRNA. Cap structures differ between bacteria and eukaryotes, and these structural differences also form the basis for the recognition of foreign mRNA by the innate immune system of higher organisms, as described below. Importantly, in eukaryotes, the 5' cap is added in the nucleus, which means that all cytosolic mRNA that does not carry the correct 5' cap is most likely of foreign (pathogenic) origin and thus can constitute a threat.

[0006] In addition to mediating the initiation of translation by ribosome binding, the 5' cap also protects the mRNA from 5' to 3' exonucleases and prevents the activation of RIG-I by 5' phosphate priming, which mediates a strong intracellular anti-viral response.

[0007] 5' cap-initiated translation is the main mechanism of translation initiation. Without the 5' cap, most mRNA molecules would lose their activity due to the lack of ribosome engagement and the subsequent mRNA scanning and translation. Only internal ribosome entry sites can substitute for this property of the 5' cap. However, mRNA produced by in vitro transcription (IVT) is usually used for translation initiated by 5' cap binding. Without the 5' cap, the 5' end is easily exploited by 5' to 3' exonucleases, such as human exonuclease Xrn1, which can digest decapped mRNA or uncapped mRNA. When uncapped, the mRNA molecule usually has 5' end triphosphate groups, which mediate the binding of RIG-I (retinoic acid-inducible gene I) and similar proteins, thereby activating the IFN-I response by this pattern recognition receptor. The main function of this response under pathophysiological conditions is to limit viral spread, to initiate and promote the innate immune response, and to activate the adaptive immune response for long-term, broad viral control. Viral spread is limited mainly by the production of pro-inflammatory cytokines and the restriction of viral protein expression.

[0008] Therefore, the introduction of uncapped mRNA into cells usually leads to low or even essentially no protein expression from the transfected mRNA and to the induction of pro-inflammatory cytokines, which cause inflammation and can lead to cell death and / or (global) inhibition of protein translation, depending on the amount of transfection. Therefore, it is crucial during mRNA production to ensure a high capping efficiency, i.e. that most or almost all mRNA molecules in a pharmaceutical mRNA preparation are capped.

[0009] Finally, eukaryotic mRNA is mostly formed as so-called pre-mRNA consisting of exons and introns. Only exons code for proteins, and therefore introns are removed during the splicing process, which also takes place in the nucleus. In addition, single nucleobases can be chemically modified by enzymatic mechanisms, which is another way to distinguish foreign mRNA from self mRNA. Only fully mature mRNA can leave the nucleus and is expressed in the cytoplasm.

[0010] Considering (i) that mRNA can code for almost any protein, (ii) that the presence of mRNA in the cytoplasm is transient and therefore does not pose a risk of permanent changes to the genome, and (iii) that mRNA, unlike DNA, does not have to enter the nucleus to be expressed and can therefore also be applied to non-dividing differentiated cells, the introduction of synthetic mRNA into cells can be considered as a very interesting way of expressing cellular proteins. In 1990, it was first shown that the injection of naked mRNA leads to an immune response against the protein encoded by the mRNA, thus demonstrating that mRNA can be used as a messenger for protein production in vivo (Wolff JA, et al. Science. 1990; 247: 1465-1468). Two years later, it was also shown that the in vivo delivery of mRNA can express biologically active proteins (Jirikowski GF, et al. Science. 1992; 255: 996-998).

[0011] However, it took more than two decades for the full potential of mRNA as a highly valuable molecular form to be recognized more widely in the fields of medicine and biotechnology. The reason for this delay was the general belief that mRNA is a biologically very unstable molecule that is rapidly degraded by the ubiquitous presence of RNases in the body and on the skin, thus complicating the handling of mRNA. In addition, since mRNA is large and negatively charged, delivery vehicles had to be developed that could form complexes with mRNA, thus protecting mRNA from degradation and mediating the entry of mRNA into cells and cytoplasmic delivery. Finally, while people began to explore mRNA as a therapeutic, innate immune mechanisms were also discovered, showing that exogenously added mRNA can act as a potent immune-stimulating molecule, triggering inflammatory reactions and inhibiting the biosynthesis of cellular proteins, as described above.

[0012] It is therefore apparent that in vitro transcribed mRNA is more challenging than initially thought, as only mRNA that mimics as closely as possible the endogenous mRNA will avoid recognition by innate immune receptors, resulting in high protein expression with no side effects. Therefore, innovations that improve in vitro synthesis of mRNA, including improving the yield of synthesis, and structural changes that reduce innate immunity, have great economic value, such as patents containing chemically modified bases (PCT / US06 / 32372 - RNA containing modified nucleosides and methods of use thereof) and patents containing modified 5' cap structures (US7074596B2 - Synthesis and use of anti-reverse transcription mRNA cap analogs).

[0013] The mRNA-based SARS-CoV2 vaccines Comirnaty (sold by Pfizer / BioNTech) and SpikeVax (sold by Moderna) have achieved a breakthrough success, thanks to the above-mentioned in-depth understanding of mRNA biology, RNA (in particular mRNA) is considered one of the fastest growing molecular modalities in the field of therapeutic development and biotechnology, with billions of dollars in market, the final size is still difficult to predict, but largely depends on the cost of goods and safety. The cost of goods is directly related to the manufacturing method, and safety is related to the avoidance of byproducts, which also includes 5' incompletely capped mRNA. Especially for medical applications that require the application of large amounts of mRNA more frequently, such as enzyme replacement therapy, in which mRNA encodes proteins missing in patients due to genetic defects, both factors are crucial for the long-term success of therapeutic mRNA.

[0014] Therefore, there is a need to provide improved in vitro transcription of RNA that enables in vitro transcription of mRNA at a lower cost and higher 5' end capping yield than the prior art. Given the enormous economic significance of in vitro transcribed mRNA in the medical and biotechnology fields, those skilled in the art will recognize the enormous economic potential of the invention described herein. In addition, there is also a need to develop new devices to achieve improved synthesis of in vitro transcribed mRNA, more specifically, to enable the addition of 5' end cap structures on in vitro transcribed mRNA by a variety of methods. The resulting mRNA product should have a higher degree of 5' end cap structure, while reducing the consumption of expensive reagents and simplifying the purification process, thereby reducing the cost of in vitro transcribed mRNA and reducing the activation of innate immune response by 5' uncapped mRNA to improve protein expression and reduce adverse side effects of in vitro transcribed mRNA in in vitro or in vivo applications.

[0015] The inventors have worked to improve RNA in vitro transcription with higher 5’ end capping yields. As mentioned above, the 5’ cap is an important feature of mRNA molecules, but unfortunately, it is also one of the most costly components, regardless of the method of introduction. Generally, there are two methods of introducing the 5’ cap on nascent mRNA molecules, including:

[0016] (i) Post-transcriptional capping by enzymes such as vaccinia virus capping enzymes or Fausto capping enzymes, which are typically used in the second step after mRNA in vitro transcription due to incompatible buffer requirements (salts) between different enzymes. In addition, some of the methyl donor molecules, such as S-adenosyl methionine (SAM), are unstable and can therefore limit the effective duration of the IVT reaction. These enzymes modify the 5’ end of the nascent RNA, recruiting and coupling GTP through a series of intermediate steps, and then methylating the inversely coupled (5’-5’ coupling) GTP to form the mature cap. Under the right conditions, and using uncomplicated 5’ ends (limited or weak secondary structure formation), the above enzymes are typically highly efficient, and the cap will only be added in the correct orientation; and

[0017] (ii) Co-transcriptional capping by cap analogs, including mCap (TriLink Biotechnologies, Inc), anti-reverse cap analog (ARCA - ThermoFisher Scientific), and trinucleotide cap analogs. In this process, cap analogs that mimic the 5’ cap (i.e., dinucleotides with 5’-5’ linkage or trinucleotides with 5’-5’ linkage) are mixed into the IVT reaction and are introduced by the RNA polymerase as the first nucleotide instead of the competing nucleotide (e.g., rGTP in the case of ARCA). Using mCAP results in about 50% of the cap being introduced in the wrong orientation, resulting in 50% of the mRNA molecules being inactive. The anti-reverse cap analog (ARCA) is modified to the sugar backbone to prevent introduction in the wrong orientation. Likewise, the trinucleotide cap analog can only be introduced in the correct orientation.

[0018] The underlying principle of introducing and competing for the initiation nucleotide is the same for each of the above cases. Typically, in a batch in vitro transcription reaction involving co-transcriptional capping, a fixed ARCA:GTP (or other cap analog:competing nucleotide) ratio is set at the beginning of the reaction, while a fixed amount of several other reagents, including nucleoside triphosphates (NTPs), magnesium, RNA polymerase, deoxyribonucleic acid (DNA), and optionally pyrophosphatase, are set. During the course of the reaction, NTPs are consumed, and the first to be depleted limits the yield of the reaction. In order to achieve reasonable capping efficiency, the concentration of the nucleotide that competes with the cap analog (e.g., rGTP with ARCA, for being introduced as the first nucleotide of the mRNA) and that produces uncapped mRNA after being introduced is limited. Alternatively, the cap analog can be added at an elevated concentration, but this strategy is limited by the cost and solubility of the cap analog.

[0019] Thus, a balance must be struck between mRNA yield and capping efficiency of the IVT reaction. The most commonly used protocol employs a 4:1 ratio of cap analog (e.g., ARCA) and competing nucleotide (e.g., rGTP), which, if the two molecules have identical tendencies to be introduced as the first nucleotide, results in a theoretical capping efficiency of about 80%. In such a protocol, the amount of limiting nucleotide (e.g., rGTP) is limited to 1 / 4 of the concentration of the other nucleotides (ATP, CTP, UTP, and / or their chemically modified counterparts), thereby limiting the yield to about 1 / 4.

[0020] Thus, according to the inventors’ calculations, it can be determined that, when performing an IVT reaction involving co-transcriptional capping by a cap analog, the amount of cap analog consumed is a fraction of the amount of (competing) nucleotide consumed. In fact, for a given length of mRNA, the relative amount of cap analog consumed relative to nucleotide can be expressed as 1 / mRNA length, or, more accurately, the amount of cap analog consumed relative to competing nucleotide can be expressed as 1 / (mRNA length the fraction of competing nucleotide in that mRNA). Most (therapeutically relevant) mRNA products are at least 500 nt long, and thus the fraction is at least 1 / 500, and up to 1 / 20,000 for the longest constructs. Thus, it can be inferred that the concentration of cap analog remains relatively constant during the course of a batch reaction, and the unconsumed cap analog constitutes a significant expense and waste.

[0021] To address this problem, fed-batch reactions in the prior art add a feed solution containing the fresh amount of nucleotides consumed during the course of the reaction, thereby restoring the original concentration of these nucleotides. Such feed solutions typically contain a mixture of all consumed nucleotides, and optionally the relative proportions are adjusted according to empirical (WO2020185811) or calculated reaction relative consumption.

[0022] To achieve high capping efficiency in co-transcriptional capping IVT reactions, a high ratio of cap analogue to competing nucleotides must be maintained to prevent the formation of undesirable uncapped RNA contaminants due to the introduction of competing nucleotides. The theoretical capping efficiency at any given moment can be expressed as: Capping efficiency = 100 ([hat-like items]) t / [Sum of competing nucleotides] t) Where t = a specific time point, assuming the introduction rates of such capping analogs and competing nucleotides are equal or similar. As previously mentioned, in batch reactions (see Table 1), the capping analog / competing nucleotide (e.g., ARCA / rGTP) ratio increases as the reaction proceeds because the consumption of competing nucleotides is higher than that of capping analogs. Assuming the competing nucleotide is the yield-limiting nucleotide and drops to 0 mM, and assuming the introduction rates are equal or similar, the capping efficiency can be expressed as: Capping efficiency = 100 ([hat-like items]) 开始 / ([Sum of competing nucleotides]) 开始 / 2)).

[0023] Table 1 - Cap efficiency and component ratios for batch reactions

[0024]

[0025] However, in batch feed reactions (see Table 2), capping efficiency depends not only on the initial concentration ratio of the capping analogue and competing nucleotides, but also on the timing, concentration, and proportion of each component in the feed solution.

[0026] Table 2 - Cap efficiency and component ratios for fed-batch reactions

[0027]

[0028] As shown in Tables 1 and 2, obtaining high capping levels of mRNA requires an extremely high cap analog to competing nucleotide ratio. This is typically achievable only by using relatively low concentrations of competing nucleotides (e.g., to achieve >95% capping with 10 mM ARCA, an average concentration of 500 μM rGTP is required). Generally, the lower the concentration of the rate-limiting nucleotide, the slower the reaction rate. These principles remain valid when using trinucleotide cap analogs with increased introduction rates into mRNA, and when using engineered RNA polymerase variants that favor cap analog introduction at the head position of nascent mRNA compared to non-competitive nucleotide introduction, although the ratios may be more favorable.

[0029] It is also understandable that a higher number of feeds is required to obtain the same RNA yield compared to a similar reaction using the same amount of each NTP. The combination of a slower reaction rate and a higher number of feeds significantly prolongs the reaction duration. Therefore, maximizing the transcription rate is highly beneficial. Generally, more frequent feeds to replenish the rate-limiting nucleotide, while maintaining an average addition rate versus a consumption rate, result in the highest reaction rate for a given desired capping efficiency, as illustrated in the theoretical model below. In light of this, the inventors have recognized that continuous feeding is a desirable feature for any batch feed system designed to achieve high capping efficiency.

[0030] refer to Figure 6 In high-frequency or continuous feed concentrations (to avoid over-diluting reactants), fine fluid control of small volumes is required. This can be challenging depending on the total reaction volume and the associated tolerance feed volume. For example, for small-scale reactions (e.g., 20 µl, typically producing 100–200 µg mRNA in batches with no co-transcriptional capping and 5–10 mM per NTP), the fluid scale becomes too small to achieve sufficient precision, as shown in Table 3 below. For this application, the practical limits for microfluidic feed NTPs are in the range of 0.4 µl / min to >100 mL / min. For this application, the practical limits for macrofluidic feed NTPs are in the range of >60 µl / min to >100 L / min.

[0031] Table 3 - Cap reaction yields achieved by continuous feed of reagents quantity

[0032]

[0033] Besides the difficulty in fluid control, dilution of IVT reactions through repeated or continuous feed solutions is another challenge. Industrial batch IVT reactions utilize 5-10 mM of each NTP (except competing nucleotides) to drive high-speed, high-efficiency reactions; these NTPs are present at the start of the reaction. On the other hand, industrial batch-feed IVT reactions provide the same total amount of NTPs as batch reactions, distributed across multiple or continuous feeds. When using a 100 mM concentrated feed stock solution, each nucleotide accounts for 5-10% of the total reaction volume. When more than one NTP is provided, multiple amounts of this concentration are added to the reaction, potentially leading to unnecessarily diluted remaining reaction components (including RNAP, DNA, etc.), up to 40%. The dilution problem is exacerbated when using lower concentration feed stocks.

[0034] Finally, continuous feed batch reactions using concentrated feed solutions rely on (continuous) vigorous solution mixing to distribute the components in the feed solution throughout the reaction volume. Vigorous mixing, especially prolonged mixing, can damage proteins and reduce system productivity. Weaker mixing, on the other hand, can result in prolonged periods of unmixed or incompletely mixed solutions.

[0035] Despite recent advancements, current co-transcriptional IVT protocols still fail to achieve sufficiently high capping efficiency while simultaneously achieving high reaction yields and / or high consumption of expensive cap analogs. Solutions relying on existing batch feed protocols cannot achieve precise control over the feed rate, thus failing to maintain a constant, extremely low level of competing nucleotides at a given (extreme) cap analog to competing nucleotide ratio to sustain optimal reaction rates.

[0036] Therefore, there is an urgent need for improved methods and related devices to achieve a combination of high yield and high (even higher) capping efficiency for the economical production of high-quality mRNA.

[0037] The inventors have devised a novel IVT method, particularly for co-transcriptional capping of RNA using cap analogs, wherein a high ratio of cap analog to competing nucleotides is selected (to obtain therapeutically relevant capping efficiency). However, the result is that the yield of such reactions is too low, making it neither practical nor economical. Therefore, they have provided a batch feed scheme to address the yield problem. However, one problem with using the batch feed method is that using a concentrated stock solution for feeding to prevent over-dilutement of the reaction results in excessively low volumes, making adequate fluid control difficult and often leading to large fluctuations in reaction conditions. Conversely, feeding a diluted stock solution results in over-dilutement of the reaction. Therefore, the method of the present invention relies on using a diluted stock solution to obtain sufficiently precise control and simultaneously / subsequently removing excess fluid to correct for dilution. Furthermore, the inventors have used a system for adding the feed solution in a highly dispersed manner to the reaction volume to prevent localized time deviations in the overall reaction composition.

[0038] Therefore, in a first aspect of the invention, a method for preparing capped ribonucleic acid (RNA) molecules is provided, wherein the method comprises performing a feed batch transcription reaction in a substantially constant volume in the presence of a cap analogue to produce substantially capped RNA molecules.

[0039] In the second aspect, capped ribonucleic acid (RNA) molecules obtained or available through the methods of the first aspect are provided.

[0040] Thirdly, an apparatus for producing capped RNA is provided, the apparatus comprising: a reaction chamber in which transcription occurs in the presence of a cap analogue; and a feeding means for feeding a feed solution containing one or more reagents required for producing capped RNA, wherein the apparatus is configured to, in use, feed the reagents into the reaction chamber in a batch feed mode via the feeding means and perform transcription in the reaction chamber at a substantially constant volume, thereby producing substantially capped RNA molecules.

[0041] In another aspect, a method for preparing capped ribonucleic acid (RNA) molecules is provided, wherein the method comprises performing a feed-batch transcription reaction in the presence of a cap analogue to produce substantially capped RNA molecules.

[0042] Advantageously, the method and apparatus of the present invention achieve unexpectedly higher 5' capping yields compared to batch or standard feed batch methods, and the product cost is lower than that of the prior art. The capped RNA molecules of the second aspect of the present invention have a higher degree of 5' cap structure, while reducing the consumption of expensive reagents and simplifying the purification process, thereby significantly reducing the cost of in vitro RNA transcription. Furthermore, the increased 5' capping level results in higher protein expression, and fewer unwanted side effects from in vitro transcribed mRNA in in vitro or in vivo applications. For example, when 5' uncapped RNA is administered to subjects, for example as a vaccine, they induce less activation of the innate immune response.

[0043] The phrase "essentially constant volume" can refer to the volume of the transcription reaction being maintained, remaining essentially fixed or the same in a steady state. That is, the volume neither increases nor decreases during the reaction, and can therefore be described as volume equilibrium. This differs from the batch feeding method used in the prior art, which increases the reaction volume during the reaction.

[0044] However, it should be understood that very small volume fluctuations may occur in the method of the present invention. Preferably, any increase or decrease in the reaction volume is less than 5%, 4%, or 4%. More preferably, any increase or decrease in the reaction volume is less than 3%, 2%, or 1%. Even more preferably, any increase or decrease in the reaction volume is less than 0.5%, 0.4%, or 0.3%. Most preferably, any increase or decrease in the reaction volume is less than 0.2%, 0.1%, or 0.05%.

[0045] The method of the present invention can be performed in vitro or ex vivo. However, most preferably, the method is performed in vitro. Preferably, the method includes in vitro transcription (IVT).

[0046] RNA can be single-stranded or double-stranded. RNA can be coding or non-coding. RNA can be selected from the following RNA molecules: messenger RNA (mRNA), microRNA (miRNA), interfering RNA (RNAi), short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA, RNA aptamers, self-amplifying RNA (saRNA), coding RNA, non-coding RNA, and circular RNA.

[0047] Preferably, the RNA includes mRNA. The mRNA may contain a basic element cap, a 5' UTR, a 3' UTR, an IRES, a coding sequence, and a variable-length poly(A) tail.

[0048] The length of the RNA molecule (preferably mRNA) can be at least 50 bases, at least 60 bases, at least 75 bases, at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, or at least 900 bases. Those skilled in the art will understand that when the RNA is double-stranded, such as double-stranded RNA, "base length" refers to the length of the base pairs.

[0049] The length of the RNA molecule (preferably mRNA) can be at least 1000 bases, at least 2000 bases, at least 3000 bases, at least 4000 bases, at least 5000 bases, at least 6000 bases, at least 7000 bases, at least 8000 bases, at least 9000 bases, at least 10000 bases, at least 11000 bases, or at least 12000 bases.

[0050] The method preferably includes contact with: (i) a template nucleic acid sequence, (ii) an RNA polymerase, (iii) multiple nucleotide triphosphates (NTPs), and (iv) a cap analog, wherein the RNA polymerase uses the multiple NTPs and the cap analog to transcribe the template nucleic acid, thereby forming a capped RNA molecule, preferably in a single one-pot reaction.

[0051] The term "fed-batch" is well known to those skilled in the art and can refer to a process in which one or more reaction substrates (i.e., NTPs, capped analogs, and / or optional buffer solutions and / or Mg2+ ions) are fed into a reactor during a reaction, and the product (i.e., capped RNA molecules) is retained in the reactor until the end of the run. This contrasts with a "batch" reaction, in which all substrates are provided to the reactor at the start of the reaction and then the reaction proceeds to completion. A fed-batch reaction also differs from a "continuous" reaction, in which the substrate is continuously fed into the reactor and the product is continuously removed from the reactor.

[0052] The template nucleic acid sequence is preferably transcribed by RNA polymerase to produce an RNA molecule. Therefore, preferably, the template nucleic acid sequence comprises DNA. Preferably, the DNA contains a promoter suitable for RNA polymerase, wherein the promoter is suitably located upstream of the nucleic acid sequence serving as the template for the desired RNA molecule. The template nucleic acid can be prepared synthetically, for example by PCR or doggybone DNA. The template nucleic acid can comprise a vector, preferably a plasmid. The template nucleic acid can contain restriction sites or other suitable linearization sites to allow run-off transcription by RNA polymerase.

[0053] Preferably, the method includes using an RNA polymerase, selected from T7, T3, SP6, KP34, Syn5, or other DNA-dependent RNA polymerases, or any mutant variants of these RNA polymerases. Each of these RNA polymerases is capable of synthesizing capped RNA according to the method of the invention. Preferably, the method includes using T7 RNA polymerase or a variant thereof. In one embodiment of the invention, more than one RNA polymerase is used simultaneously in the method (preferably an IVT reaction) to transcribe multiple RNA sequences from multiple DNA templates or the same RNA sequence as multiple RNA molecules with different properties.

[0054] Preferably, the various nucleotide triphosphates (NTPs) are selected from ATP, GTP, CTP, and / or UTP, or modified variants thereof, including pseudoUTP, N1-methyl-UTP, m5CTP, m6ATP, m1ATP, inosine triphosphate, hm5CTP, m1GTP, m7GTP, or m6AmTP. NTPs are building blocks of RNA. Preferably, the method includes using substantially equal proportions of each of the ATP, CTP, and / or UTPs, and adjusting the amount of GTP to the amount of a cap analogue according to the desired capping efficiency. However, in some embodiments, the method may include different proportions of each of the ATP, GTP, CTP, and / or UTPs. This will depend on the sequence of the template nucleic acid and the resulting RNA molecule to be transcribed.

[0055] Preferably, the cap analogue is selected from the following cap analogues: mCap (m7G(5')ppp(5')G, TriLink Biotechnologies, Inc); anti-reverse cap analogue (an mCap version modified by 3' OH methylation on m7G, ARCA – ThermoFisher Scientific); and trinucleotide cap analogues (e.g., Cleancap – TriLink Biotechnologies); and modifications and / or combinations thereof. Those skilled in the art will readily understand the different types of modifications that can be made to these cap analogues.

[0056] It should be understood that the cap analogue mimics the 5' cap (i.e., a dinucleotide with a 5'-5' linkage, or a trinucleotide with a 5'-5' linkage, wherein the 5'-5' linked nucleotide is a guanosine or a variant thereof methylated at the 7' position) and is included in the reaction mixture and introduced by RNA polymerase as the first nucleotide of the nascent RNA rather than the corresponding competing nucleotide.

[0057] For example, in an embodiment where the cap analogue is ARCA, the competing nucleotide is rGTP. Furthermore, in an embodiment where the cap analogue is mCap, the competing nucleotide is rGTP. In an embodiment where the cap analogue is a trinucleotide cap analogue, the competing nucleotides are (i) rGTP (if the cap analogue binding sequence is GG) and (ii) rATP (if the cap analogue binding sequence is AG), wherein the cap analogue binding sequence is defined as the nucleotide that binds to the template nucleic acid sequence (preferably DNA) and is the first two introduced nucleotides.

[0058] Preferably, the method comprises contacting the template nucleic acid sequence, RNA polymerase, various nucleotide triphosphates (NTPs), and cap analogs in the presence of magnesium ions. Preferably, the concentration of magnesium ions is 1-5 mM higher than the total concentration of NTPs present at the time and / or added to the reaction. For example, a feed batch reaction containing 10 mM of each NTP may preferably contain >40 mM Mg2+; a feed batch reaction initially containing 5 mM of each NTP may initially contain >20 mM Mg2+, and after an additional 5 mM of each NTP is fed, an additional 20 mM Mg2+ is added. Since excess Mg2+ increases the tendency of RNA polymerase to produce dsRNA through RNA-templated RNA synthesis, excess Mg2+ should be minimized throughout the reaction. Therefore, it is preferable to add any additional Mg2+ required during or immediately after the addition of NTPs, rather than providing excess Mg2+ at the start of the reaction. As a general formula, the Mg2+ concentration can preferably be: ((the sum of the total amount of each NTP and cap analogue provided / reaction volume) + 1-5 mM of Mg2+ in the reaction volume).

[0059] Preferably, the method includes contacting the reagent with a buffer. In a preferred embodiment, the buffer is Tris (tris(hydroxymethyl)aminomethane) having a suitable counterion (preferably hydrochloride). The concentration of the buffer is preferably 10-50 mM Tris-HCl with a pH set at 7.8-7.9 (25 °C); more preferably 20-40 mM Tris-HCl with a pH set at 7.8-7.9 (25 °C); and most preferably 40 mM Tris-HCl with a pH set at 7.8-7.9 (25 °C).

[0060] Preferably, the method includes contacting the reaction reagents with a base. Preferably, the base is used for reactions with long durations and frequent feeds, wherein the buffering capacity of the initially provided buffer is excessive. The concentration of the base is preferably equal to or greater than the amount of any acid added to or generated during the reaction. However, in a preferred embodiment, a base is not required because the buffering capacity of the initially provided buffer is sufficient to maintain the pH value required for the reaction ±0.1 units.

[0061] Preferably, the method includes contacting the reagent with a reducing agent. The reducing agent may be selected from dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol, and tris(2-carboxyethyl)phosphine (TCEP). However, DTT is preferred. The concentration of the reducing agent may be 0.5-5 mM, more preferably 1-3 mM, and even more preferably 1 mM. In IVT reactions involving prolonged incubation and exposure to intermittent or continuous gas flows (e.g., for evaporating excess liquid, as described herein), higher reducing agent concentrations may be required to offset the increased oxidation (and deactivation) of the reducing agent and / or reaction components. In this case, it is preferable to maintain the amount of active, unoxidized reducing agent at 1 mM by adding additional reducing agent. Besides DTT, dithioerythritol (DTE), β-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP) may also be used as reducing agents at similar concentrations.

[0062] Preferably, the method includes contacting the reagent with spermidine. The concentration of spermidine can be 0.1-10 mM, more preferably 1-5 mM, and even more preferably 2 mM. In a preferred embodiment, the concentration of spermidine is related to the amount of DNA and / or RNA polymerase in the reaction.

[0063] NTP and cap analogs (and optionally buffer and Mg2+) are preferably included in the feed solution. Advantageously and preferably, RNA is synthesized and the degree of correct capping during co-transcriptional capping is high when the feed solution is provided in a more diluted form relative to a more concentrated form. Therefore, preferably, the method includes feeding the reagents in a diluted feed solution. Preferably, the diluted feed solution may contain a concentration of feed components such that, if fluid removal is not employed or will not be employed, the total number of feeds increases by >25% of the initial reaction volume, resulting in a >25% dilution of components present in the initial reaction volume, but absent or present at a lower concentration in the feed solution.

[0064] In a preferred embodiment, the diluted feed solution may contain a concentration of feed components such that, without or without fluid removal, the total number of feeds increases the initial reaction volume by >20%, more preferably >40%, and even more preferably >100%. Alternatively, the diluted feed solution is defined as a feed solution containing one or more components, said components being concentrated to a degree greater than 2 times, more preferably greater than 4 times, and even more preferably greater than 10 times below their respective solubility limits and / or technical manufacturability limits. For example, the maximum solubility of GTP has been reported to be about 100 mg / ml (169.74 mM), therefore a 100 mM stock solution is typically the highest concentration stock solution available. Feeding with a 100 mM stock typically results in a 5-10% dilution of the initial reaction volume (5-10 mM total for each NTP) without removing excess fluid. Therefore, a 2x dilution corresponds to 50 mM and 10-20% reaction dilution (5-10 mM total for each NTP), a 4x dilution corresponds to 25 mM and 20-40% reaction dilution (5-10 mM total for each NTP), and a 10x dilution corresponds to 10 mM and 50-100% reaction dilution (5-10 mM total for each NTP).

[0065] In a preferred embodiment, the diluted feed solution is defined as the dilution of the cap analog competing nucleotide compared to the most concentrated stock solution of the competing nucleotide available, and is at least 2x, more preferably 5x, even more preferably 10x, optionally more than 25x, and optionally more than 100x. Preferably, not all components in the feed solution are diluted or diluted equally, but are added in the amount required to maintain stable reaction conditions associated with that component.

[0066] In a preferred embodiment, the method includes feeding (or replenishing) a competing nucleotide (e.g., rGTP) that competes with a nucleotide-capped analogue (e.g., ARCA), the feed rate being adapted to maintain the ratio of the competing nucleotide (e.g., rGTP) to the capped analogue (e.g., ARCA) between predetermined minimum and maximum thresholds. The feeding (or replenishment) can be continuous or intermittent. The minimum threshold for the nucleotide-capped analogue:competing nucleotide (e.g., ARCA:rGTP, or another capped analogue, and / or another competing nucleotide) can be determined by the desired capping efficiency, while the maximum threshold for the nucleotide-capped analogue:competing nucleotide (e.g., ARCA:rGTP, or another capped analogue, and / or another competing nucleotide) can be determined by the desired minimum reaction rate and / or the (final) yield of the reaction.

[0067] In a preferred embodiment, the method comprises feeding only competing nucleotides (e.g., rGTP in the case of using ARCA as a cap analogue), rather than a mixture of nucleotides (which would be consumed). The feeding can be continuous or intermittent.

[0068] Preferably, the method comprises continuously feeding only competing nucleotides; less preferably, the number of feeds is the same as the ratio of non-competitive nucleotides to competing nucleotides (e.g., if the ratio is 9:1, then 9x feeds) and the volume is equal to the feed stock concentration. The amount of competing nucleotides before consumption.

[0069] Preferably, the method includes a feed of >=4x, with each feed stock containing >=10% of the reaction volume, every 1-4 hours. More preferably, the method includes a feed of >=10x, with each feed stock containing >=10% of the reaction volume, every 2-4 hours. Even more preferably, the method includes a feed of >=20x, with each feed stock containing >=5% of the reaction volume, every 3-4 hours.

[0070] In such an implementation, it is preferable to add the maximum desired concentration of each non-competitive nucleotide to the reaction at the start of the reaction to achieve the highest possible transcription rate, and the transcription rate is limited only by cap analog competing nucleotides provided at concentrations significantly lower than those of the non-competitive nucleotides and / or cap analogs.

[0071] In a preferred embodiment, the reaction composition at the start of the reaction comprises 5 mM, more preferably 6 mM, more preferably 7 mM, of each of rATP, rCTP, rUTP or their derivatives (e.g., pseudo-UTP, N1-methyl-UTP, m5CTP, m6ATP, m1ATP, inosine triphosphate, hm5CTP, m1GTP, m7GTP or m6AmTP) or mixtures thereof. In another embodiment, the reaction composition at the start of the reaction comprises 8 mM, more preferably 9 mM, even more preferably 10 mM, of each of rATP, rCTP, rUTP or their derivatives (e.g., pseudo-UTP, N1-methyl-UTP, m5CTP, m6ATP, m1ATP, inosine triphosphate, hm5CTP, m1GTP, m7GTP or m6AmTP) or mixtures thereof.

[0072] In such an implementation, the initial and / or average concentration of the competing nucleotide (e.g., rGTP, preferably when ARCA is a capping analog) is selected as a fraction of capping analog concentration sufficient to achieve the desired capping efficiency, preferably 1:9, more preferably 1:10, and even more preferably 1:20.

[0073] It should be understood that a 1:9 ratio corresponds to a 90% capping efficiency, which the inventors consider to be the lowest useful capping efficiency. Furthermore, a 90% capping efficiency is significantly higher than the 80% capping efficiency commonly used with a 4:1 ratio. A 1:10 ratio is even more preferable due to the improved quality; while a 1:20 ratio achieves >95% capping efficiency and a low initial concentration of 0.5 mM GTP. Concentrations of any nucleotide below 0.5 mM significantly reduce the reaction rate.

[0074] Preferably, the initial and / or average concentration of the competing nucleotide (e.g., rGTP when ARCA is used as a capping analog) is selected as a fraction of the capping analog concentration sufficient to achieve the desired capping efficiency, preferably 1:50, more preferably 1:75, and even more preferably 1:100.

[0075] Therefore, advantageously and preferably, the method and apparatus comprise a feed-batch in vitro transcription (IVT) method with volume balance (i.e., a substantially fixed steady-state volume) that allows for high-yield RNA transcription and high capping efficiency through co-transcriptional capping with a high ratio of cap analogs and competing nucleotides.

[0076] In a preferred embodiment, the feed solution contains competing nucleotides in RNA-free water, along with optional nucleotide counterions. Preferably, the water does not contain any buffer or salt components. In another embodiment, the feed solution contains competing nucleotides in water (e.g., RNA-free water) without the addition of buffers or salt components, except for any counterions of the nucleotides and matching amounts of Mg2+ and their appropriate counterions (e.g., chlorides, acetates).

[0077] In one embodiment, the initial concentration of the cap analog (e.g., ARCA, mCAP, or a trinucleotide cap analog, etc.) is high enough to allow the competing nucleotide (e.g., rGTP) concentration to be sufficient to maintain a suitable reaction rate, preferably >5 mM, more preferably >8 mM, and even more preferably >10 mM. In such an embodiment, a suitable transcription rate is defined as >1% of the desired yield per hour, more preferably >5% of the desired yield per hour, and even more preferably >10% of the desired yield per hour.

[0078] In a preferred embodiment, the method includes reducing the volume of the transcription reaction during or after feeding to compensate for the additional feed solution volume added thereto. Preferably, the volume reduction maintains the desired volume and / or concentration of the transcription reaction and increases or sustains the reaction rate. This volume reduction can be performed after feeding or substantially continuously. Therefore, preferably, the method includes feeding only competing nucleotides while reducing the volume, more preferably, at a high cap analog to competing nucleotide ratio.

[0079] Therefore, in a preferred embodiment, competing nucleotides and / or any other reaction components (e.g., other nucleotides, bases, buffers, salts (NaCl or TrisCl), Mg2+, etc.) are fed to allow for simultaneous or subsequent addition and removal of fluids. Preferably, the addition and removal of fluids are matched so as not to change the reaction volume and thus not to cause the concentration of non-feed reaction components (e.g., RNA polymerase and / or template DNA) to exceed predetermined parameters. In a preferred embodiment, the addition and removal of fluids are matched to maintain substantially the same reaction volume. A steady state is preferred. Furthermore, fluid removal is carried out in such a manner that only water is removed without removing any significant amount of dissolved reaction components or products.

[0080] In a preferred embodiment, excess fluid (preferably water) is removed from the reaction by evaporation, thereby maintaining a substantially constant reaction volume.

[0081] The evaporation of water from the water surface depends primarily on water temperature, air temperature, air humidity, and air velocity above the water surface. The amount of water evaporating per unit time can be expressed as:

[0082] ,

[0083] in:

[0084] Table 4 - Evaporation parameters = Water evaporation rate per hour (kg / h)

[0085] = ( 25 + 19 v) = Evaporation coefficient (kg / m2h)

[0086] v = Air velocity above the water surface (m / s)

[0087] A = Water surface area (m2),

[0088] Figure 7 = The maximum humidity ratio of saturated air at the same surface temperature as water (kg / kg) (kg H2O / kg dry air); and

[0089] x = Air humidity ratio (kg / kg) (kg H2O / kg dry air).

[0090] For the purposes of this invention, the practical limit for the removal of excess evaporative water is considered to be: ~0.25 µl / min / cm² (0.5 m / s air velocity, 20 o C air temperature, 37 o(C water temperature, 50% humidity) to ~15µl / min / cm2 (10m / s air velocity, 20 o C air temperature, 37 o (C water temperature, 30% humidity).

[0091] Figure 7

[0092]

[0093] like Figure 7 As shown, the feed rate of the feed solution depends strongly on the concentration of the stock solution of fresh NTP (preferably GTP when using mCap and / or ARCA as cap analogs) used for the IVT reaction and the total reaction volume, according to a linear relationship. Figure 7 Figure A shows the feasible feed rate range for microfluidics within the gray shaded area; assuming an 8-hour continuous feed protocol, all volume-feed stock concentration combinations overlapping this gray area are theoretically achievable. The figure illustrates that for small volumes typically used for screening purposes, impractical feed rates are required if concentrated volumes are used. Conversely, as mentioned above, controllable feed rates can be achieved using diluted feed stocks.

[0094] Similarly, based on the minimum and maximum continuous evaporation rates for each volume in Table 4, Figure 7 B shows that using cubic (all dimensions approximately equal) or surface-optimized reaction vessels (minimum height fluid layer, maximum surface area) can achieve matching or exceed [the required parameters]. Figure 7 The evaporation rate of feed rate A is assumed to be a maximum evaporation rate of 15 µl / min / cm². The gray-marked area represents all combinations of feed rate / feed stock concentration, which can be compensated for by evaporation using a cubic design device. The dark gray area ( Figure 7 B) shows a combination of feed rate / feed stock concentration that requires an evaporation rate >15 µl / min / cm² or an increased surface area of ​​the reaction volume in contact with the gas stream that causes evaporation to achieve enhanced evaporation. Figure 8A C combines Figure 8B The results from A and 7B, and further considering hybrid dynamics, indicate that for volumes <1 ml, feed stock concentrations between 1 mM and 10 mM provide ideal conditions for rapid and complete mixing, adequate feed rate control (via microfluidic devices), and matched evaporation. For volumes >1 ml, the ideal feed stock concentration is approximately 10 mM for rapid and complete mixing, as well as precise control of the feed rate and matched evaporation.

[0095] Therefore, in a preferred embodiment, excess water is evaporated by applying a controlled gas flow over the reaction mixture. This gas can be, for example, nitrogen or air. However, air is preferred. Preferably, the magnitude of the gas flow is precisely controlled. Preferably, the gas flow is in direct contact with the surface of the reaction volume and preferably does not contact the feed solution flow. To avoid contamination, the gas flow is preferably sterilized, more preferably filtered. For example, the gas flow (preferably an air flow) can be sterilized by passing it through a HEPA filter.

[0096] To improve the effectiveness of evaporation, the gas flow velocity can be at least 0.5 m / s, at least 2 m / s, at least 3.5 m / s, at least 5 m / s, at least 10 m / s, or >10 m / s. However, it is understood that care should be taken to avoid foaming and splashing of the reaction mixture.

[0097] In another embodiment, as an alternative or supplementary method to improve evaporation efficiency, the humidity of the gas can be reduced before the gas flow (preferably an air flow) comes into contact with the surface of the reaction mixture. In a preferred embodiment, the humidity is below 50%, more preferably below 40%, and even more preferably below 30%. The humidity can be adjusted in any way known to those skilled in the art.

[0098] In some embodiments, to improve the effectiveness of evaporation, the temperature of the gas flow (preferably an air flow) can be between 4°C and 50°C. In a preferred embodiment, the gas temperature is maintained at the same temperature as the reaction mixture to avoid cooling the fluid. In another embodiment, the gas temperature is maintained at room temperature (e.g., 19-23°C).

[0099] In another embodiment of the invention, excess fluid (preferably water) is removed by osmosis by contacting a sufficiently large surface area of ​​the reaction volume with a semipermeable or water-selective membrane (e.g., a PDMS membrane, a dialysis membrane, or a molecular sieve). Preferably, the membrane has a pore size smaller than that of the protein, DNA, and RNA components in the reaction, but larger than that of NTPs, buffer components, salts, reducing agents, and / or spermidine (thus allowing these components to pass through).

[0100] In such an implementation, the driving force for water removal is a significantly higher osmotic pressure on the membrane receiver side than on the IVT reaction components. This increase in osmotic pressure can be achieved through high concentrations of salts (e.g., NaCl), polymers, and other molecules that interact with water. Preferably, membrane selectivity is used to prevent the transfer of permeate across the membrane, thereby avoiding alteration of the reaction components.

[0101] Preferably, the method includes feeding pressurized air (optionally filtered, for example with a HEPA filter) into the reaction chamber to provide sufficient pressure to drive a predictable and / or sufficiently large volume of feed solution through a semipermeable membrane.

[0102] In a preferred embodiment of the invention, the method for removing excess water is (continuously) adjusted and / or fine-tuned by a PID (proportional-integral-derivative) controller loop that responds to measurements of fluid volume and / or osmotic pressure of the IVT reaction volume.

[0103] In a preferred embodiment of the invention, the method includes measuring the level of the reactive fluid, preferably by optical measurement of the fluid height. This optical measurement can be performed in a substantially vertical direction, thereby detecting reflections from the fluid surface. This measurement can be achieved, for example, by measuring the angle of rotation using infrared methods. Alternatively, the optical measurement can also be performed in a horizontal direction, detecting changes in the fluid surface's obstruction of the light path or fluid absorption. Various wavelengths are suitable for measuring the fluid level.

[0104] In another embodiment, the reaction fluid level can be measured using an electronically conductive probe. This probe can be inserted into the container holding the IVT reaction. The conductive probe functions by emitting a signal when the reaction fluid is in direct contact with the probe located directly above the desired fluid level, or by emitting a proportional signal when the probe is partially or fully immersed in the reaction fluid.

[0105] In another embodiment, the reaction fluid level can be measured by continuously weighing the entire system and subtracting the empty weight of the reaction vessel and the weight of the feed solution before it has been introduced into the reaction volume. The remaining weight directly corresponds to the volume of the IVT reaction. The excess weight compared to the theoretical or initial weight of the solution is determined as excess fluid and can be used for calculations.

[0106] In another implementation, the reflection of sound waves on the liquid surface can be detected using a probe positioned significantly above the fluid surface, thereby measuring the fluid level via acoustic distance sensing. A variety of wavelengths are suitable for measuring fluid levels.

[0107] In another embodiment, the method may include calculating excess liquid from solution osmotic pressure measurements and comparing it to the expected osmotic pressure of the solution at that IVT reaction stage. The method relies on the existence of a measurable difference between the osmotic pressure of the feed solution and the osmotic pressure of the IVT reaction at certain stages of the IVT reaction. Methods for measuring osmotic pressure are well known to those skilled in the art.

[0108] In one embodiment of the invention, the method includes determining the feed rate of the feed solution based on empirical evidence or theoretical calculations of the consumption rate of a known amount of RNA polymerase with known activity against competing nucleotides (and cap analogs) in the same or similar reaction compositions, taking into account the RNA sequence (preferably mRNA) composition. In a preferred embodiment, the feed rate is initiated at the start of the reaction and substantially matched to the consumption rate to maintain the ratio of cap analogs to competing nucleotides. Alternatively, feeding may begin after a certain period of time.

[0109] In another embodiment, the method includes adjusting (preferably continuously adjusting) the feed rate of the feed solution based on real-time or intermittent measurements of the reaction substrate (e.g., one or more nucleotides, preferably competing nucleotides) and / or the reaction product (preferably mRNA).

[0110] In a preferred embodiment of the invention, the method includes feeding (or replenishing) a nucleotide (e.g., rGTP) that competes with a nucleotide cap analog (e.g., ARCA) into the reaction by feeding a competing nucleotide (e.g., rGTP) into the IVT reaction mixture (preferably from an external reservoir). The feeding (or replenishment) can be continuous or intermittent. In this case, the method preferably includes continuously or intermittently mixing the reaction reservoir to address local differences in the concentration of the competing nucleotide (e.g., rGTP concentration), thereby addressing local differences in the ratio of the cap analog to the competing nucleotide (e.g., the ARCA:rGTP ratio). Preferably, this mixing is performed in a manner that does not impair protein / enzyme function due to shear stress or otherwise disrupt the IVT reaction.

[0111] In a preferred embodiment of the invention, the method includes feeding the feed solution to the reaction in a distributed manner, thereby preventing the formation of temporary high local concentrations in the feed solution, and optionally undesirable cap analogue to competing nucleotide ratios. This distributed feeding preferably includes the simultaneous or sequential release of the feed solution at spatially separated locations within the reaction volume. In a preferred embodiment, advantageously, distributed feeding minimizes the flow / droplet / solid particles relative to the surrounding transcriptional reaction fluid, thereby enabling faster diffusion, mixing, and / or dissolution, and minimizing local and temporal deviations in desired reaction conditions, particularly the cap analogue to competing nucleotide ratio.

[0112] Preferably, the method includes intermittently or continuously feeding a competing nucleotide and / or any other reaction component that competes with the nucleotide-cap analog into the reaction through a semipermeable membrane, wherein the semipermeable membrane comprises a plurality of axially, radially, and / or longitudinally spaced microscopic openings and / or pores through which the feed solution can pass.

[0113] In a preferred embodiment of the invention, the method includes feeding a competing nucleotide (e.g., rGTP) and / or any other reaction component that competes with a nucleotide-cap analog (e.g., ARCA) into the reaction via a microfluidic channel. Preferably, the microfluidic channel comprises a plurality of axially, radially, and / or longitudinally spaced microfluidic openings through which the feed solution (preferably the competing nucleotide) can pass. The reaction vessel is preferably intermittently or continuously mixed to distribute these freshly added nucleotides throughout the IVT reaction mixture. Feeding can be continuous or intermittent. In a preferred embodiment, the reaction vessel is part of or leak-free anchored to the microfluidic channel of the feed nucleotide.

[0114] In another embodiment of the invention, the method includes feeding a competing nucleotide (e.g., rGTP) that competes with a nucleotide cap analog (e.g., ARCA) and / or any other reaction component into the reaction via one or more needles, pipette tips, robotic probes, serum pipettes, Pasteur pipettes, or other suitable probes. Feeding can be continuous or intermittent.

[0115] Preferably, the one or more needles, pipette tips, robotic probes, serum pipettes, Pasteur pipettes, or other suitable probes comprise a plurality of axially, radially, and / or longitudinally spaced microfluidic openings through which a feed solution (preferably a competing nucleotide) can pass. The one or more needles, pipette tips, robotic probes, serum pipettes, Pasteur pipettes, or other suitable probes can be operated by direct displacement (e.g., solid material plunger), immiscible fluid pumping, pneumatic pumping (e.g., via a (robotic) pipette or (continuous pressure source)), magnetism, or gravity. The probe can contain a portion or all volume of a competing nucleotide (e.g., rGTP) or a mixture of a competing nucleotide (e.g., rGTP) and a cap analogue (e.g., ARCA). Alternatively or additionally, the probe can be directly connected via cannula, tubing, microfluidic channel, or any other suitable means to an external reservoir containing the nucleotide (e.g., rGTP or a mixture of rGTP and ARCA) to be fed into the reaction.

[0116] The probes, or each probe and / or the associated reservoir, can be cooled (for preservation) and / or (pre)heated (for adding a preheated solution). Preferably, the probes, or each probe, are preheated, and the reservoirs are cooled, to maintain the highest stability of the nucleotide / nucleotide mixture during storage until just before its addition to the reaction mixture. Advantageously, preheating the solution containing competing nucleotides and / or ARCA prevents temperature fluctuations in the reaction mixture and provides more stable synthetic conditions.

[0117] In one embodiment of the invention, the method includes feeding a competing nucleotide (e.g., rGTP) that competes with a nucleotide-cap analog (e.g., ARCA) into the reaction via one or more needles, pipette tips, robotic probes, serum pipettes, Pasteur pipettes, or other suitable probes, wherein such probes contain multiple independent doses of the nucleotide.

[0118] In a preferred embodiment, the individual doses are separated by an air gap or other suitable immiscible medium. In such embodiments, the probe can remain in contact with the reaction vessel and there is no need to refill between doses, thereby maintaining a closed system and limiting the possibility of introducing contamination. Feeding can be continuous or intermittent.

[0119] In one embodiment of the invention, the method includes feeding competing nucleotides and / or any other reactive components onto the surface of the reaction volume via spraying, atomizing, or any other method that produces significantly small droplets. Preferably, the droplets are distributed substantially uniformly over the fluid containing the reaction volume. The sprayed or atomized fluid may be applied to a portion or the entire surface of the reaction volume. In a preferred embodiment, the sprayed or atomized fluid is applied to a portion of the reaction volume surface that is separated (i.e., spaced apart) from the reaction volume surface exposed to the gas flow for evaporating excess fluid.

[0120] In one embodiment of the invention, the method includes dissolving a competing nucleotide (e.g., rGTP) that competes with a nucleotide-cap analog (e.g., ARCA) in a suitable liquid medium (e.g., buffer, (RNA-free) (deionized) water, or other aqueous solution) or as a solid (e.g., dry powder), optionally bound to a carrier, to feed into the reaction. When added as a solid (preferably a fine dry powder), it dissolves directly in the IVT reaction mixture. The addition rate allows the solid to dissolve immediately and is limited to avoid the formation of localized high concentrations of nucleotides. The feed can be continuous or intermittent.

[0121] In another embodiment of the invention, the method includes feeding a competing nucleotide (e.g., rGTP) and / or any other reaction component that competes with the nucleotide-cap analog (e.g., ARCA) into the reaction by dissolution, rupture, swelling, degradation, exchange, or other release from the carrier, wherein the carrier is added to the reaction at the start of the reaction / incubation and / or during the reaction and / or is fed intermittently and / or continuously. Thus, feeding can be continuous or intermittent. Some carriers may exhibit one or more types of release mechanisms (e.g., swelling and degradation). Such carriers may be in the form of microbubbles, liposomes, solids, salts, polymers, matrices, hydrogels, or other suitable carriers to allow for the slow and / or controlled release of the nucleotide of interest into solution.

[0122] The process or reaction that leads to the release of the nucleotide of interest can be autocatalytic, temperature-induced, induced by a specific pH or pH change, catalyzed by an external factor (e.g., light of a specific wavelength), or, in a preferred embodiment, catalyzed by any IVT product or metabolite.

[0123] In one embodiment of the invention, the method includes feeding a competing nucleotide in combination with other (rate-limiting or production-limiting) nucleotides into a reaction.

[0124] In one embodiment of the invention, the method includes feeding a competitive nucleotide in combination with a matching amount of a magnesium salt (e.g., chloride or acetate) into the reaction to maintain a stable amount of free magnesium ions in the solution.

[0125] In another embodiment of the invention, the method includes feeding a competing nucleotide into a reaction by adding a liquid amount matching the amount of evaporation since the last feed / IVT reaction began.

[0126] In another embodiment of the invention, the method includes feeding a competing nucleotide into the reaction by adding it to a reaction buffer, the concentration of which maintains the concentration of the buffer and the total reactant ions at a stable / ideal level.

[0127] In one embodiment of the invention, the competing nucleotide (e.g., rGTP) and the cap analog itself can both be present at relatively low concentrations, resulting in a depletion or reduction in their concentrations. In such cases, the method comprises feeding the competing nucleotide and cap analog into the reaction at appropriate relative proportions and rates. In a preferred embodiment, the relative proportions of the competing nucleotide and cap analog in the solution fed into the reaction are substantially analogous to the relative consumption of each component during transcription of a given RNA sequence (e.g., 1 ARCA to 250 rGTP for a 250 G mRNA sequence). The rate of nucleotide (as a mixture) supply depends on the reaction rate of the IVT reaction and the concentration of the nucleotide (mixture) in the feed solution.

[0128] In another embodiment, in addition to the competing nucleotide, the feed solution preferably also contains one or more components of the IVT reaction, such as buffer and / or one or more other NTPs. By feeding these reaction components other than the rate-limiting nucleotide, the reaction conditions can be maintained within predetermined parameters even if other components in the IVT mixture are diluted by the feed solution.

[0129] In a preferred embodiment of the invention, the method includes dissolving a cap analog competing nucleotide (e.g., GTP) in (un)buffered RNase-free water. Preferably, the method includes directly feeding the dissolved competing nucleotide into the IVT reaction at a concentration and rate suitable for maintaining a desired ratio of the rate-limiting nucleotide to the cap analog. The concentration of the rate-limiting nucleotide (e.g., rGTP) in the feed solution is preferably such that the volume of feed solution added to the IVT reaction is matched to the evaporation rate of the IVT reaction in a given reaction vessel, thereby preventing significant dilution or concentration of the IVT mixture. The evaporation rate of the IVT mixture can be controlled or modified by changing the shape, volume, or material of the reaction vessel, as well as by changing the volume of air in contact with the solution surface, the ambient temperature (including air temperature), the reaction temperature, the relative humidity of the air or gas in contact with the fluid surface, the presence of hygroscopic solids, and other methods known to those skilled in the art.

[0130] In one embodiment of the invention, the method includes generating multiple RNA sequences in a single IVT reaction. The multiple RNA sequences may include modified and unmodified RNA, preferably mRNA. Preferably, for each (modified) (m)RNA sequence, a separate cap analogue is used. For example, an ARCA with the first binding nucleotide G on the first RNA sequence, and a cap analogue complementary to T on the second RNA sequence, and so on. In this case, the method of the invention can be applied to simultaneously or independently feeding two rate-limiting nucleotides.

[0131] The apparatus of the third aspect is preferably used to perform the method of the first aspect.

[0132] Therefore, in one embodiment, the reaction chamber includes a first section into which a feed solution is fed, and a second section into which a transcription reaction takes place, wherein the first and second sections are separated by a semipermeable membrane. Preferably, the semipermeable membrane is a dialysis membrane. Preferably, the pore size of the semipermeable membrane is smaller than that of the protein, DNA, and RNA components in the reaction, but larger than that of NTPs, buffer components, salts, reducing agents, and / or spermidine, thus allowing these components to pass through.

[0133] In one implementation scheme (such as) Figure 8C As shown), the first part is the external volume of the reaction chamber, and the second part is the internal volume of the reaction chamber, which is at least partially surrounded by a semi-permeable membrane. However, in another embodiment (such as...), Figure 8D As shown), the first part is the internal volume of the reaction chamber, and the second part is the external volume of the reaction chamber, which is at least partially surrounded by a semipermeable membrane.

[0134] Preferably, the feeding means is configured to feed the feed solution into a first part of the reaction chamber, thereby contacting one side of the semipermeable membrane, wherein the positive pressure generated by the feed fluid drives the feed solution and its contained components through the semipermeable membrane. It is understood that the semipermeable membrane provides a distributed feeding solution mixing method.

[0135] In one embodiment, the apparatus may include a gas inlet configured to feed pressurized gas into a first portion of the reaction chamber, thereby providing additional pressure to the feed solution. The gas may be air or nitrogen; air is preferred. The gas inlet may include a filter, such as a HEPA filter, to prevent contamination. Advantageously, the continuous pressure supplied to the feed solution by the pressurized gas forces it to pass through the membrane at a rate greater than the diffusion rate, preventing transcriptional reaction components from transferring from the second portion to the first portion, thus creating unidirectional flow.

[0136] The apparatus preferably includes a gas (preferably air) outlet configured to release excess pressure within the reaction chamber. Advantageously, this allows the feed solution to flow continuously from the first section to the second section.

[0137] To maintain a substantially constant volume of the transcription reaction under steady-state conditions, thereby keeping the proportions of various reagents (especially cap analogs: the proportion of competing nucleotides) constant, excess fluid is removed from the reaction by evaporation. Therefore, preferably, the reaction chamber includes an evaporation zone located above the region where the transcription reaction occurs, and fluid (preferably water) evaporates in this zone to compensate for the additional volume provided by the feed, thus maintaining a substantially constant reaction volume. Preferably, the apparatus includes a gas inlet configured to feed gas into the evaporation zone to facilitate fluid evaporation. The gas can be air or nitrogen. Air is preferred. The gas inlet may include a filter, such as a HEPA filter, to prevent contamination. Preferably, the gas inlet and gas outlet are in fluid communication.

[0138] In another implementation scheme (such as) Figure 8E As shown), the feeding means preferably includes a probe having a distal outlet through which the feed solution can pass. Preferably, the outlet is configured to extend into the reaction fluid within the reaction chamber. The probe can be a needle, pipette tip, pasteurized pipette, or serum pipette, or any other probe suitable for adding the feed solution. The apparatus may include a magnetic or physical stirrer, which may optionally be connected to the probe.

[0139] In yet another implementation scheme (such as) Figure 1A As shown), the probe includes multiple axially, radially, and / or longitudinally spaced outlets through which the feed solution preferably enters the fluid.

[0140] In another implementation (such as) Figure 1BAs shown, the probe includes a spray device or atomizer configured to generate droplets of the feed solution in the reaction chamber. Preferably, the spray device or atomizer is mounted above the surface of the reaction fluid. Preferably, most (preferably all) of the spray droplets are combined with the reaction fluid.

[0141] Preferably, the apparatus includes a mixer, such as a stirrer, configured to mix the feed solution in the second portion. Alternatively, the apparatus may include an oscillating platform on which the reaction chamber is placed for stirring the reaction volume.

[0142] All features described herein (including any appended claims, abstract, and drawings) and / or all steps of any disclosed method or process may be combined with any of the foregoing aspects in any combination, unless at least some of these features and / or steps are mutually exclusive in the combination.

[0143] To better understand the present invention and to demonstrate how to implement embodiments of the invention, reference will now be made to the accompanying drawings by way of example, wherein:

[0144] Figure 2 The yield and nanoluciferase reporter mRNA activity in IVT reactions using different nucleotide compositions (10 mM each NTP, 10 mM CTP / ATP / UTP and 2.5 mM GTP, or 2.5 mM each NTP) and DNA templates containing differentiation transcription initiation sites (TSS) (GGG…, GGA…, GAA…) are shown. The effect of nucleotide composition and TSS on IVT reaction yield over time was measured by extracting, purifying, and measuring the absorbance of purified samples collected at the indicated time points. Each aliquot was 20 µl. Figure 3A The luciferase activity of the reporter mRNA produced by the co-transcriptional IVT reaction of (A) is shown.

[0145] Figure 3B The diagram illustrates the mRNA yield of a feed-batch implementation of the method of the present invention, wherein all non-competitive nucleotides are supplied at 10 mM at the start of the reaction, and the competing nucleotide (GTP) is supplied at 2.5 mM. Then, every 2 hours, GTP equivalent to 2.5 mM (final concentration) is re-fed to increase the reaction yield while maintaining a high ratio of cap analogue (ARCA) to competing nucleotide (GTP). 20 µl samples were collected after 15, 30, 45, 60, 90, 120, 180, 240, 300, 360, and 480 minutes, quenched with EDTA, purified by silica column chromatography, and the concentration was measured by UV absorption.

[0146] Figure 3 shows the effect of increasing the ratio of cap analogue (ARCA) to competing nucleotide (GTP) on yield and mRNA activity when using a DNA template with GGG TSS and 10 mM ATP, CTP, UTP, and ARCA, as well as different amounts of GTP, at the start of the reaction. At 37... o After incubation at C for 2 hours, the reaction was terminated by quenching with EDTA in 20 µl, purified by silica gel column chromatography, and the yield was then measured using UV absorbance at 230 / 260 / 280 nm. Figure 4A The effect of the ARCA-GTP ratio on the reaction yield after 2 hours of incubation was shown. Figure 4B The effect of the ARCA-GTP ratio on reporter mRNA activity after transfection of HeLa cells was shown.

[0147] Figure 4 shows the effect of feed solution concentration containing 1-100 mM GTP on mRNA yield and activity from feed batch IVT reactions. The reaction was run for 30 hours with a 3-hour feed interval and an initial ARCA to GTP ratio of 20:1. Figure 5A The reaction yield is shown compared to the theoretical reaction yield. Figure 5B The mRNA activity of different samples prepared with different feed solution concentrations is shown.

[0148] Figure 5 shows the yield and mRNA activity of the reaction fed nine times with a diluted feed solution containing 5 mM GTP in a closed container, a container open to the environment, and a container connected to a dry N2 gas flow (with the evaporation volume set to be similar to the volume added during the 3-hour feed process). Figure 6 The output is displayed. Figure 6 The activity of luciferase mRNA was shown 24 hours after transfection into HeLa cells.

[0149] Figure 6 Three embodiments for feeding a nucleotide-containing feed solution are shown, namely... Figure 6 A: In batch reactions, a single feed (competitive nucleotide) causes a large fluctuation in the ratio of capping analog to competing nucleotide, which in turn causes a large fluctuation in capping efficiency; Figure 6 B: Similar to A, batch reactions with multiple feeds of competing nucleotides result in smaller fluctuations in the ratio of capped analogues to competing nucleotides, thus leading to smaller fluctuations in capping efficiency. Interestingly, theoretical models show that the more feeds, the faster the average reaction rate, allowing for more than two feeds per 2x larger feed in A while maintaining the same capping efficiency; Figure 7 C: By continuously feeding competitive nucleotides at the consumption rate of competitive nucleotides, a stable ratio of cap analogues to competitive nucleotides is produced, resulting in a stable capping efficiency, because the system has higher productivity. Figure 7D shows the reaction productivity using feed methods A, B, or C, indicating that the average productivity of the IVT reaction per unit time increases with increasing feed frequency, provided the competing nucleotides are fed at a consumption rate. Although the average is faster, higher feed frequencies are initially slower due to the higher initial transcription rate, which is associated with increased levels of the rate-limiting competing nucleotides. These results only apply to cases where the competing nucleotides are present on average and / or consistently at the rate-limiting concentration. Once the concentration of the competing nucleotides exceeds the rate-limiting concentration, the productivity differences for each method (A, B, or C) become negligible.

[0150] Figure 7 The theoretical modeling results for feed rate, evaporation rate, and hybrid dynamics are shown for various total reaction volumes and feed stock concentrations. Figure 7 A: Shows the feed rate required to maintain the desired capping efficiency for each volume during an 8-hour continuous feeding process. The gray area shows the range of feed rates that can be practically achieved using microfluidics according to Table 3. Figure 4B B: Shows the feed rate required to maintain the desired capping efficiency for each volume similar to A during an 8-hour continuous feed process. The gray area shows the range of feed rates that can be compensated for by evaporation in a cubic device, assuming a maximum evaporation rate of 15 µl / min / cm². The dark gray area shows the feed rate that requires optimization of the reaction surface area to compensate for the evaporation flow rate, assuming a maximum evaporation rate of 15 µl / min / cm². Example C: Shows the feed rate required to maintain the desired capping efficiency for each volume similar to A during an 8-hour continuous feeding process. Information from 7A and 7B is consistent with... Example 1 - Batch IVT reactions to determine the effect of nucleotide composition on productivity and mRNA quality The inferred hybrid dynamics estimates are combined to show the optimal combination of feed rate, feed stock, and total volume, in which high-quality mRNA can be produced in high yield and constant volume via co-transcriptional capping IVT.

[0151] Figure 8 illustrates six different implementation schemes for reactor designs used to carry out the method of the present invention.

[0152] Figure 1A

[0153] The inventors have devised a novel in vitro transcription (IVT) method for mRNA that achieves lower cost and higher yield of 5'-capped mRNA compared to existing technologies. Considering the significant economic implications of in vitro transcription of mRNA in the medical and biotechnology fields, those skilled in the art will understand the substantial economic potential of this invention. Furthermore, this invention relates to an apparatus for improving in vitro transcribed mRNA synthesis via the aforementioned method, and more specifically to adding a 5' cap structure to in vitro transcribed mRNA. The resulting mRNA product has a higher degree of 5' cap structure, while reducing the consumption of expensive reagents and simplifying the purification process. This lowers the cost of in vitro mRNA transcription and reduces the activation of innate immune responses by 5'-uncapped mRNA, thereby improving protein expression and reducing adverse side effects of in vitro transcribed mRNA for in vitro or in vivo applications.

[0154] Figure 1B

[0155] Add 200 ng each of the PCR-generated DNA template (containing a 5' T7 promoter and a 3' encoding a 150 nt poly-A tail) to a 200 µl IVT reaction mixture. The DNA template is identical except for the first three nucleotides after the T7 promoter (the so-called transcription start site (TSS)).

[0156] Template A: T7_starter-GGG-remaining 5'UTR-CDS-3'UTR-poly-A-tail,

[0157] Template B: T7_starter-GGA-remaining 5'UTR-CDS-3'UTR-poly-A-tail,

[0158] Template C: T7_starter-GAA-remaining 5'UTR-CDS-3'UTR-poly-A-tail.

[0159] Three variants of the batch IVT reaction were prepared as follows: 2 µl M11 T7RNAP (RiboPro), 2 µl Hiscribe reaction buffer (NEB), 1 µl RNase inhibitor (NEB), 1 µl IPP (NEB), and varying amounts of ATP, CTP, UTP, and GTP (from a 100 mM stock solution, NEB) were mixed to obtain reaction composition 1 (containing 10 mM of each NTP), reaction composition 2 (containing 10 mM of UTP, CTP, and ATP, and 2.5 mM of GTP), and reaction composition 3 (containing 2.5 mM of each NTP). All reactions contained 10 mM ARCA (Jena Bioscience) from a 50 mM stock solution.

[0160] After mixing, the IVT reaction was incubated directly in a sealed PCR tube at 37°C for 3 hours to minimize or eliminate evaporation. At the indicated time points, 20 µl of sample was immediately mixed with 80 µl of 25 mM EDTA (Sigma-Aldrich) to quench the reaction. Subsequently, the sample was purified using a standard silica column (Monarch kit, NEB) according to the manufacturer's protocol, followed by double elution with RNase-free water (Invitrogen). The absorbance at 230 / 260 / 280 nm was then measured using an iD3 reader (Molecular Devices) in a clear-bottomed, black-walled 384-well plate (Greiner).

[0161] Following the manufacturer's instructions, the purified mRNA was then mixed with PBS and pre-diluted Lipofectamine MessengerMax (Invitrogen). 100 ng of the purified mRNA was transfected into 80% confluence HeLa cells in DMEM / F12 medium + 10% FCS. After 24 hours of incubation, the medium was collected and detected on black 384-well plates (Greiner) using a secretory nanoluciferase assay (Promega) on an iD3 plate reader (Molecular Devices) at a moderate sensitivity setting, collecting all wavelengths. Cell samples treated only with Lipofectamine and buffer were used for background subtraction.

[0162] like Example 2 - Fed-batch IVT reactions to improve reaction yields As shown, the results clearly demonstrate that higher total NTP levels lead to higher yields, and the amount of the most limiting NTP is almost perfectly correlated with mRNA yield. IVT reactions containing 10 mM of each NTP theoretically achieve yields >220 µg; however, in this assay, the maximum yield was approximately 180–195 µg, likely due to limited incubation time and potential losses during purification. IVT reactions containing all NTPs or only 2.5 mM GTP showed a maximum yield of approximately 50 µg. Interestingly, despite similar final yields, reactions containing 10 mM UTP, CTP, and ATP, and 2.5 mM GTP showed higher yields at shorter time points (especially <90 minutes) and reached saturation earlier than IVT reactions containing 2.5 mM of each NTP. There was no difference in final yield between different DNA templates containing different TSSs.

[0163] Surprisingly, as Figure 2As shown, the mRNA activity produced in all reactions showed an increasing trend, indicating that higher quality mRNA was produced near the end of the co-transcriptional IVT reaction. This effect was more pronounced in samples containing 2.5 mM of competing nucleotides (rGTP, competing with ARCA, introduced as the first nucleotide in the reaction), suggesting higher co-transcriptional capping using ARCA near the end of the reaction. Regardless, lower concentrations of rGTP significantly affected mRNA activity. Interestingly, this effect correlated with the TSS used; GGG and GGA initiation sites showed similar mRNA activity under all corresponding reaction conditions; however, for GAA templates, especially reactions containing 10 mM UTP, ATP, and CTP, and 2.5 mM GTP, the activity was lower than reactions containing 2.5 mM of each NTP. One possible explanation is that the T7 RNAP polymerase may skip the first nucleotide of the DNA template and introduce the second templated nucleotide. For templates containing GGG and GGA, GTP or ARCA is still introduced depending on the ratio of GTP to ARCA at that time. However, in the GAA template, a higher concentration of ATP can replace GTP or ARCA, although it is less favorable for the initiation of transcription of the enzyme.

[0164] These results indicate that reactions containing a smaller amount of cap analog competing nucleotides can produce higher capping efficiency and higher quality mRNA.

[0165] Example 3 - High cap analog to competing nucleotide ratios improve mRNA activity but reduce yield.

[0166] The IVT reaction was prepared according to Example 1, except that only DNA template A (GGG TSS) was used and the volume was increased to 250 µl by scaling up the components. For the reaction containing 10 mM CTP, UTP, and ATP, and 2.5 mM GTP, a control containing 10 mM each of the NTPs and 10 mM ARCA was used. Immediately after preparation, the IVT reaction was incubated at 37 °C, and 20 µl samples were removed at the indicated time points and processed in a manner similar to Example 1. After 2 hours of incubation, samples C, D, and E received an additional dose (feed fractions) of 2.5 mM (final concentration) GTP, gently mixed into the reaction via pipette, and were then incubated again at 37 °C. After another 2 hours of incubation, samples D and E received an additional dose (feed fractions) of 2.5 mM (final concentration) GTP and were further incubated at 37 °C. After another 2 hours of incubation, sample E was given the final dose of 2.5 mM (final concentration) GTP (feed in batches) and incubated for another 2 hours. The reaction time was the same for all samples, for a total of 8 hours.

[0167] Figure 3AThe results clearly demonstrate the advantages of the batch feed method, which improves the reaction cycle and overall yield. Each addition of GTP increased the IVT reaction yield. Using up to three GTP feeds (at a final concentration equivalent to 2.5 mM), the total amount of GTP added was the same as the initial amount added to the control reaction, and the overall yield was also close to that of the control reaction. Interestingly, for each subsequent GTP dose, the increase in (m)RNA production was slightly lower than that at the previous dose, and the transcription rate was also slightly lower, resulting in a final yield of 10% less for sample E than for sample A.

[0168] Example 4 - Fed-batch reactions using concentrated and dilute stocks

[0169] Batch IVT reactions were prepared according to Example 1, except that only DNA template A (GGG TSS) was used and the volume was reduced to 20 µl by scaling down the components. All reactions contained 10 mM CTP, UTP, ATP, and ARCA, as well as varying amounts of GTP. After preparation, the IVT reactions were immediately incubated at 37 °C for 2 hours, and then processed in a similar manner to Example 1.

[0170] like Figure 4A As shown, the results indicate that the yield of the IVT response decreased in a dose-dependent manner, which deviates from the theoretical prediction. A higher ARCA:GTP ratio indicates slower transcription (when the GTP concentration is reduced). This can be compensated for by extending the incubation time. Interestingly, mRNA activity also increased in a dose-dependent manner; a higher ARCA:GTP ratio resulted in higher mRNA activity. Surprisingly, the increase in activity exceeded the theoretical increase in capping efficiency.

[0171] Figure 4B

[0172] Based on the high activity of mRNA produced in the IVT reaction due to the increased ARCA:GTP ratio, a ratio of ARCA to GTP of 20:1 was chosen for subsequent experiments.

[0173] The IVT reactions were prepared according to Example 1, except that only DNA template A (GGG TSS) was used, and the volume was reduced to 100 µl by scaling down the components. All reactions contained 10 mM CTP, UTP, and ATP, and 0.5 mM GTP. Immediately after preparation, the IVT reactions were incubated at 37 °C. Sample A received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 100 mM stock solution (0.5 µl), Sample B received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 10 mM stock solution (5 µl), and Sample C received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 1 mM stock solution (50 µl). After each addition, the added volume was gently mixed into the reaction using a pipette, and then the reaction was incubated again at 37 °C. To avoid altering the salt concentration of the IVT reaction, a diluted stock solution was prepared using 1x IVT reaction buffer. After nine doses, the reaction was terminated by adding excess EDTA and then processed in a manner similar to that in Example 1.

[0174] Manually adding 0.5 µl of 100 mM GTP proved quite challenging. However, as... Example 5 - Fed-batch reactions with dilute stocks with compensatory water evaporation As shown, using a more diluted feed solution reduced the reaction yield, likely due to the dilution of the reactants. Surprisingly, as... Figure 5A As shown, IVT reactions using a slightly diluted feed solution resulted in higher mRNA activity.

[0175] Figure 5B

[0176] Based on the results of Example 4, the inventors next explored whether the combination of diluting the feed solution and compensatory water evaporation, used to maintain a constant reaction volume, resulted in optimized yield and activity of the obtained mRNA. This approach enables continuous automated feeding without (significantly) altering the reaction concentration due to dilution, as the volume remains substantially constant under steady-state conditions.

[0177] The IVT reaction was prepared according to Example 4. Sample A (control) received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 100 mM stock solution (0.5 µl) in a closed tube. Sample B (control) received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 5 mM stock solution (10 µl) in a closed tube. Sample C received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 5 mM stock solution (10 µl) in an open tube. Sample D received a dose of GTP equivalent to 0.5 mM final concentration every 3 hours from a 5 mM stock solution (10 µl) in an open tube. Dry N2 gas was blown into the open tube at a gas flow rate of 3.3 µl / h evaporation, as shown in the pilot experiment, to maintain a stable volume.

[0178] Interestingly, such as Example 6 - IVT reactor design for implementing fixed volume fed-batch IVT As shown, when excess fluid evaporation is allowed, feeding from the concentrated stock (100 mM - Sample C) or the diluted stock (5 mM - Sample D) resulted in very similar yields. Furthermore, surprisingly, as... Figure 8A As shown, the IVT reaction using diluted stock (sample D - 5 mM) increased the activity of the generated mRNA compared to the IVT reaction using a more concentrated stock (sample C - 100 mM).

[0179] Figure 8B

[0180] refer to Figure 8A The image shows a first embodiment of a reactor 2 for carrying out the IVT method of the present invention. The reactor 2 includes a reaction vessel 4 consisting of an outer chamber 5 and an inner reaction volume 6, in which the IVT reaction is carried out and surrounded by a semipermeable membrane 8 (preferably a dialysis membrane 8), the pore size of which is smaller than that of the protein, DNA, and RNA components in the reaction, but larger than that of NTPs, buffer components, salts, reducing agents, and / or spermidine (thus allowing these components to pass through).

[0181] During reaction setup, a feed solution 10 containing IVT reaction feed components is fed into the outer chamber 5 of the reaction vessel 4 via feed pipe 12, thereby contacting the outside of the semi-permeable membrane 8 surrounding the inner reaction volume 6. The positive pressure generated by the feed fluid 10 entering the originally closed system drives the feed solution 10 and its components from the outer chamber 5 through the semi-permeable membrane 8 (in the direction of arrow A), thereby expanding the volume of the inner reaction volume 6.

[0182] If the pressure generated by the feed solution 10 itself is insufficient to drive a predictable and / or sufficiently large volume of feed solution 10 through the semipermeable membrane 8, additional pressure can be applied to the feed solution 10 by feeding pressurized air 14 into the outer chamber 5 via the inlet pipe 16. To prevent contamination, the pipe 16 providing the additional pressure is optionally equipped with a HEPA filter 20. The use of the semipermeable membrane 8 provides a distributed mixing method for the feed solution 10. By providing continuous pressure to the feed solution 10, the feed solution 10 can be forced through the membrane 8 (from the outer chamber 5 into the inner reaction volume 6) at a rate greater than the diffusion rate, preventing IVT reaction components from transferring from the reaction volume 6 into the feed solution 10, thereby effectively creating unidirectional flow. Excess pressure in the system is released through the air outlet 18 connected to the top of the reaction vessel 4, thereby effectively pumping the feed fluid 10 from the outer chamber 5 of the reactor into the inner reaction volume 6 in the reaction vessel 4.

[0183] To maintain a substantially constant volume of the IVT reaction in a steady state, ensuring that the proportions of various reagents (particularly cap analogs: the proportion of competing nucleotides) remain constant, excess fluid is removed from the reaction by evaporation. Therefore, the reactor 2 for the reaction includes an evaporation zone 22, which is an air pocket located above the internal reaction volume 6 in which the IVT reaction takes place and contained within a solid wall 23, at least a portion of which is optionally supplied by an air inlet 16. Fresh air 24 (optionally conditioned and / or filtered air) is fed into the evaporation zone 22 via an air conduit 26, and the evaporation zone 22 is also connected to an air outlet 18. To ensure optimal mixing of the feed solution 10 within the internal reaction volume 6, the reaction vessel 4 is optionally equipped with a magnetic stirrer 28. Alternatively, the entire reactor 2 may be placed on an oscillating platform (not shown), optionally moving in a figure-eight or other mixing pattern to effectively stir the reaction volume 6 along the interior of the semipermeable membrane 8 (and the internal reaction volume 6).

[0184] refer to Figure 8A This illustrates a second embodiment of reactor 2. In this embodiment, Figure 8B The arrangement of the internal reaction volume 6 and the external chamber 5 containing the feed solution 10 in contact with the semipermeable membrane 8 is reversed. Therefore, in this embodiment of reactor 2, the internal chamber 30 defined by the semipermeable membrane 8 contains the feed solution 10 supplied via the feed pipe 12. The feed solution 10 is then pushed outwards by excess pressure (in the direction of arrow B) through the semipermeable membrane 8 into the external container 32 containing the reaction volume 34.

[0185] To achieve controlled evaporation in evaporation zone 22 and maintain a substantially constant reaction volume, an air inlet pipe 16 providing conditioning air 14 is connected to the reaction vessel 32, such that the conditioning air 14 contacts the upper surface of the reaction volume 34, circulates around the internal chamber 30 containing the feed solution 10, and flows towards the air outlet pipe 18. Figure 8C Compared to the first implementation scheme shown, Figure 8A The advantage of the second embodiment of the reactor 2 shown is that the shape of the internal chamber 30 surrounded by the semipermeable membrane 8 is maintained by the pressure applied to the semipermeable membrane 8 without any additional structure.

[0186] refer to Figure 8B The image shows a third embodiment of reactor 2. In this embodiment, the single reaction vessel 36 comprises a single chamber (which is different from...). Figure 8C and Figure 8A (Different embodiments are shown), the chamber contains a reaction volume 38 and an added feed solution 10. The feed solution 10 is supplied to the closed system via a probe 40, which in some embodiments may be a needle, pipette tip, pasteurized pipette, or serum pipette, or any other probe suitable for adding the feed solution 10. A magnetic or physical stirrer 28 (optionally connected to the probe 40) may be introduced into the reaction vessel 36 to mix the reaction volume with the feed solution. Alternatively, a magnetic or physical stirrer 28 may be introduced into the reaction vessel 36 to mix the reaction volume with the feed solution. Figure 8C The device is placed on an oscillating platform to mix the contents of the device. Figure 8D and 8B similar, Figure 8C The embodiment includes an air inlet duct 26 for adding air 24 (optionally conditioned air) to the reaction vessel 36, such that the air 14 interacts with the surface of the reaction volume 38 and is discharged through an air outlet 18. Each air outlet is optionally equipped with a HEPA filter 20 to prevent contamination. The evaporation zone 22 is clearly shown in the figure as the space above the reaction volume 38, ensuring the removal of excess fluid through evaporation.

[0187] refer to Figure 8C The fourth embodiment of reactor 2 is shown. It follows the same principle as... Figure 8E The same design principle applies, except that the probe 40 (through which the feed solution 10 is fed into the reaction volume 38) is modified to feed the feed solution 10 in a distributed manner, preferably through multiple axially, radially, and / or longitudinally spaced microfluidic openings 42. The mixing and evaporation of the IVT reagent to maintain a constant reaction volume and a stable state are performed in a manner similar to... Figure 8C The implementation plan.

[0188] refer to Figure 8FThe fifth embodiment of reactor 2 is shown. This embodiment follows the same principle as... Figure 8A and 8D The embodiment shown has the same design, except that the feed probe 40 is a spray device or atomizer 44 installed at a suitable position above the surface of the reaction volume 6, so that most (preferably all) of the spray droplets 46 are combined with the reaction volume 38, thus providing another method for the distributed addition of the feed solution 10.

[0189] Final Reference Conclusion The sixth embodiment of reactor 2 is shown. In this embodiment, pipes 10 and 16 are shown as separate from each other, rather than as... ​ They are connected together as shown.

[0190] It should be understood that the dimensions of the components in device 2 of the present invention can be adjusted, scaled, or otherwise modified to accommodate desired volumes, feed rates, mixing rates, and evaporation rates. Furthermore, relative dimensions can be modified to give the device an optimized volume-to-surface area ratio or other advantageous shape. Finally, those skilled in the art will understand that conduits can be permanently or temporarily fixed to the device by direct ligation, threading, pressure clamping, force fitting, or any other method known to those skilled in the art. Additionally, conduits for feeding and discharging fluids and / or air can be bundled together or individually connected to the container. The various aspects of the device can be symmetrical, but may also be asymmetrical, unlike the figures.

[0191] ​

[0192] The inventors have devised a novel IVT method, particularly for co-transcriptional capping of RNA using cap analogs, selecting a very high ratio of cap analog to competing nucleotides (for treatment-related capping efficiency). However, this results in very low yields, making such reactions impractical and uneconomical. Therefore, they have provided a batch feed scheme to address the yield problem. However, a problem with this scheme is that feeding from a concentrated stock solution to prevent over-dilution leads to excessively low volumes, making proper fluid control challenging and causing significant fluctuations in reaction conditions. Conversely, feeding from a diluted stock solution results in over-dilution. Therefore, a preferred embodiment of the method relies on using a diluted stock solution to achieve sufficiently precise control, and simultaneously / subsequently removing excess fluid to correct for dilution. Furthermore, the inventors have used a highly distributed system for adding the feed solution to the reaction volume to prevent temporary local deviations in the overall reaction composition.

Claims

1. A method for preparing capped ribonucleic acid (RNA) molecules, wherein the method comprises performing a feed-batch transcription reaction in the presence of a substantially constant volume of a cap analogue to produce substantially capped RNA molecules.

2. The method of claim 1, wherein the method comprises in vitro transcription (IVT).

3. The method according to claim 1 or claim 2, wherein the RNA is selected from the following RNA molecules: messenger RNA (mRNA), microRNA (miRNA), interfering RNA (RNAi), short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA, RNA aptamer, self-amplifying RNA (saRNA), coding RNA, non-coding RNA, and circular RNA.

4. The method according to any one of the preceding claims, wherein the RNA comprises mRNA.

5. The method according to any one of the preceding claims, wherein the method comprises contacting the following: (i) Template nucleic acid sequence, optionally DNA, (ii) An RNA polymerase, optionally selected from T7, T3, SP6, KP34, Syn5 or other DNA-dependent RNA polymerases or any mutant variant of these RNA polymerases. (iii) Multiple nucleotide triphosphates (NTPs), optionally selected from ATP, GTP, CTP and / or UTP or modified variants thereof, including pseudoUTP, N1-methyl-UTP, m5CTP, m6ATP, m1ATP, inosine triphosphate, hm5CTP, m1GTP, m7GTP and m6AmTP, and (iv) Hat-like items, The RNA polymerase therein uses the various NTPs and the cap analogue to transcribe the template nucleic acid, thereby forming a capped RNA molecule, preferably in a single one-pot reaction.

6. The method according to any one of the preceding claims, wherein the cap analogue is selected from the following cap analogues: mCap (m7G(5')ppp(5')G, TriLink Biotechnologies, Inc); anti-reverse cap analogue (an mCap version modified by 3' OH methylation on m7G, ARCA – ThermoFisher Scientific); and trinucleotide cap analogues (e.g., Cleancap – TriLink Biotechnologies); and modifications and / or combinations thereof.

7. The method according to any one of the preceding claims, wherein the cap analogue mimics a 5' cap and is contained in the reaction mixture and is introduced by RNA polymerase as the first nucleotide of the nascent RNA rather than the corresponding competing nucleotide.

8. The method according to any one of claims 5 to 7, wherein the method comprises contacting the template nucleic acid sequence, RNA polymerase, multiple nucleotide triphosphates (NTPs), and cap analogs in the presence of magnesium ions, wherein the concentration of magnesium ions is 1-5 mM higher than the total concentration of NTPs present and / or fed into the reaction up to this point, optionally wherein the method comprises adding any additional Mg2+ required during or immediately after the feeding, rather than providing an excess of Mg2+ at the start of the reaction.

9. The method according to any one of the preceding claims, wherein the method comprises contacting the reagent with a buffer, wherein the concentration of the buffer is preferably 10-50 mM Tris-HCl, and the pH is set to 7.8-7.9 (at 25 °C).

10. The method according to any one of the preceding claims, wherein the method comprises contacting the reaction reagent with a base, wherein the concentration of the base is equal to or exceeds the amount of any acid added in the reaction or generated during the reaction.

11. The method according to any one of the preceding claims, wherein the method comprises contacting the reagent with a reducing agent, wherein the reducing agent is selected from dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol and tris(2-carboxyethyl)phosphine (TCEP), and optionally wherein the concentration of the reducing agent is 0.5-5 mM or 1-3 mM or about 1 mM.

12. The method according to any one of the preceding claims, wherein the method comprises contacting the reagent with spermidine, wherein the concentration of spermidine is 0.1-10 mM or 1-5 mM or 2 mM.

13. The method according to any one of the preceding claims, wherein the method comprises feeding the reagent in a diluted feed solution, wherein, The diluted feed solution contains a certain concentration of feed components such that if fluid removal is not used or will not be used, the total feed volume increases by >25% of the initial reaction volume, resulting in a >25% dilution of the components present in the initial reaction volume, but which are absent or present at a lower concentration in the feed solution.

14. The method of claim 13, wherein the diluted feed solution contains a feed component of a concentration such that if fluid removal is not used or will not be used, the total feed increases by >20% of the initial reaction volume, or the total feed increases by >40% of the initial reaction volume, or the total feed increases by >100% of the initial reaction volume.

15. The method of claim 13 or claim 14, wherein the diluted feed solution is defined as a feed solution containing one or more components, the concentration of which is >2 times, >4 times, or >10 times lower than their respective solubility limits and / or technical manufacturability limits.

16. The method according to any one of claims 13 to 15, wherein the diluted feed solution is defined as the dilution of the cap analog competing nucleotide compared to the maximum concentrated stock solution available for the competing nucleotide, and is at least 2x, 5x, 10x, optionally more than 25x or more than 100x.

17. The method according to any one of the preceding claims, wherein the method comprises continuously or intermittently feeding or replenishing the competing nucleotide competing with the nucleotide cap analog at a rate suitable for maintaining the ratio between the competing nucleotide and the nucleotide cap analog between a predetermined minimum threshold and a maximum threshold, wherein the minimum threshold of the ratio of the nucleotide cap analog to the competing nucleotide is determined by the desired capping efficiency, and the maximum threshold of the ratio of the nucleotide cap analog to the competing nucleotide is determined by the desired minimum reaction rate and / or reaction yield.

18. The method according to any one of the preceding claims, wherein the method comprises continuously or intermittently feeding only the competing nucleotides and not feeding a mixture of nucleotides.

19. The method of claim 18, wherein the maximum desired concentration of each non-competitive nucleotide is added to the reaction at the start of the reaction to achieve the highest possible transcription rate, and the transcription rate is limited only by cap analog competing nucleotides provided at concentrations significantly lower than those of the non-competitive nucleotides and / or cap analogs.

20. The method according to any one of the preceding claims, wherein, The composition of the reaction comprises 5 mM, 6 mM or 7 mM of each of rATP, rCTP, rUTP or derivatives thereof at the start of the reaction, optionally further comprising pseudoUTP, N1-methyl-UTP, m5CTP, m6ATP, m1ATP, inosine triphosphate, hm5CTP, m1GTP, m7GTP, m6AmTP or mixtures thereof.

21. The method according to any one of the preceding claims, wherein, The composition of the reaction comprises, at the start of the reaction, 8 mM, 9 mM, or 10 mM of each of rATP, rCTP, rUTP, or derivatives thereof, wherein the derivatives optionally include pseudoUTP, N1-methyl-UTP, m5CTP, m6ATP, m1ATP, inosine triphosphate, hm5CTP, m1GTP, m7GTP, m6AmTP, or mixtures thereof.

22. The method according to any one of the preceding claims, wherein, The initial and / or average concentrations of the competing nucleotides are selected as fractions of capped analog concentrations sufficient to achieve the desired capping efficiency: (i) 1:9, 1:10, or 1:20; or (ii) 1:50, 1:75, or 1:

100.

23. The method according to any one of the preceding claims, wherein the feed solution contains competing nucleotides and optionally counterions of nucleotides in RNase-free water.

24. The method according to any one of the preceding claims, wherein the initial concentration of the cap analog is high enough to allow the competing nucleotide concentration to be sufficient to maintain a suitable reaction rate, wherein the initial concentration of the cap analog is >5 mM or >8 mM or >10 mM.

25. The method according to any one of the preceding claims, wherein the method comprises reducing the volume of the transcription reaction during and after feeding to compensate for the additional volume of feed solution added thereto.

26. The method according to any one of the preceding claims, wherein the method comprises feeding only competing nucleotides while reducing volume, optionally using a high cap analog to competing nucleotide ratio.

27. The method according to any one of the preceding claims, wherein, Feeding of competing nucleotides and / or any other reactive components allows for the simultaneous or subsequent addition and removal of fluids.

28. The method according to claim 27, wherein, The addition and removal of fluids are matched so as not to change the reaction volume and therefore not to cause the concentration of non-feed reactants to exceed predetermined parameters.

29. The method of claim 27 or claim 28, wherein the addition and removal of fluids are matched to maintain substantially the same reaction volume, preferably wherein the removal of fluids is carried out in a manner that removes only water without removing any significant amount of dissolved reaction components or products.

30. The method according to any one of claims 25 to 29, wherein excess fluid is removed from the reaction by evaporation, thereby maintaining the reaction volume substantially constant.

31. The method of claim 30, wherein the evaporation of excess fluid is achieved by applying a controlled gas flow (preferably a controlled air flow) over the reaction mixture, wherein the gas flow is in direct contact with the surface of the reaction volume and not with the feed solution flow.

32. The method of claim 31, wherein the magnitude of the airflow (preferably an airflow) is controlled, and wherein the airflow velocity is at least 0.5 m / s, at least 2 m / s, at least 3.5 m / s, at least 5 m / s, at least 10 m / s, or >10 m / s.

33. The method according to any one of claims 31 or 32, wherein the air humidity is reduced before the airflow comes into contact with the surface of the reaction mixture, optionally wherein the air humidity is less than 50%, less than 40%, or less than 30%.

34. The method according to any one of claims 31 to 33, wherein the temperature of the gas (preferably air) is maintained at the same temperature as the reaction mixture to avoid cooling the fluid.

35. The method according to any one of claims 25 to 34, wherein excess fluid is removed by osmosis by contacting a sufficiently large surface of the reaction volume with a semipermeable membrane or a water-selective membrane, wherein the pore size of the membrane is smaller than that of the protein, DNA, and RNA components of the reaction, but larger than that of NTPs, buffer components, salts, reducing agents, and / or spermidine.

36. The method of claim 35, wherein the method comprises feeding pressurized air into the reaction chamber to provide sufficient pressure to drive a predictable and / or sufficiently large volume of feed solution through the semipermeable membrane.

37. The method according to any one of claims 25 to 36, wherein the method for removing excess fluid is adjusted and / or fine-tuned by a PID loop in response to measurements of fluid volume and / or osmotic pressure of the IVT reaction volume.

38. The method according to any one of the preceding claims, wherein the method comprises measuring the level of the reactive fluid, preferably by optical measurement of the fluid height, wherein such optical measurement is performed in the direction of: (i) a substantially vertical direction to detect reflections on the fluid surface; or (ii) a substantially horizontal direction to detect obstruction of the light path by the fluid surface, or to detect changes in fluid absorption.

39. The method of claim 38, wherein the reaction fluid level is measured by an electronic conductivity probe or by continuously weighing the entire system and subtracting the empty weight of the reaction vessel and the weight of the feed solution not yet added to the reaction volume.

40. The method according to any one of the preceding claims, wherein the method comprises calculating excess fluid based on osmotic pressure measurements of the solution and comparing it with the required osmotic pressure of the solution for that stage of the IVT reaction.

41. The method according to any one of the preceding claims, wherein the method comprises determining the feed rate of the feed solution based on empirical evidence or theoretical calculations of the consumption rate of a known amount of RNA polymerase having known activity in the same or similar reaction composition for competing nucleotides and cap analogs, and taking into account the composition of the RNA sequence (preferably mRNA), optionally wherein the feed rate is initiated at the start of the reaction and substantially matched with the consumption rate to maintain the ratio of cap analog to competing nucleotide.

42. The method according to any one of the preceding claims, wherein the method comprises adjusting the feed rate of the feed solution based on real-time or intermittent measurements of the reaction substrate and / or reaction products.

43. The method according to any one of the preceding claims, wherein the method comprises continuously or intermittently mixing the reaction reservoir to address local differences in the concentration of competing nucleotides, thereby addressing local differences in the ratio between the cap analogue and the competing nucleotides.

44. The method according to any one of the preceding claims, wherein the method comprises feeding the feed solution into the reaction in a distributed manner to prevent the formation of temporary local high concentrations of the feed solution and optionally undesirable cap analog to competing nucleotide ratios, wherein distributed feeding comprises releasing the feed solution simultaneously or sequentially at spatially separated locations within the reaction volume.

45. The method of claim 44, wherein the method comprises intermittently or continuously feeding a competing nucleotide and / or any other reaction component competing with the nucleotide cap analog into the reaction through a semipermeable membrane, wherein the semipermeable membrane comprises a plurality of axially, radially and / or longitudinally spaced microscopic openings and / or pores through which the feed solution passes.

46. ​​The method of claim 44, wherein the method comprises intermittently or continuously feeding a competing nucleotide and / or any other reaction component that competes with the nucleotide cap analog into the reaction through a microfluidic channel, wherein the microfluidic channel comprises a plurality of axially, radially, and / or longitudinally spaced microfluidic openings through which the feed solution passes.

47. The method of claim 44, wherein the method comprises intermittently or continuously feeding a competing nucleotide and / or any other reaction component that competes with the nucleotide cap analog into the reaction through one or more needles, pipette tips, robotic probes, serum pipettes, Pasteur pipettes or other suitable probes.

48. The method according to any one of the preceding claims, wherein the method comprises feeding the competing nucleotides and / or other reaction components onto the surface of the reaction volume by spraying, atomizing or any other means of generating significantly small droplets, optionally wherein the droplets are substantially uniformly distributed over the fluid containing the reaction volume.

49. The method of claim 47, wherein the sprayed or atomized fluid is applied to a portion of the entire reaction volume surface, or wherein the sprayed or atomized fluid is applied to a portion of the reaction volume surface separated from the reaction volume surface exposed to the airflow to evaporate excess fluid.

50. The method according to any one of the preceding claims, wherein the method comprises feeding a competing nucleotide and / or other reaction component competing with the nucleotide cap analogue into the reaction by dissolving, breaking, swelling, degrading, exchanging or otherwise releasing it from a carrier, wherein the carrier is added at the start of the reaction / incubation, and / or added during the reaction, and / or fed intermittently, and / or continuously into the reaction.

51. A capped ribonucleic acid (RNA) molecule, which is obtained by the method of any one of claims 1 to 50 or may be obtained by the method of any one of claims 1 to 50.

52. An apparatus for producing capped RNA ribonucleic acid (RNA), the apparatus comprising: The transcription reaction occurs in the reaction chamber in the presence of a cap analogue. And a feeding means for feeding a feed solution containing one or more reagents required for the production of capped RNA, wherein the device is configured to, in use, feed the reagents into a reaction chamber in a feed batch mode via the feeding means and to perform transcription in the reaction chamber at a substantially constant volume, thereby producing substantially capped RNA molecules.

53. The apparatus of claim 51, wherein the apparatus is used to perform the method of any one of claims 1 to 50.

54. The apparatus according to claim 52 or 53, wherein the reaction chamber comprises a first part and a second part, the feed solution is fed into the first part, and a transcription reaction occurs in the second part, wherein the first part and the second part are separated by a semipermeable membrane, preferably, wherein the semipermeable membrane is a dialysis membrane.

55. The apparatus of claim 54, wherein the pore size of the semipermeable membrane is smaller than that of the reacting protein, DNA, and RNA components, but larger than that of NTPs, buffer components, salts, reducing agents, and / or spermidine, thus allowing them to pass through.

56. The apparatus of claim 54 or 55, wherein the feeding means is configured to feed the feed solution into a first portion of the reaction chamber, thereby causing contact with one side of the semipermeable membrane, wherein the positive pressure generated by the feed fluid drives the feed solution and its contained components through the semipermeable membrane.

57. The apparatus according to any one of claims 54 to 56, wherein the apparatus includes a gas inlet, preferably an air inlet, configured to feed pressurized gas into a first portion of the reaction chamber, thereby providing additional pressure to the feed solution.

58. The apparatus according to any one of claims 52 to 57, wherein the apparatus includes a gas outlet, preferably an air outlet, configured to release excess pressure in the reaction chamber.

59. The apparatus according to any one of claims 52 to 58, wherein the reaction chamber includes an evaporation zone disposed above the portion where the transcription reaction occurs, and the fluid evaporates in the evaporation zone to compensate for the additional volume provided by the feed, thereby maintaining a substantially constant reaction volume.

60. The apparatus of claim 59, wherein the apparatus includes a gas inlet, preferably an air inlet, configured to feed gas (preferably air) into the evaporation zone to promote the evaporation of the fluid, optionally wherein the gas inlet is in fluid communication with the gas outlet.

61. The apparatus according to any one of claims 52 to 60, wherein the feeding means comprises a probe having a distal outlet through which the feed solution is passed, optionally wherein the probe is a needle, pipette tip, pasteurized pipette, serum pipette, or any other probe suitable for adding the feed solution.

62. The apparatus of claim 61, wherein the outlet is configured to extend into the reaction liquid in the reaction chamber.

63. The apparatus of claim 61 or 62, wherein the probe comprises a plurality of axially, radially, and / or longitudinally spaced outlets through which the feed solution preferably enters the liquid.

64. The apparatus according to any one of claims 61 to 63, wherein the probe comprises a spray device or atomizer configured to generate droplets of feed solution in the reaction chamber, optionally wherein the spray device or atomizer is mounted above the surface of the reaction liquid.

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