In vitro production and purification of therapeutic MRNA

Abstract

The present invention comprises novel systems, methods, and compositions for the in vitro production of polynucleotides, particularly mRNA for use in therapeutic applications.

Description

Technical Field

[0001] This international PCT application claims the benefit and priority of U.S. Provisional Application No. 63 / 011133, filed on April 16, 2020, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, the entire disclosure of which is incorporated herein by reference. The ASCII copy was created on April 16, 2021, named "90125-00181-Sequence-Listing-AF.txt", and is 26.9 kilobytes in size.

[0003] The present invention generally relates to the in vitro production of polynucleotides, particularly to the field of production of mRNA for therapeutic use.

Background Art

[0004] Messenger RNA (mRNA) is a template molecule that is transcribed from a cell's DNA and translated into an amino acid sequence, i.e., a protein, in the ribosomes within the cells of an organism. To control the expression level of the encoded protein, mRNA has untranslated regions (UTRs) adjacent to the actual open reading frame (ORF) that contains the genetic information encoding the amino acid sequence. Such UTRs are referred to as the 5'-UTR and 3'-UTR, respectively, and are regions of the mRNA located before the start codon and after the stop codon. Further, mRNA includes a poly(A) tail (poly(A) strand) region, which is a long sequence of adenine nucleotides that facilitates the transport of mRNA from the nucleus and protects the mRNA from degradation to some extent. Due to the progress of recent science and technology, mRNA has become a promising candidate for various applications, including diagnostic uses and therapeutic products such as vaccines.

[0005] Many approaches are being developed for the large-scale production of mRNA, driven by the growing demand from the medical community for not only personalized medicine but also emergency response in epidemic crisis situations, as seen in the recent COVID-19 pandemic. Most current methods utilize fermentation to synthesize mRNA from self-replicating DNA templates in culture, and then use volatile organic solvents to isolate the total RNA as raw material. These processes are costly, dangerous, and generate a hazardous waste stream that must be mediated, while production rates depend heavily on the performance of the producing strain and its ability to remove impurities from diverse tRNA, rRNA, and host mRNA. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] As will be seen, there has long been a need for an effective in vitro mRNA production process that does not require volatile organic solvents, does not generate a series of hazardous wastes, and is significantly less expensive than its fermentation-based counterpart, while producing homogeneous and pure mRNA suitable for therapeutic use. [Means for solving the problem]

[0007] One aspect of the present invention includes a novel in vitro method for the production of polynucleotides and, in particular, mRNAs that may be targeted for one or more diagnostic or therapeutic applications. In a preferred aspect, the present invention includes a fully recombinant, stable, reliable, and functional in vitro system for batch or continuous flow production of polynucleotides and, preferably, RNA. In this preferred aspect, the current improved in vitro system can create an in vitro environment configured to mimic the production of polynucleotides that occurs in vivo by utilizing components involved in polynucleotide transcription.

[0008] Another aspect of the present invention includes a novel in vitro method for mRNA production. In this embodiment, an in vitro bioreactor (and preferably a batch bioreactor) or continuous flow bioreactor may be configured to combine isolated RNA polymerase (RNAP), a nucleotide template (and preferably a linear non-self-replicating DNA template), together with a plurality of nucleotide triphosphates (NTPs) (which may be incorporated into the mRNA molecule synthesized through the action of RNAP), and an energy source (e.g., a novel inorganic polyphosphate energy regeneration system outlined in PCT application PCT / US2018 / 012121 by Koglin and Humbert (its description, figures, examples, sequences, and claims are incorporated herein by reference in their entirety)). In a preferred embodiment, the synthesized mRNA can then be purified and used for a variety of downstream purposes, including diagnostic or therapeutic applications such as vaccines targeting selected target pathogens.

[0009] In a preferred embodiment, the production of macromolecules using the recombinant cell-free system of the present invention can be achieved in a bioreactor system. As used herein, “bioreactor” may be any form of sealed device configured to maintain an environment conducive to the production of macromolecules, and preferably polynucleotide macromolecules, and more preferably mRNA transcribed in vitro from a DNA template. The bioreactor may be configured to operate on a batch, continuous, or semi-continuous basis, for example, by a feed fluid or supply solution. In one embodiment, the present invention may further include a bioreactor configured to produce mRNA. In this embodiment, the present invention may be particularly suitable for operation with a continuous flow bioreactor system, which may include one or more hollow continuous flow conduits (e.g., made of fibrous material that are in fluid communication with an external bioreactor compartment). In this embodiment, the hollow continuous flow conduit is formed as an exchange medium for in vitro transcripts or polynucleotides (and preferably mRNA).

[0010] Another aspect of the present invention involves the synthetic biological production of polynucleotides (and preferably mRNA via various reaction types). In one embodiment, mRNA can be produced in vitro using a batch reaction. In this preferred embodiment, isolated RNAPs, DNA templates, and NTPs can all be combined in a batch-feed reaction chamber and incubated until the reactants are consumed. To avoid the scaling limitations of batch reactions, in another preferred embodiment, mRNA can be produced in vitro using a continuous-flow bioreactor. The continuous-flow bioreactor may be configured to operate continuously so that new input material can be injected while producing large quantities of mRNA that may be output between or after the reaction process.

[0011] Another aspect of the present invention may include novel systems and methods for the isolation and purification of polynucleotides (and preferably mRNA produced in the in vitro production system of the present invention). In a preferred embodiment, the reaction output containing the synthesized mRNA can be subjected to a stepwise isolation and purification process that allows for the sequential removal of reaction mixture components, namely RNAP, template DNA, free NTPs, and buffer, from the mRNA output without the use of harmful organic solvents or expensive disposable purification kits. In one embodiment, the present invention can utilize a purification column cascade, where the reaction material from either a batch or continuous flow bioreactor is washed on a protein affinity resin, followed by a DNA affinity resin, or vice versa. This step can remove all protein and DNA template reaction components except for unreacted free nucleotides. The free NTPs and buffer can be removed by alcohol precipitation, leaving only the isolated precipitated mRNA material. The mRNA can then be resolubilized for desired downstream applications or dried for long-term storage or reduced-volume delivery.

[0012] Another aspect of the present invention may include pharmaceutical compositions for novel mRNA-based vaccines targeting a target pathogen such as COVID-19, produced by one or more of the in vitro mRNA production methods described herein, as well as the use thereof for treating subjects in need of treatment.

[0013] Another aspect of the present invention may include novel systems, methods, and compositions for multi-step in vitro production of mRNA. In one preferred embodiment, mRNA production from a DNA template is divided into poly(A) tail addition or poly(A) tailing. For example, mRNA is produced from a DNA template in a first bioreactor (and preferably a continuous flow bioreactor of the present invention). Production in this first stage may or may not be coupled with the addition of a 5' cap to the mRNA transcript. In a second stage, the mRNA transcript may be introduced into a second bioreactor (and preferably a continuous flow bioreactor of the present invention) and further modified to include a poly(A) tail. Optionally, the mRNA may be modified to include a 5' prime cap, assuming it was not included in the first bioreactor as described above.

[0014] Another aspect of the present invention may include novel systems, methods, and compositions for multi-step in vitro production of mRNA. In one preferred embodiment, mRNA production from a DNA template is divided into poly(A) tail addition or poly(A) tailing. For example, mRNA is produced from a DNA template in a first bioreactor (and preferably a continuous flow bioreactor of the present invention). Production in this first stage may or may not be coupled with the addition of a 5' cap to the mRNA transcript. In a second stage, the mRNA transcript may be introduced into a second bioreactor (and preferably a continuous flow bioreactor of the present invention) and further modified to include a poly(A) tail. Optionally, the mRNA may be modified to include a 5' prime cap, assuming it was not included in the first bioreactor as described above. This multi-step in vitro mRNA production has several advantages. For example, since the generation of poly(A) tails after transcription is separated from RNA synthesis, operators can limit the undesirable synthesis of double-stranded RNA (dsRNA). The multi-step production system of the present invention also reduces manual handling of mRNA and further reduces the risk of shearing. Finally, the multi-step production system of the present invention enables one-step isolation / purification of mRNA, for example, via only poly(A)-tailed RNA bound to polydT resin.

[0015] Another aspect of the present invention may include novel systems, methods, and compositions for multi-step in vitro production of mRNA having a Cap1 enzyme treatment system. In one preferred embodiment, the Cap0 / Cap1 enzyme treatment system can act as a checkpoint recognizing Cap0 on the RNA, after which enzymatic methylation generates Cap1, followed by the generation of a poly(A) tail. This choice of poly(A) tail ensures that the RNA is also completely capped during production, resulting in an improved yield of fully functional mRNA.

[0016] Another aspect of the present invention may involve multi-step in vitro production of novel mRNA in which mRNA synthesis and poly(A) tailing are separated. In this aspect, a reaction mixture having a DNA template (e.g., a linear plasmid, a PCR product, an amine-functional surface-tethered PCR product), a first amount of RNA polymerase, and a reaction buffer is introduced into a first bioreactor, which may include the batch or continuous flow bioreactor of the present invention or other suitable bioreactors (e.g., the hollow fiber reactor described herein). In a preferred embodiment, the reaction mixture is introduced into and passed through an internal reaction cell of the first bioreactor. This internal reaction cell may be in fluid communication with a supply chamber holding a supply solution containing nucleotides (NTPs), a reaction buffer that may contain components necessary to catalyze and drive mRNA synthesis as described herein, and a pyrophosphatase that catalyzes the hydrolysis of pyrophosphate to inorganic phosphate. In this embodiment, the supply solution may be circulated in a counterflow flow relative to the reaction mixture. In this embodiment, the supply chamber and reaction cell may be configured to include the continuous flow configuration outlined herein.

[0017] As described above, the compartmentalization and counterflow of the feed solution and reaction mixture create a gradient so that free NT from the feed solution, upon passing through the feed chamber, is drawn into the internal compartment of the reaction cell where it can react with the components of the reaction mixture. In this embodiment, RNAP can associate with a DNA template having a target sequence encoding a target mRNA and enzymatically catalyze the incorporation of NT into the target mRNA nucleotide.

[0018] The reaction mixture from the internal reaction cell may be extracted after one or more reaction cycles and introduced into a mixed cell where the newly synthesized mRNA is diluted in a buffer (and preferably a high-salt buffer) in a ratio of, for example, 1:5. Dilution and addition of the high-salt buffer produce a high-concentration RNA solution, inactivating RNAP, and further adjusting the buffer conditions to enable the enzymatic activity of poly(A) polymerase in the second poly(A) tailing step.

[0019] In a preferred embodiment, enzymes for 5' capping and poly(A) tailing of mRNA transcripts may also be provided in dilution buffer. This new reaction mixture is then introduced into a reaction cell of a second hollow fiber reactor having a supply chamber containing additional components such as nucleotide triphosphates (e.g., ATP and GTP) and S-adenosylmethionine (SAM). As described above, these components may be placed in a supply chamber that forms a gradient in the reaction cell so that they pass through a porous barrier, such as the hollow fiber barrier (34) described herein, enter the reaction cell, and act as substrates for 5' capping and poly(A) tailing of mRNA transcripts. In this configuration, mRNA is capped and poly(A) tailed, and the reaction mixture from the reaction cell of the second hollow fiber reactor is diluted in a high-salt buffer to allow slow binding of the poly(A) tailed RNA to the polydT resin. Other components pass over the resin and are recovered. In a particular embodiment, recombinant enzymes from the reaction mixture are captured by affinity resin in a column and then washed over the polydT resin. The reaction mixture is thoroughly washed on polydT resin, and after the poly(A)-modified RNA has bound to the resin, it is washed with a high-salt buffer. After washing, the vaporized poly(A)-modified RNA is released from the resin with distillate water. The final product of the multi-step in vitro production system is concentrated capped and poly(A)-tailed RNA in distilled water, which can be subjected to further processing, such as encapsulation in lipid nanoparticles (LNPs) for therapeutic applications.

[0020] Further aspects of the technology of the present invention will become apparent from the following specification, drawings, and claims.

[0021] The aspects, features, and advantages of this disclosure will be better understood from the following detailed description in conjunction with the accompanying drawings. All of these are given merely as examples and are not intended to limit the embodiments of this disclosure. [Brief explanation of the drawing]

[0022] [Figure 1]Shows a schematic diagram of a continuous flow mRNA production system in one embodiment. [Figure 2] Shows a schematic diagram of an mRNA batch production system in one embodiment. [Figure 3] Shows a schematic diagram of an mRNA purification and isolation system in one embodiment. [Figure 4] Shows an exemplary 1.0% agarose gel demonstrating mRNA production reactions performed on March 30, 2020, April 1, 2020, and April 3, 2020. A marker ladder (purchased from New England Biolabs (NEB)) is loaded in lane 1. A total of 10 ul of unpurified reaction (bulk) and purified reaction from March 30, 2020, are loaded in lanes 2 and 3 on the gel. A total of 10 ul of unpurified reaction (bulk) and purified reaction from April 1, 2020, are loaded in lanes 4 and 5 on the gel. Lanes 6 - 11 show 10 ul samples taken from the purification process outlined herein. Lane 6 is the unpurified reaction (bulk), lane 7 is the material after applying the reaction mixture to a protein affinity column, lane 8 shows the product after applying the reaction mixture to a DNA affinity column, lane 9 shows the reaction mixture after being washed via alcohol precipitation and then dissolved in sterile water, lane 10 shows the precipitated and isolated product after sterile filtration, and lane 10 shows the precipitated and isolated mRNA product after being purified through an RNA purification column (provided by NEB) and optionally concentrated. The gel is visualized by illumination at 365 nm on a UV table. [Figure 5A] Shows a schematic diagram of a multi - stage mRNA production system in one embodiment. [Figure 5B] Shows a schematic diagram of a multi - stage mRNA production system utilizing multiple hollow fiber bioreactors in one embodiment. [Figure 6]Bioanalyzer analysis. Three different mRNA product samples were capped and tailed using the system outlined in Figure 5, and the pre / post samples were electrophoresed on an Agilent Bioanalyzer 2100 instrument using an Agilent RNA 6000 Nano kit according to the manufacturer's instructions. Analysis of the samples shows a visual size shift due to tailing and a small population of non-tailed mRNA. 200 ng of raw mRNA versus capped and tailed mRNA on the Bioanalyzer 2100. Expected column sizes: m001 is approximately 1650 nt, m002 is approximately 1675 nt, m003 is approximately 2600 nt. Approximately 250 - 300 nt is added by tailing. [Figure 7] Denaturing agarose gel analysis. In vitro transcribed large (11000 nt) mRNA was produced, purified, and analyzed on a denaturing agarose gel according to the system described in Figure 5. 1% agarose was dissolved, and a denaturing gel was poured using 2.5% formaldehyde and 0.01% GelRed to analyze the long mRNA samples. 1 μg of mRNA sample was run alongside a commercially available RNA ladder (RiboRuler HR). The denaturing gel prevents most secondary structure formation, but small smears of various mRNA populations will still be visible. The success of producing such large mRNA species demonstrates the feasibility of the system of the invention described herein.

Mode for Carrying Out the Invention

[0023] Generally referring to Figure 1, in one embodiment, the technology of the present invention may include a novel continuous flow bioreactor (1) configured for the scaled in vitro production of polynucleotides (particularly mRNA). Generally referring to Figure 1, a continuous flow conduit (3) may pass through a continuous flow reaction chamber (2). In this embodiment, the continuous flow conduit (3) can continuously recirculate a supply solution. The supply solution may include one or more of the following components: - A first amount of isolated NTP, - A certain amount of reaction buffer, -Optionally, components of the inorganic polyphosphate energy regeneration system described in PCT / US2018 / 012121, and - One or more cofactors for mRNA polynucleotide production, optionally.

[0024] In this embodiment, the continuous flow reaction chamber (2) may hold a reaction mixture for the production of polynucleotides (particularly mRNA). This input reaction mixture may include one or more of the following components: - First amount of isolated RNAP enzyme, - The first amount of DNA template, - A certain amount of reaction buffer, -Optionally, an initial amount of isolated NTPs.

[0025] In particular, the continuous flow conduit (3) can be in fluid communication with the continuous flow reaction chamber (2). Specifically, as shown in Figure 1, the continuous flow conduit (3) can be in fluid communication with the continuous flow reaction chamber (2) through a plurality of conduit openings (4) configured to allow one or more components of the feed solution to pass from the plurality of conduit openings (4) into the continuous flow conduit (3). In one preferred embodiment, the continuous flow conduit (3) may include a continuous flow conduit (3) made from hollow fibers. In this embodiment, the continuous flow conduit (3) may be made from an MWCO PES membrane having pores between 5 kDa and 20 kDa in size. This fiber membrane may be further treated to reduce protein and nucleotide binding. This treatment may include the deposition of RNA-free acetylated BSA on the outside of the conduit, and purified RNA polymerase may be placed on the inside of the conduit where mRNA can be produced. In this configuration, the multiple conduit openings (4) create a gradient so that free NT from the supply solution, as it passes through the continuous flow reaction chamber (2), is drawn into the internal compartment of the continuous flow reaction chamber (2) and can react there with the components of the reaction mixture. In this embodiment, RNAP can associate with a DNA template (and preferably a linear DNA template) having a target sequence encoding a target mRNA (14), and enzymatically catalyze the incorporation of NTP into the target mRNA (14) nucleotide.

[0026] In certain embodiments, the target mRNA(14) may be configured to produce a three-dimensional structural shape of a hairpin configuration (e.g., a dsRNA configuration), which can be used to induce an RNA interference pathway in a subject that requires its induction; however, in further embodiments, the target mRNA(14) may be used as a pharmaceutical composition. For example, in one embodiment, the target mRNA(14) may encode an antigenic peptide (e.g., one against a viral protein). In this embodiment, the mRNA may be administered to a subject that requires it, translated to form an antigenic protein, which can then induce an immune response.

[0027] For example, in one preferred embodiment, the continuous flow bioreactor (1) may be configured to produce mRNA encoding one or more antigenic peptides directed to induce an immune response against COVID-19 coronavirus in a subject requiring it. More specifically, in this embodiment, the continuous flow bioreactor (1) may be configured to produce mRNA encoding one or more polyvalent COVID-19 coronavirus constructs described in U.S. Patent Application No. 62 / 992,072 by Koglin and Humbert (the specification, figures, sequences, and construct configurations of which are specifically incorporated herein by reference).

[0028] In some embodiments, at least one coding region of the mRNA produced according to the present invention encodes at least two, three, four, five, six, seven, eight, or more antigenic peptides or proteins comprising or consisting of the COVID-19 coronavirus protein or a fragment or variant thereof. More preferably, at least one coding region encodes at least two, three, four, five, six, seven, eight, or more antigenic peptides or proteins comprising or consisting of the COVID-19 coronavirus spike protein subunit 1 (S1), ii) the receptor-binding motif (RBM) of S1, and ii) the nucleocapsid protein (NCP) and fragments or variants thereof, or any one fragment or variant of those proteins (these may be further bound to a signal peptide, preferably an IgE signal peptide (incorporating SEQ ID NO: 9)). More preferably, at least one coding region encodes at least two, three, four, five, six, seven, eight, or more amino acid sequences selected from the group consisting of the incorporated sequence numbers 1-6In, or any one fragment or variant of these amino acid sequences. In particular, the incorporated sequences are identified by the "In" designation.

[0029] Referring again to Figure 1, the components of the reaction mixture may be loaded into the internal compartment of the continuous flow reaction chamber (2) before the start of the reaction, or may be replenished during operation as needed. The feed solution may also be loaded into the continuous flow conduit (3) before the start of the reaction. For example, as shown in Figure 1, the feed solution may be added to the continuous flow conduit (3) from an input reservoir (5) coupled to an input valve (7) configured to allow real-time injection of the feed solution into the continuous flow conduit (3). In certain embodiments, the addition of the feed solution may be achieved manually, while in alternative embodiments, the process may be automated. In this latter embodiment, the feed solution may be added to the system based on a predetermined schedule or based on a predetermined threshold (e.g., the concentration of NTP in the feed solution, the concentration of mRNA synthesized in the continuous flow reaction chamber (2), or another parameter such as the energy consumption of the reaction). Such parameters may be measured by one or more sensors communicating with a computer system configured to run a computer executable program in response to changes in one or more parameters as outlined herein.

[0030] In particular, once NTPs are incorporated into newly synthesized mRNA after the start of the reaction in the internal compartment of the continuous flow reaction chamber (2), fresh NTPs can be continuously supplied to the continuous flow conduit (3) due to other factors. Such metric production allows for more efficient use of reaction enzymes and energy, for example, in the form of energy hydrolysis of nucleotide triphosphates (e.g., one or more nucleotide triphosphates selected from the group consisting of adenine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP)).

[0031] The continuous flow bioreactor (1) of the present invention may be further configured to include an inorganic polyphosphate energy regeneration system, which generally includes a cellular adenosine triphosphate (ATP) energy regeneration system. In this embodiment, the continuous flow reaction chamber (2) is - A certain amount of isolated adenosyl kinase enzyme (and preferably Gst AdK derived from thermophilic bacteria), -A certain amount of isolated polyphosphate kinase enzyme (Taq PPK from thermophilic bacteria), - A certain amount of inorganic polyphosphate (PPi) derived from thermophilic bacteria, - May contain a certain amount of adenosine monophosphate (AMP).

[0032] In this embodiment, the adenosyl kinase (AdK) and polyphosphate kinase (PPK) enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP. More specifically, as generally shown in Figure 8 of Application 121 (incorporated herein by reference), in another preferred embodiment, isolated and purified Gst AdK (SEQ ID NO: 8In of Application 121 (incorporated herein by reference)) and / or Taq PPK (SEQ ID NO: 11In of Application 121 (incorporated herein by reference)) may be added to this cell-free expression system along with a certain amount of inorganic polyphosphate. In one embodiment, this amount of inorganic polyphosphate may comprise an optimal polyphosphate concentration range. In this preferred embodiment, such an optimal polyphosphate concentration range is generally defined as the concentration of inorganic polyphosphate (PPi) that stably maintains the equilibrium of the reaction. In this preferred embodiment, the optimal polyphosphate concentration range may be about 0.2 to 2 mg / ml of PPi.

[0033] As described above, PPK can synthesize ADP from polyphosphate and AMP. In this preferred embodiment, the conjugation of Gst AdK and PPK can remove adenosine diphosphate (ADP) from the system by converting two ADPs into one ATP and one adenosine monophosphate (AMP).

number

[0034] This reaction may be fast enough to drive the equilibrium reaction of PPK toward ADP production.

number

[0035] In this system, the presence of higher concentrations of AMP may further drive the reaction of Taq PPK toward ADP.

[0036] The mRNA output (9) containing a portion of the reaction mixture and newly synthesized mRNA in the continuous flow reaction chamber (2) can be extracted for further modification. Referring again to Figure 1, the mRNA output (9) can be extracted into the continuous flow reaction chamber (2) into an output reservoir (6) coupled to an output valve (8) configured to allow extraction of the mRNA output (9) from the continuous flow reaction chamber (2). In certain embodiments, the extraction of the mRNA output (9) can be achieved manually, while in alternative embodiments, the process can be automated. In this latter embodiment, the mRNA output (9) can be extracted from the system based on a predetermined schedule or based on a predetermined threshold (e.g., the concentration of NTP in the supply solution, the concentration of mRNA synthesized in the continuous flow reaction chamber (2), or another parameter such as the energy consumption of the reaction). Such parameters can be measured by one or more sensors communicating with a computer system configured to run a computer executable program in response to changes in one or more parameters as outlined herein.

[0037] Referring generally to Figure 2, in one embodiment, the technique of the present invention may include a novel batch-feed reaction chamber (10) configured for the scaled in vitro production of polynucleotides (particularly mRNA). In this embodiment, the batch-feed reaction chamber (10) may hold a reaction mixture for the production of polynucleotides (particularly mRNA). This input reaction mixture may include one or more of the following components: - First amount of isolated RNAP enzyme, - The first amount of DNA template, - A certain amount of reaction buffer, - First amount of isolated NTP, -Optionally, components of the inorganic polyphosphate energy regeneration system described in PCT / US2018 / 012121, and - One or more cofactors for mRNA polynucleotide production, optionally.

[0038] In this configuration, free RNAP can associate with a DNA template (and preferably a linear DNA template) having a target sequence encoding the target mRNA(14), and enzymatically catalyze the incorporation of NTP into the target mRNA(14) nucleotide within the batch-feed reaction chamber (10). Referring again to Figure 1, one or more components of the reaction mixture can enter the batch-feed reaction chamber (10) through the input reservoir (5).

[0039] In certain embodiments, the target mRNA (14) generated in the batch-feed reaction chamber (10) may be configured to produce a three-dimensional structural shape of hairpin configuration (e.g., dsRNA configuration), which may be used to induce RNA interference pathways in subjects requiring such induction. In further embodiments, the target mRNA (14) may be used as a pharmaceutical composition. For example, in one embodiment, the target mRNA (14) may encode an antigenic peptide (e.g., one against a viral protein). In this embodiment, the mRNA may be administered to a subject requiring it, translated to form an antigenic protein, which may then trigger an immune response.

[0040] For example, in one preferred embodiment, the batch-feed reaction chamber (10) may be configured to produce mRNA encoding one or more antigenic peptides directed to induce an immune response to COVID-19 coronavirus in subjects requiring it. More specifically, in this embodiment, the continuous-flow bioreactor (1) may be configured to produce mRNA encoding one or more polyvalent COVID-19 coronavirus constructs described in U.S. Patent Application No. 62 / 992,072 by Koglin and Humbert (the specification, figures, sequences, and construct configurations of which are specifically incorporated herein by reference).

[0041] In some embodiments, at least one coding region of the mRNA produced according to the present invention encodes at least two, three, four, five, six, seven, eight, or more antigenic peptides or proteins comprising or consisting of the COVID-19 coronavirus protein or a fragment or variant thereof. More preferably, at least one coding region encodes at least two, three, four, five, six, seven, eight, or more antigenic peptides or proteins comprising or consisting of the COVID-19 coronavirus spike protein subunit 1 (S1), ii) the receptor-binding motif (RBM) of S1, and ii) the nucleocapsid protein (NCP) and fragments or variants thereof, or any one fragment or variant of those proteins (these may be further bound to a signal peptide, preferably an IgE signal peptide (incorporating SEQ ID NO: 9)). More preferably, at least one coding region encodes at least two, three, four, five, six, seven, eight, or more amino acid sequences selected from the group consisting of the incorporated SEQ ID NOs. 1 to 6In, or any one fragment or variant of these amino acid sequences.

[0042] In particular, once the NTP is incorporated into the newly synthesized mRNA after the start of the reaction in the batch-feed reaction chamber (10), the synthesis reaction may proceed for a predetermined time, or until a threshold such as a predetermined concentration of newly synthesized mRNA is met, or until the enzyme or energy source of the reaction mixture is consumed. As described above, in one embodiment, the batch-feed reaction chamber (10) may include an energy source in the form of energy hydrolysis of nucleotide triphosphates (e.g., one or more nucleotide triphosphates selected from the group consisting of: adenine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). Finally, the batch-feed reaction chamber (10) of the present invention may be further configured to include the inorganic polyphosphate energy regeneration system outlined above.

[0043] The technology of the present invention further includes systems and methods for isolating mRNA output (9) produced via either a batch-feed reaction chamber (10) system or a continuous-flow bioreactor (1) outlined above. Referring generally to Figure 3, in one embodiment, the mRNA output (9) may pass through a protein affinity column (11) configured to capture the protein fraction (15) of the mRNA output (9), which may include free RNAP or other proteins that may be present in the mRNA output (9). In embodiments, the captured RNAP can be eluted from the protein affinity column (11) and reused in a subsequent in vitro mRNA production reaction.

[0044] As further shown in Figure 3, after the protein fraction (15) has been removed, the mRNA output (9) may pass through a DNA affinity column (12) configured to capture the DNA fraction (16) of the mRNA output (9), which may contain free DNA templates that may be present in the mRNA output (9).

[0045] Again, as shown in Figure 3, after removing the protein fraction (15) and DNA fraction (16), the mRNA output (9) may pass through a nucleotide precipitate (13) configured to remove the NTP fraction (17) and any excess buffer or other components of the mRNA output (9). In a preferred embodiment, the free NTP fraction (17) may be separated and removed from the target mRNA (14) via one or more rounds of alcohol precipitation and washing to extract the target mRNA (14) that may be present in the mRNA output (9). The finally isolated target mRNA (14) may be further purified and optionally resolubilized for desired downstream applications or dried for long-term storage or reduced-volume delivery. As described above, the order of the protein affinity column (11) and the DNA affinity column (12) may be switched so that the DNA fraction (16) is removed first, followed by the protein fraction (15), and vice versa. Such an operating order is also applicable to the removal of the free NTP fraction (17).

[0046] The present invention may include systems, methods, and compositions for a hollow fiber bioreactor (20) that can be configured for multi-step mRNA production, as outlined below. As shown in the figure, in a preferred embodiment, the hollow fiber bioreactor (20) may include a reaction cell (31) configured to contain a reaction mixture, preferably a DNA template (e.g., a linear plasmid, a PCR product, an amine-functional surface-tethered PCR product), a first amount of RNA polymerase, and a reaction buffer, as well as other components necessary for in vitro mRNA production. The mixed reaction cell (31) may be located within a supply chamber (32). In embodiments, this internal mixed reaction cell (31) may be in fluid communication with a supply chamber (32) that may contain a supply solution containing nucleotides (NTPs), a reaction buffer that may contain components necessary to catalyze and drive mRNA synthesis as described herein, and a pyrophosphatase that catalyzes the hydrolysis of pyrophosphate to inorganic phosphate. As shown below, in a multi-step system, the supply chamber (32) may contain a supply solution containing enzymes for 5' capping and poly(A) tailing of mRNA transcripts. As described above, the components of the supply solution placed in the supply chamber (32) can pass through a porous barrier and enter the reaction cell (32), forming a gradient with the reaction cell (32) to act as substrates for mRNA synthesis and / or 5' capping and poly(A) tailing of mRNA transcripts.

[0047] In a preferred embodiment, the reaction cell (32) is separated from the supply chamber (32) by a porous barrier (which may be a hollow fiber barrier (34) that includes an MWCO PES membrane having pores between 5 kDa and 20 kDa in size). This fiber membrane may be further treated to reduce protein and nucleotide binding. This treatment may include RNA-free acetylated BSA deposited on the outside of the conduit, with purified RNA polymerase placed on the inside of the conduit where mRNA can be produced.

[0048] As outlined above, the multi-step in vitro production system (30) of the present invention may include a plurality of bioreactors configured to separate mRNA synthesis and poly(A) tailing of mRNA transcripts and optionally 5' capping. In this embodiment, mRNA is produced in a first bioreactor, and poly(A) tailing of mRNA transcripts is carried out in a second bioreactor. As further outlined below, the 5' prime capping and modification of the cap may be carried out simultaneously with mRNA synthesis in the first bioreactor, or separately from the mRNA synthesis step, during poly(A) tailing in the second bioreactor.

[0049] In the schematic flow diagrams provided in Figures 5A-B, the multi-step in vitro production system (30) of the present invention may include a first bioreactor, which in this embodiment is shown as a first hollow fiber reactor (20a) configured for the synthesis of mRNA macromolecules and optionally for 5' capping of the transcript. In particular, the use of the first hollow fiber reactor (20a) is preferred but not required, as any in vitro mRNA bioreactor system may be used with the present invention. Referring again to Figures 5A-B, the first hollow fiber reactor (20a) may include a reaction cell (31) configured to contain a first-step reaction mixture (21), preferably a DNA template (e.g., linear plasmid, PCR product, amine-functional surface-tethered PCR product), a first amount of RNA polymerase (SEQ ID NO: 1), and a reaction buffer. This reaction cell (31) of the first hollow fiber reactor (20a) may be in fluid communication with a supply chamber (32) holding a first-step supply solution (22) containing nucleotides (NTPs), a reaction buffer which may contain components necessary to catalyze and drive mRNA synthesis as described herein or assisted herein, and a pyrophosphatase that catalyzes the hydrolysis of pyrophosphate to inorganic phosphate. In this embodiment, the first-step supply solution (22) may be circulated in a counterflow compared to the first-step reaction mixture (21). In this embodiment, the supply chamber (32) and the reaction cell (33) may be configured individually or collectively for continuous or batch inflow and outflow of components as outlined herein.

[0050] As described above, the partitioning and counterflow of the first-stage feed solution (22) and the first-stage reaction mixture (21) generate a gradient so that the free NT from the first-stage feed solution (22) can be drawn into the reaction cell (31) as it passes through the feed chamber (32), where it can react with the components of the first-stage reaction mixture (21) so that RNAP can associate with a DNA template having a target sequence encoding the target mRNA (14) and enzymatically catalyze its incorporation into the target mRNA nucleotide that forms the a of NT. This reaction cycle can be carried out for a predetermined period (preferably 3-4 hours) or until a certain amount of mRNA is produced in the reaction cell (31). The consumed first-stage feed solution (23) can be extracted from the feed chamber (32) during or after the cycle is completed.

[0051] In this embodiment, the second-step reaction mixture (24) containing a poly(A) tail-less mRNA transcript and a 5' cap may be extracted from the reaction cell (32) of the first hollow fiber bioreactor (20a), and the newly synthesized mRNA may be introduced into a mixed cell (33) diluted with a high-salt buffer, preferably in a 1:5 ratio. Dilution and addition of the high-salt buffer produce a high-concentration RNA solution, inactivating RNAP, and further adjustment of the buffer conditions enables the enzymatic activity of poly(A) polymerase in the second-step poly(A) tailing step.

[0052] In particular, to produce mature mRNA prepared for efficient translation by ribosomes, two key modifications (the 5' cap structure and the poly(A) tail) must be present. The m7G cap structure consists of 7-methylguanosine triphosphate ligated to the 5' end of mRNA via a 5'→5' triphosphate bond (m7G cap). The m7G cap (also known as the Cap0 structure) is essential for most protein translation in vivo. The m7G cap also protects mature mRNA from degradation, enables a regulated degradation mechanism, enhances preRNA splicing, and directs nuclear export. In vivo, the Cap0 structure can be further modified to the Cap1 structure by adding a methyl group at the 2'O position of the mRNA's start nucleotide. 2'O methylation in the Cap1 structure helps mRNA evade innate immune responses in vivo and is particularly important for mRNA produced for therapeutic applications. More specifically, 5' capping is the first step in co-transcriptional premRNA processing, and in many eukaryotes, the capping mechanism is directly linked to the phosphorylated C-terminal domain of RNAP. The biosynthesis of the Cap0 structure requires three consecutive enzymatic activities: hydrolysis of the 5' triphosphate terminus of the nascent transcript to diphosphate by RNA triphosphatase; capping of the diphosphate with GMP by RNA guanylyltransferase using GTP as a substrate and GMP covalently bound to the lysine site as an intermediate; and finally, methylation of the 5' guanine base at the N7 position by RNA methyltransferase (MT).

[0053] Referring again to Figures 5A-B, the modification reaction mixture (25) may be introduced into the second step reaction mixture (24) in the mixing cell (33), or alternatively, into the second hollow fiber reactor (20b). As described above, the modification reaction mixture (25) may optionally contain an enzyme for 5' capping, as well as a specific enzyme for poly(A) tailing of the mRNA transcript. Additional enzymes may be added to initiate polyadenylation and extend the pol(A) tail. The second step reaction mixture (24) may contain in some amounts one or more of the following: - mRNA triphosphatase enzyme, -RNA guanylyltransferase enzyme, - mRNA methyltransferase enzyme, -Poly(A) polymerase enzyme, -Polyadenylation initiation enzyme, and - Polyadenylation elongation enzyme.

[0054] In a preferred embodiment, the second-step reaction mixture (24) may contain in some amounts one or more of the following: - mRNA triphothphatase enzyme based on the amino acid sequence of Sequence ID No. 2, - RNA guanylyltransferase enzyme based on the amino acid sequence of Sequence ID No. 3, - mRNA cap guanine-N7 methyltransferase enzyme based on the amino acid sequence of SEQ ID NO: 4, -Poly(A) polymerase enzyme based on the amino acid sequence of Sequence ID No. 5, -Polyadenylation initiator enzymes based on the amino acid sequence of SEQ ID NO: 6, and - A polyadenylation elongation enzyme based on the amino acid sequence of Sequence ID No. 7.

[0055] In this configuration, Cap1-producing methyltransferase (SEQ ID NO: 4) recognizes and binds to Cap0-GTP on the mRNA transcript, generating Cap1 methylation. Poly(A) polymerase recognizes and binds to Cap1-methyltransferase, forming a complex, and the enzymes begin to generate poly(A) tails so that they work cooperatively as a selective tool to ensure that all poly(A)-tailed RNA is also capped. In particular, the five enzymes required for 5' capping of mRNA transcripts may alternatively be added to the first-step supply solution (22) and contained in the first-step reaction mixture (21) containing the corresponding capping components, so that mRNA synthesis and 5' capping are coupled within the first hollow fiber reactor (20a), as described below.

[0056] Referring again to Figures 5A-B, the reaction mixture (24) of the mRNA transcript described above in the second step may also be supplied in a dilution buffer in a mixing cell (33) or, alternatively, to a second hollow fiber reactor (20b). This fresh solution is then introduced into the second bioreactor and preferably into the reaction cell (31) of the second hollow fiber reactor (20b), which has a supply chamber (32) containing additional components such as nucleotide triphosphates (e.g., ATP and GTP) and S-adenosylmethionine (SAM) required for poly(A) tailing and 5' capping, respectively. As described above, these components may be placed in the supply chamber (32) that forms a gradient with the reaction cell so that they pass through a porous barrier, such as the hollow fiber barrier (34) described herein, enter the reaction cell (31), and act as substrates for 5' capping and poly(A) tailing of the mRNA transcript.

[0057] During one or more capping and poly(A) tailing cycles, mRNA is capped and poly(A) tailed. The consumed modification reaction mixture (26) can be extracted from the hollow fiber reactor (20b), and the mRNA concentrate (27) from the reaction cell (31) of the second hollow fiber reactor (20b) can be extracted and introduced into a nucleotide removal device (28) to remove any free nucleotides and form a purified mRNA (29) output. In this embodiment, the mRNA concentrate (27) from the reaction cell (31) of the second hollow fiber reactor (20b) can be diluted in a high-salt buffer to allow slow binding of the poly(A) tailed RNA to the polydT resin. Other components pass over the resin and are recovered. In certain embodiments, recombinant enzymes from the reaction mixture are captured by affinity resin in a column and then washed over the polydT resin. The reaction mixture is thoroughly washed on polydT resin, and after the poly(A)-modified RNA has bound to the resin, it is washed with a high-salt buffer. After washing, the vaporized poly(A)-modified RNA is released from the resin with distillate water. The final product of the multi-step in vitro production system is concentrated capped and poly(A)-tailed RNA in distilled water (29), which can be subjected to further processing, such as encapsulation in lipid nanoparticles (LNPs) for therapeutic applications.

[0058] Therefore, in other preferred embodiments, the target mRNA(14) is purified or isolated mRNA. The terms “purified mRNA” or “isolated mRNA,” as used herein, should be understood as mRNA that has higher purity after certain purification steps (e.g., alcohol purification, and e.g., HPLC, TFF, and other polynucleotide precipitation steps) than the starting material (e.g., mRNA transcribed in vitro in a continuous flow bioreactor (1) or batch-feed reaction chamber (10)). Typical impurities that are essentially absent in purified mRNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription (e.g., RNA polymerase, RNase), BSA, pyrophosphatase, restriction endonuclease, DNase), spermidine, immature RNA sequences, RNA fragments, free nucleotides (modified nucleotides, conventional NTPs, Cap analogs), plasmid DNA fragments, buffer components (HEPES, TRIS, MgCI2), etc. Other impurities (e.g., those that may originate from the fermentation procedure) include bacterial impurities (bioburden, bacterial DNA) or impurities originating from the purification procedure (organic solvents, etc.). Therefore, in this regard, it is desirable that the "purity of RNA" be as close to 100% as possible. It is also desirable for RNA purity that the amount of full-length RNA transcript be as close to 100% as possible. Accordingly, the "purified mRNA" or "isolated mRNA" used herein has a purity of 70%, 75%, 80%, 85%, and especially 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably 99% or higher. Purity can be determined, for example, by analytical HPLC, where the above percentages correspond to the ratio between the peak area of ​​the target RNA and the total area of ​​all peaks representing by-products. Alternatively, purity can be determined, for example, by analytical agarose gel electrophoresis or capillary gel electrophoresis.

[0059] As used herein, the term “dried mRNA” should be understood as mRNA that has been freeze-dried, spray-dried, or spray-freeze-dried as defined above to obtain temperature-stable dried mRNA (powder). It should also be understood that “dried mRNA” as defined herein, and “purified mRNA” or “GMP-grade mRNA” as defined herein, may have superior stability properties and improved efficiency (e.g., better translationability of mRNA in vivo).

[0060] In further embodiments, the present invention provides compositions comprising mRNA transcribed in vitro in a continuous flow bioreactor (1), or in a batch-feed reaction chamber (10) if the mRNA encodes an antigenic peptide (particularly a polyvalent COVID-19 mRNA vaccine), and at least one pharmaceutically acceptable carrier. In particular, compositions according to the present invention preferably comprise at least one mRNA as described herein, and encode at least one antigenic peptide or protein comprising, or comprising, any one of these proteins, a fragment or variant, or any one of these fragments or variants.

[0061] The compositions according to the present invention are preferably provided as pharmaceutical compositions or vaccines. A “vaccine” is typically understood to be a prophylactic or therapeutic material that provides at least one epitope of an antigen (preferably an immunogen). In some embodiments, mRNA may encode a peptide having or providing at least one epitope. The term “provided on at least an epitope” means, for example, that the vaccine contains an epitope (or an antigen containing or providing such epitope), or that the vaccine contains, for example, a molecule encoding an epitope, or an antigen containing or providing an epitope. The antigen preferably stimulates the adaptive immune system to provide an adaptive immune response. The (pharmaceutical) compositions or vaccines provided herein may further comprise at least one pharmaceutically acceptable excipient, adjuvant, or further components (e.g., additives, auxiliary substances, etc.). In a preferred embodiment, the (pharmaceutical) composition or vaccine according to the present invention may further comprise a plurality of mRNAs transcribed in vitro in a continuous flow bioreactor (1) or batch-feed reaction chamber (10), and may further comprise a polyvalent COVID-19 mRNA vaccine as described herein. According to another embodiment, the (pharmaceutical) composition or vaccine according to the present invention may comprise an adjuvant, preferably added to enhance the immunostimulatory properties of the composition. In this context, the adjuvant can be understood as any compound suitable for supporting the administration and delivery of the composition according to the present invention. Furthermore, such an adjuvant, without being bound thereto, can initiate or increase an immune response of the innate immune system, i.e., a nonspecific immune response. In other words, when administered, the composition according to the present invention typically initiates an adaptive immune response by an antigen or fragment or variant thereof as defined herein, encoded by at least one coding sequence of the mRNA of the present invention contained in the composition of the present invention. In addition, the composition according to the present invention can generate a (supportive) innate immune response by adding an adjuvant as defined herein to the composition according to the present invention.

[0062] Similar to the (pharmaceutical) compositions of the present invention, mRNA vaccine entities produced by one or more of the methods outlined above may be provided in liquid and / or dry (e.g., lyophilized) forms. They may contain further components, in particular further components that enable their pharmaceutically acceptable use. mRNA, mRNA vaccines, or their (pharmaceutical) compositions may additionally contain, for example, pharmaceutically acceptable carriers, and / or further auxiliary substances and additives, and / or adjuvants. mRNA, mRNA vaccines, or their (pharmaceutical) compositions typically comprise a safe and effective amount of mRNA as defined herein, encoding an antigenic peptide or protein, or a fragment or variant thereof, or preferably a combination of antigens as defined herein. As used herein, “therapeutic effective dose” means an amount of mRNA sufficient to significantly induce a positive immune response, preferably preventing infection with COVID-19 coronavirus. However, at the same time, “therapeutic effective dose” is an amount small enough to avoid serious side effects, i.e., enabling a reasonable relationship between benefits and risks. The determination of these limits is typically within the realm of reasonable medical judgment. With respect to the mRNA, mRNA vaccine, or (pharmaceutical) composition of the present invention, the expression “therapeutic effective dose” preferably means an amount of mRNA, mRNA vaccine, or (pharmaceutical) composition that is suitable for stimulating the adaptive immune system, in such a manner that no excessive or harmful immune response is achieved, but preferably no such immune response is achieved below a measurable level. The mRNA in such “therapeutic effective dose” of the (pharmaceutical) composition or vaccine as defined herein may further be selected depending on the type of mRNA, e.g., monocistronic, bicistronic, or multicistronic mRNA, because bicistronic, or even more so, multicistronic mRNA may result in significantly higher expression of the encoded antigen than using an equivalent amount of monocistronic mRNA.The "therapeutic effective dose" of mRNA, mRNA vaccine, or (pharmaceutical) composition thereof as defined above will further vary within the scope of the physician's knowledge and experience in relation to the specific condition being treated, as well as the age and physical condition of the patient being treated, the severity of the condition, the duration of treatment, the nature of the accompanying therapy, the specific pharmaceutically acceptable carrier used, and similar factors. The mRNA, mRNA vaccine, or (pharmaceutical) composition thereof according to the present invention may be used, in accordance with the present invention, for human and veterinary purposes as a pharmaceutical composition or as a vaccine.

[0063] In a preferred embodiment, the mRNA of the (pharmaceutical) composition, preferably the polyvalent COVID-19 mRNA vaccine or kit of parts according to the present invention, is provided in lyophilized form. Preferably, the lyophilized mRNA is reconstituted before administration, advantageously, into a suitable buffer based on an aqueous carrier, e.g., Ringer's solution (preferably Ringer's lactate solution), phosphate buffer. In a preferred embodiment, the (pharmaceutical) composition, vaccine, or kit of parts according to the present invention contains at least one, two, three, four, five, six, or more mRNAs, preferably separately provided in lyophilized form (optionally, with at least one further additive), and preferably reconstituted separately into a suitable buffer (e.g., Ringer's lactate solution) before use to allow for the individual administration of each (monocistronic) mRNA. The vaccine or (pharmaceutical) composition according to the present invention may typically contain a pharmaceutically acceptable carrier. As used herein, the expression “pharmaceutically acceptable carrier” preferably includes liquid or non-liquid based vaccines of the present invention. When the vaccine of the present invention is provided in liquid form, the carrier is water, typically pyrogen-free water, isotonic saline, or a buffer (aqueous) solution (e.g., phosphate buffer solution, citrate buffer solution, etc.). In particular, with respect to the injection of the vaccine of the present invention, water, or preferably a buffer, more preferably an aqueous buffer, may be used, which includes a sodium salt (preferably at least 50 mM sodium salt), a calcium salt (preferably at least 0.01 mM calcium salt), and optionally a potassium salt (preferably at least 3 mM potassium salt). According to a preferred embodiment, the sodium salt, calcium salt, and optionally a potassium salt may be in the form of their halides (e.g., chloride, iodide, or bromide), their hydroxides, carbonates, bicarbonates, or sulfates, etc.Examples of sodium salts include, for example, NaCl, Na, NaBr, a2C(1 / 4), NaHCCh, a2SO4; examples of optionally selected potassium salts include, for example, KCl, KI, KBr, K2CO3, KHCO3, K2SO4; and examples of calcium salts include, for example, CaCb, Cal2, CaBr2, CaCC>3, CaSC, Ca(OH)2, but are not limited to these. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred embodiment, the buffer suitable for injection purposes as defined above may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCb), and optionally potassium chloride (KCl), and in addition to the chlorides, anions may also be present. CaCb may also be substituted with another salt such as KCl. Typically, an injection buffer The salts present in the solution are at concentrations of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCb). The injection buffer can be hypertonic, isotonic, or hypotonic with respect to a particular reference medium; that is, the buffer can have a higher, the same, or lower salt content with respect to a particular reference medium, preferably the aforementioned salts at such concentrations, which does not result in cell damage due to osmosis or other concentration effects. The reference medium is blood, lymph, cytoplasm, or other body fluids, or a medium in an "in vivo" method that is a liquid, such as a general buffer or liquid, which can be used as a reference medium in an "in vitro" method. Such general buffers or liquids are known to those skilled in the art. Ringer's lactate solution is particularly preferred as the liquid base.

[0064] However, one or more compatible solid or liquid fillers or diluents, or encapsulating compounds, that are suitable for administration to humans may also be used. As used herein, the term “compatible” means that the components of the vaccine of the present invention can be mixed with the mRNA according to the present invention as defined herein, in such a manner that no interactions occur that would substantially reduce the pharmaceutically effectiveness of the vaccine of the present invention under typical conditions of use. The pharmaceutically acceptable carriers, fillers, and diluents must, of course, be sufficiently high in purity and sufficiently low in toxicity so that they are suitable for administration to a person being treated. Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or components thereof include sugars (e.g., lactose, glucose, trehalose, and sucrose), starches (e.g., corn starch or potato starch), dextrose, cellulose and its derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, etc.), tragacanth powder, malt, gelatin, animal fat, solid lubricants (e.g., stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g., peanut oil, cottonseed oil, sesame oil, olive oil, comb oil, and cocoa-derived oils, etc.), polyols (e.g., polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol, etc.), and alginic acid.

[0065] The selection of a pharmaceutically acceptable carrier is, in principle, determined by the method by which the pharmaceutical composition or vaccine according to the present invention is administered. The composition or vaccine may be administered, for example, systemically or topically. Systemic administration routes generally include transdermal, oral, and parenteral routes, including, for example, subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal injections, and / or intranasal administration routes. Topical administration routes generally include, for example, topical administration routes, but also include intradermal, transdermal, subcutaneous, or intramuscular injections, or intralesional, intracranial, intrapulmonary, intracardiac, and sublingual injections. More preferably, the composition or vaccine according to the present invention may be administered by intradermal, subcutaneous, or intramuscular routes, preferably by injection which may be needle-free and / or needle injection. Accordingly, the composition / vaccine is preferably formulated in liquid or solid form. The appropriate amount of the vaccine or composition according to the present invention to be administered can be determined by routine experiments, for example, using animal models. Such models include, but are not limited to, rabbit, sheep, mouse, rat, dog, and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, saline, or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Pharmaceutically acceptable carriers suitable for topical application include those suitable for use in lotions, creams, gels, etc. When the compositions or vaccines of the present invention are administered orally, tablets, capsules, etc., are preferred unit dose forms. Pharmaceutically acceptable carriers for preparing unit dose forms that can be used for oral administration are well known in the prior art. The selection will depend on secondary considerations such as taste, cost, and shelf life, which are not important to the purpose of the present invention and can be easily carried out by those skilled in the art.

[0066] For clarity and readability, the following scientific background information and definitions are provided. Any technical features disclosed herein may be part of each and all embodiments of the present invention. Additional definitions and explanations may be provided in the context of this disclosure.

[0067] A poly(A) sequence, also called a "3'-poly(A) tail or poly(A) sequence," is typically a long sequence of up to about 400 adenosine nucleotides, e.g., about 25 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, and most preferably about 60 to about 250, added to the 3' end of RNA. Furthermore, poly(A) sequences or poly(A) tails can be generated in vitro by enzymatic polyadenylation of RNA using, for example, a poly(A) polymerase derived from E. coli or yeast.

[0068] Polyadenylation: Polyadenylation is typically understood as the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule (e.g., immature mRNA). Polyadenylation can be induced by a so-called polyadenylation signal. This signal is preferably located within a sequence of nucleotides at the 3' end of the nucleic acid molecule, such as the RNA molecule being polyadenylated. The polyadenylation signal typically contains a hexamer consisting of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, preferably hexamer sequences, are also possible. Polyadenylation typically occurs during the processing of premRNA (also called immature mRNA). Typically, RNA maturation (from premRNA to mature mRNA) includes a polyadenylation step.

[0069] 5' cap structure: The 5' cap is typically a modified nucleotide (cap analogue), particularly a guanine nucleotide, that is added to the 5' end of an mRNA molecule. Preferably, the 5' cap is added using a 5'-5'-triphosphate bond (also known as m7GpppN). Further examples of 5' cap structures include glyceryl, inverted deoxydebase residues (or moieties), ',5' methylene nucleotide, l-(β-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, α-nucleotide, modified base nucleotide, threopentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5'-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted debase moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted debase moieties, 1,4-butanediol phosphate, 3'-phosphoromic acid, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothio acid, phosphorodithio acid, or crosslinked or non-crosslinked methylphosphonic acid moieties. These modified 5' cap structures can be used in the context of the present invention to modify the mRNA sequence of the composition of the present invention. Further modified 5' cap structures that can be used in the context of the present invention include CAP1 (additional methylation of ribose on the nucleotide adjacent to m7GpppN), CAP2 (additional methylation of ribose on the second nucleotide downstream of m7GpppN), CAP3 (additional methylation of ribose on the third nucleotide downstream of m7GpppN), CAP4 (additional methylation of ribose on the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphothioeate-modified ARCA), inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0070] Vaccines: Vaccines are typically understood to be preventive or therapeutic materials that provide at least one antigen or antigenic function. The antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response. In the context of the present invention, the antigen-providing mRNA is typically an mRNA having at least one open reading frame that can be translated by a cell or the organism providing the mRNA. The product of this translation is a peptide or protein that can act as an antigen, preferably as an immunogen. The product may also be a fusion protein composed of multiple immunogens, for example, a fusion protein consisting of two or more epitopes, peptides, or proteins derived from the same or different viral proteins, where the epitopes, peptides, or proteins may be linked by linker sequences.

[0071] Adjuvant Components: In its broadest sense, an adjuvant or adjuvant component is typically a drug or composition that can modify (e.g., enhance) the effectiveness of other agents, such as drugs or vaccines (e.g., pharmacological or immunological). Conventionally, in the context of the present invention, this term refers to a compound or composition that functions as a carrier or auxiliary substance for immunogens and / or other pharmaceutically active compounds. This should be interpreted broadly and refers to a wide range of substances that can increase the immunogenicity of an antigen incorporated into or co-administered with the adjuvant in question. In the context of the present invention, an adjuvant preferably enhances the specific immunogenic effect of the activator of the present invention. Typically, “adjuvant” or “adjuvant component” have the same meaning and can be used interchangeably. Adjuvants can be divided into, for example, immunostimulants, antigen delivery systems, or further combinations thereof. In the context of the present invention, adjuvants such as mRNA vaccines and immunostimulatory RNA (isRNA) outlined herein may be pharmaceutical compositions.

[0072] As used herein, the terms “expression” or “expression of a coding sequence” (e.g., gene or transgene) refer to the process by which encoded information in nucleic acid transcription units (e.g., including genomic DNA or cDNA) is translated into operational, non-operational, or structural parts of a cell, often including protein synthesis. Gene expression can be influenced by external signals (e.g., exposure of cells, tissues, or organisms to drugs that increase or decrease gene expression). Gene expression can also be regulated at any point in the DNA-to-RNA and protein pathways. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediate molecules (e.g., mRNA), or through the activation, inactivation, compartmentalization, or degradation of specific protein molecules after they are produced, or a combination thereof. Gene expression can be measured at the RNA level or at the protein level by any method known in the art, including, but not limited to, Northern blotting, RT-PCR, Western blotting, or in vitro, Insights, or in vivo protein activity assays.

[0073] The terms “nucleic acid” or “nucleic acid molecule” include single-stranded and double-stranded forms of DNA, single-stranded forms of RNA, and double-stranded forms of RNA (dsRNA). The terms “nucleotide sequence” or “nucleic acid sequence” refer to both the sense and antisense strands of nucleic acids, either as individual single-stranded or double-stranded molecules. The term “ribonucleic acid” (RNA) includes iRNA (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (microRNA), hpRNA (hairpin RNA), tRNA (transfer RNA) (whether charged or discharged with the corresponding acetylated amino acid), and cRNA (complementary RNA). The term “deoxyribonucleic acid” (DNA) includes cDNA, genomic DNA, and DNA-RNA hybrids. The terms “nucleic acid segment” and “nucleotide sequence segment,” or more generally “segment,” will be understood by those skilled in the art as functional terms encompassing both genome sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller, manipulated nucleotide sequences (those that code for or can be adapted to code for peptides, polypeptides, or proteins).

[0074] The terms “gene” or “sequence” refer to a coding region operably linked to a suitable regulatory sequence that can regulate the expression of a gene product (e.g., polypeptide or functional RNA) in some way. A gene includes the untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) before (upstream) and after (downstream) the coding region (open reading frame, ORF), and, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). As used herein, the term “structural gene” is intended to mean a DNA sequence that is transcribed into mRNA and then translated into an amino acid sequence characteristic of a particular polypeptide. Note that any reference to an SEQ ID NO or sequence specifically includes that sequence and all corresponding sequences that correspond to its first sequence. For example, with respect to any identified amino acid sequence, it specifically includes all compatible nucleotide (DNA and RNA) sequences that produce that amino acid sequence or protein, and vice versa.

[0075] Nucleic acid molecules may contain either or both naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. Nucleic acid molecules may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution with one or more analogues of naturally occurring nucleotides, internucleotide modifications: e.g., uncharged bonds: e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc., charged bonds: e.g., phosphorothioates, phosphorodithioates, etc., side chain portions: e.g., peptides, intercalators: e.g., acridine, psoralens, etc., chelating agents; alkylating agents, and modifying bonds: e.g., α-anomeric nucleic acids, etc. The term “nucleic acid molecule” also includes any topological structure, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, cyclic, and padlock structures.

[0076] In certain embodiments, the present invention may encompass the in vitro production of artificial mRNA and wild-type mRNA. Artificial mRNA (sequence) can typically be understood as an mRNA molecule that does not exist in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to their individual sequences (which do not occur in nature) and / or due to other modifications (e.g., structural modifications of nucleotides that do not occur in nature). Typically, an artificial mRNA molecule may be designed and / or produced by a genetic engineering method to correspond to a desired artificial sequence (heterogeneous sequence) of nucleotides. In this context, the artificial sequence is usually a sequence that cannot exist in nature, i.e., at least one nucleotide differs from the wild-type sequence. The term “wild-type” can be understood as a sequence that exists in nature. Furthermore, the term “artificial nucleic acid molecule” is not limited to “a single molecule” but is typically understood to include an ensemble of identical molecules. Thus, it may refer to multiple identical molecules contained in an aliquot.

[0077] In certain embodiments, the present invention may typically encompass the in vitro production of bicistronic / multicistronic mRNA: mRNA that may have two or more (bicistronic) or (multicistronic) open reading frames (ORFs) (coding regions or coding sequences). In this context, an open reading frame is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such mRNA yields two (bicistronic) or more (multicistronic) different translation products (provided the ORFs are not identical). In the case of expression in eukaryotes, such mRNA may, for example, contain an internal ribosome entry site (IRES) sequence.

[0078] In one embodiment, in vitro-produced mRNA is configured to be translated in a host organism (e.g., a mammal or human subject requiring it) to form a peptide. A peptide is a polymer of amino acid monomers. Typically, the monomers are linked by peptide bonds. The term "peptide" does not limit the length of the amino acid polymer chain. In some embodiments of the present invention, a peptide may contain, for example, fewer than 50 monomer units. Longer peptides, also called polypeptides, typically have 50 to 600 monomer units, more specifically 50 to 300 monomer units.

[0079] In one embodiment, the in vitro method described herein can produce stabilized polynucleotides, preferably stabilized mRNAs, which typically exhibit modifications that increase resistance to in vivo degradation (e.g., degradation by exonucleases or endonucleases) and / or ex vivo degradation (e.g., manufacturing processes before vaccine administration, e.g., during the preparation of the vaccine solution to be administered). RNA stabilization can be achieved, for example, by providing a 5' cap structure, a poly(A) tail, or any other UTR modification. This can also be achieved by chemical modification or modification of the G / C content of the nucleic acid. Various other methods are known in the art and are assumed in the context of the present invention.

[0080] "Pharmaceutical composition" may include the vaccine of the present invention and a drug (e.g., a carrier) that can be typically used in the pharmaceutical composition or vaccine to facilitate the administration of the pharmaceutical composition or components of the vaccine to an individual.

[0081] As used herein, “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase, and Thermus aquaticus (Taq) DNA polymerase. “RNA polymerase” catalyzes the polymerization of ribonucleotides. The DNA polymerases in the aforementioned examples are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase is an example of an RNA-dependent DNA polymerase, including viral polymerases encoded by retroviruses. Known examples of RNA polymerases ("RNAP") include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase. The aforementioned examples of RNA polymerases are also known as DNA-dependent RNA polymerases. The polymerase activity of any of the above enzymes can be determined by means well known in the art.

[0082] The terms “approximately” or “about” mean that one or more values, such as concentration, length, molecular weight, pH, time frame, temperature, pressure, or volume, are within a statistically significant range. Such values ​​or ranges may be within one order of magnitude of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The acceptable variation encompassed by “approximately” or “about” depends on the specific system under study. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise stated.

[0083] The enumeration of value ranges in this specification is intended to serve merely as a simplified reference to each distinct value contained within the range, including the endpoint boundaries defining the range, and each distinct value is invoked herein as if it were individually enumerated herein, unless otherwise indicated herein.

[0084] Sequence List Sequence ID 1 AA / DNA RNA polymerase T7 bacteriophage MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQNLKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEML IESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTK GLLTLAKGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTDTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLRLDILESDFAFA sequence number 2 amino acid mRNA_triPase [insert] 1 1Note: Add the name of the organism from which mRNA tripase originates. YRNVPIWAQKWKPTIKALQSINVKDLKIDPSFLNIIPDDLTKSVQDWVYATIYSIAPELRSFIELEMKFGVIIDAKGPDRVNPPVSSQCVFTELDAHLTPNIDASLFKELSKYIRGISEVTENTGKFSIIESQTRDSVYRVGLSTQRPRFLRM STDIKTGRVGQFIEKRHVAQLLLYSPKDSYDVKISLNLELPVPDNDPPEKYKSQSPISERTKDRVSYIHNDSCTRIDITKVENHNQNSKSRQSETTHEEVELEINTPALLNAFDNITNDSKEYASLIRTFLNNGTIIRRKLSSLSYEIFEGSKKVM Sequence ID 3 amino acid mRNA guanilyltransferase Chlorella virus MVPPTINTGKNITTERAVLTLNGLQIKLHKVVGESRDDIVAKMKDLAMDDHKFPRLPGPNPVSIERKDFEKLKQNKYVVSEKTDGIRFMMFFTRVFGFKVCTIIDRAMTVYLLPFKNIPRVLFQGSIFDGELCVDIVEKKFAFVLFDAVVVSGVTVSQMDLASRF FAMKRSLKEFKNVPEEDPAILRYKEWIPLEHPTIIKDHLKKANAIYHTDGLIIMSVDEPVIYGRNFNLFKLKPGTHHTIDFIIMSEDGTIGIFDPNLRKNVPVGKLDGYYNKGSIVECGFADGTWKYIQGRSDKNQANDRLTYEKTLLNIEENITIDELLDLFKWE Sequence ID 4 amino acid mRNA cap guanine-N7 methyltransferase Encephalitozoon cuniculi MEGKKEEIREHYNSIRERGRESRQRSKTINIRNANNFIKACLIRLYTKRGDSVLDLGCGKGGDLLKYERAGIGEYYGVDIAEVSINDARVARNMKRRFKVFFRAQDSYGRHMDLGKEFDVISSQFSFHYAFSTSESLDIA QRNIARHLRPGGYFIMTVPSRDVILERYKQGRMSNDFYKIELEKMEDVPMESVREYRFTLLDSVNNCIEYFVDFTRMVDGFKRLGLSLVERKGFIDFYEDEGRRNPELSKKMGLGCLTREESEVVGIYEVVVFRKLVPESDA Sequence ID 5 amino acid Poly(A) polymerase Saccharomyces cerevisiae MSSQKVFGITGPVSTVGATAAENKLNDSLIQELKKEGSFETEQETANRVQVLKILQELAQRFVYEVSKKKNMSDGMARDAGGKIFTYGSYRLGVHGPGSDIDTLVVVPKHVTREDFFTVFDSLLRERKELDEIA PVPDAFVPIIKIKFSGISIDLICARLDQPQVPLSLTLSDKNLLRNLDEKDLRALNGTRVTDEILELVPKPNVFRIALRAIKLWAQRRAVYANIFGFPGGVAWAMLVARICQLYPNACSAVILNRFFIILSEWNWP QPVILKPIEDGPLQVRVWNPKIYAQDRSHRMPVITPAYPSMCATHNITESTKKVILQEFVRGVQITNDIFSNKKSWANLFEKNDFFFRYKFYLEITAYTRGSDEQHLKWSGLVESKVRLLVMKLEVLAGIKIAHP FTKPFESSYCCPTEDDYEMIQDKYGSHKTETALNALKLVTDENKEEESIKDAPKAYLSTMYIGLDFNIENKKEKVDIHIPCTEFVNLCRSFNEDYGDHKVFNLALRFVKGYDLPDEVFDENEKRPSKKSKRKNLE Sequence ID 6 amino acid VP39 Vaccinia virus MDVVSLDKPFMYFEEIDNELDYEPESANEVAKKLPYQGQLKLLLGELFFLSKLQRHGILDGATVVYIGSAPGTHIRYLRDHFYNLGVIIKWMLIDGRHHDPILNGLRDVTLVTRFVDEEYLRSIKKQLHPSKIILISDVRSKRGGNEPSTADLLSNYALQNVMISI LNPVASSLKWRCPFPDQWIKDFYIPHGNKMLQPFAPSYSAEMRLLSIYTGENMRLTRVTKSDAVNYEKKMYYLNKIVRNKVVVNFDYPNQEYDYFHMYFMLRTVYCNKTFPTTKAKVLFLQQSIFRFLNIPTTSTEKVSHEPIQRKISSKNSMSKNRNSKRSVRSNK Sequence ID 7 amino acid VP55 Vaccinia virus MNRNPDQNTLPNITLKIIETYLGRVPSVNEYHMLKLQARNIQKITVFNKDIFVSLVKKNKKRFFSDVNTSASEIKDRILSYFSKQTQTYNIGKLFTIIELQSVLVTTYTDILGVLTIKA PNVISSKISYNVTSMEELARDMLNSMNVAVIDKAKVMGRHNVSSLVKNVNKLMEEYLRRHNKSCICYGSYSLYLINPNIRYGDIDILQTNSRTFLIDLAFLIKFITGNNIILSKIPYLRN YMVIKDENDNHIIDSFNIRQDTMNVVPKIFIDNIYIVDPTFQLLNMIKMFSQIDRLEDLSKDPEKFNARMATMLEYVRYTHGIVFDGKRNNMPMKCIIDENNRIVTVTTKDYFSFKKCLV YLDENVLSSDILDLNADTSCDFESVTNSVYLIHDNIMYTYFSNTILLSDKGKVHEISARGLCAHILLYQMLTSGEYKQCLSDLLNSMMNRDKIPIYSHTERDKKPGRHGFINIEKDIIVF

Claims

1. A continuous flow recombination system for in vitro production of messenger RNA (mRNA) polynucleotides, - A continuous flow bioreactor, - At least one continuous flow reaction chamber configured to hold an input reaction mixture having a DNA template, and - Having at least one continuous flow conduit configured to circulate the supply solution and to be in fluid communication with the continuous flow reaction chamber through a series of conduit openings that form a concentration gradient between the input reaction mixture and the supply solution, - A continuous flow bioreactor comprising the input reaction mixture and the supply solution containing all the necessary components for in vitro generation of target mRNA transcribed from the DNA template, - A protein removal component configured to remove the protein fraction of the mRNA output, - A DNA removal component configured to remove the DNA fraction of the mRNA output, A continuous flow recombination system comprising: a nucleotide precipitation component configured to remove the nucleotide triphosphate (NTP) fraction of the mRNA output.

2. The system according to claim 1, wherein the DNA template includes a linear DNA template or a circular DNA template.

3. The system according to claim 1 or 2, wherein the DNA template encodes an antigenic polypeptide.

4. The system according to claim 3, wherein the antigenic polypeptide comprises an antigenic polypeptide from the COVID-19 coronavirus.

5. The input reaction mixture - A first amount of isolated RNA polymerase (RNAP) enzyme, and - A certain amount of reaction buffer, The system according to claim 1, comprising one or more components selected from the group consisting of the following.

6. The supply solution is - A first amount of isolated NTP, and - A certain amount of reaction buffer, The system according to claim 1, comprising one or more components selected from the group consisting of the following.

7. The system according to claim 1, wherein the protein removal component includes a protein affinity column configured to remove the protein fraction from the mRNA output.

8. The system according to claim 1, wherein the DNA removal component includes a DNA affinity column configured to remove the protein fraction from the mRNA output.

9. The system according to claim 1, wherein the nucleotide precipitation component includes an alcohol precipitation system configured to isolate the target mRNA from the mRNA output.

10. The system according to claim 1 or 9, comprising a system for resolubilizing or drying isolated target mRNA.

11. The system according to claim 9, wherein the isolated target mRNA includes stabilized mRNA.

12. The system according to claim 9 or 11, wherein the isolated target mRNA is a vaccine.

13. The system according to any one of claims 9, 11, or 12, comprising a system for incorporating the isolated target mRNA into a pharmaceutical composition.

14. The system according to claim 1, further comprising an input reservoir coupled with an input valve configured to enable real-time injection of a supply solution into the continuous flow conduit.

15. The system according to claim 1, further comprising an output reservoir coupled to an output valve configured to enable extraction of the mRNA output from the continuous flow reaction chamber.

16. A method for continuous flow recombination production of messenger RNA (mRNA), - A step of establishing a continuous flow bioreactor, wherein the continuous flow bioreactor is - At least one continuous flow reaction chamber configured to hold an input reaction mixture having a DNA template, and - Having at least one continuous flow conduit configured to circulate the supply solution and to be in fluid communication with the continuous flow reaction chamber through a series of conduit openings that form a concentration gradient between the input reaction mixture and the supply solution, - Establishing that the input reaction mixture and the supply solution contain all the necessary components for the in vitro generation of the target mRNA transcribed from the DNA template, - A step of circulating the supply solution through the continuous flow conduit, wherein the components of the supply solution pass through the conduit opening and there interact with the components of the input reaction mixture that synthesize the target mRNA indicated by the DNA template, - A step of extracting mRNA output containing the target mRNA from the continuous flow reaction chamber, - A step of removing the protein fraction from the mRNA output, - A step of removing the DNA fraction of the mRNA output, A method comprising the step of removing the nucleotide triphosphate (NTP) fraction of the mRNA output.

17. The method according to claim 16, wherein the DNA template includes a linear DNA template or a circular DNA template.

18. The method according to claim 16 or 17, wherein the DNA template encodes an antigenic polypeptide.

19. The method according to claim 18, wherein the antigenic polypeptide comprises an antigenic polypeptide from COVID-19 coronavirus.

20. The input reaction mixture - A first amount of isolated RNA polymerase (RNAP) enzyme, and - A certain amount of reaction buffer, The method according to claim 16, comprising one or more components selected from the group consisting of the following.

21. The supply solution is - A first amount of isolated NTP, and - A certain amount of reaction buffer, The method according to claim 16, comprising one or more components selected from the group consisting of the following.

22. The method according to claim 16, wherein the step of removing the protein fraction from the mRNA output includes removing the protein fraction from the mRNA output by passing it through a protein affinity column configured to remove the protein fraction from the mRNA output.

23. The method according to claim 16, wherein the step of removing the DNA fraction from the mRNA output includes removing the DNA fraction from the mRNA output by passing it through a DNA affinity column configured to remove the DNA fraction from the mRNA output.

24. The method according to claim 16, wherein the step of removing the NTP fraction from the mRNA output includes the step of precipitating the NTP fraction from the mRNA output.

25. The method according to claim 16 or 24, further comprising the steps of isolating a target mRNA and resolubilizing or drying the isolated target mRNA.

26. The method according to claim 16 or 24, comprising the step of isolating a target mRNA, wherein the isolated target mRNA includes stabilized mRNA.

27. The method according to claim 24 or 26, comprising the step of isolating a target mRNA, wherein the isolated target mRNA is a vaccine.

28. The method according to any one of claims 24, 26, or 27, further comprising the steps of isolating a target mRNA and incorporating the isolated target mRNA into a pharmaceutical composition.

29. The method according to claim 16, further comprising the step of injecting the supply solution from the input reservoir into the continuous flow conduit.

30. The method according to claim 16, further comprising the step of extracting the mRNA output from the continuous flow reaction chamber to an output reservoir.

31. The method according to claim 16, wherein the output reservoir includes a DNA extraction component, a protein extraction component, or an NTP extraction component.

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