Single-pot in vitro transcription of synthetic capped mRNA

Abstract

The present invention concerns the field of optimized mRNA synthesis and stability. Specifically, the invention relates to a method and kit for a single-pot in vitro transcription of synthetic capped mRNA.

Description

[0001] P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0002] “Single-pot in vitro transcription of synthetic capped mRNA”

[0003] FIELD OF THE INVENTION

[0004] The present invention concerns the field of optimized mRNA synthesis and stability. Specifically, the invention relates to a method and kit for a single-pot in vitro transcription of synthetic capped mRNA.

[0005] STATE OF THE ART

[0006] In recent years, mRNA-based therapeutics have emerged as a rapidly expanding class of biologies with the potential to encode any protein directly in patients, offering promising treatments for a wide array of diseases. This innovative approach harnesses the body’s own cellular machinery to produce therapeutic proteins, enabling personalized medicine at an unprecedented scale.

[0007] One of the most significant advantages of mRNA therapeutics is their ability to be swiftly developed and deployed. This agility stems from the capability to synthesize RNA in vitro using recombinant enzymes, bypassing the lengthy and complex processes typically associated with traditional cell culture methods. As a result, mRNA platforms can be designed and scaled up more efficiently, which is particularly valuable in urgent situations such as pandemics or emerging health crises.

[0008] Numerous platforms for large-scale mRNA production have been established, each offering unique advantages. However, despite the apparent simplicity and speed of mRNA production through these existing systems, several challenges remain. Key bottlenecks, including high consumable costs and the technical complexity of the processes involved, continue to hinder the widespread adoption and manufacturing of mRNA biologies on a global scale.

[0009] One particular challenge in the mRNA manufacturing process is the 5’ capping of the mRNA transcript, which is crucial for the stability and translational efficacy of the final product. While co-transcriptional cap analogues have been identified and applied successfully, their high costs limit their widespread utilization in manufacturing. As a result, the industry still relies heavily on a double enzymatic reaction strategy that combines in vitro transcription with subsequent enzymatic capping. P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0010] Although this dual-step method offers certain advantages, such as greater control over the capping process and the potential for higher yields, it is also regarded as a complex and expensive procedure. This approach necessitates the use of additional bioreactors and extensive purification steps, which can further complicate the manufacturing workflow and contribute to increased costs. Consequently, addressing these challenges is critical for the advancement of mRNA therapeutics, as optimizing the production process will be essential for maximizing their accessibility and effectiveness in treating diseases.

[0011] There exists a need to enhance the efficiency and yield of mRNA synthesis processes by simplifying the workflow to minimize the number of operational steps. Additionally, there is a requirement to reduce the overall manufacturing costs, including those associated with consumables, to facilitate broader accessibility and scalability of mRNA therapeutics.

[0012] SUMMARY OF THE INVENTION

[0013] Herein is described a novel “single pot” capping method and kit that utilizes T7 RNA polymerase, and the vaccinia or faustovirus capping system to synthesize and cap mRNA in a single pot reaction, thus eliminating any additional purification step.

[0014] In particular, an integrated reaction buffer that supports both enzymatic capping and in vitro transcription processes was surprisingly identified, enabling one-pot, two-step capped mRNA synthesis.

[0015] In a first aspect the invention therefore describes a method for single-pot in vitro transcription of synthetic capped mRNA, said method comprising the steps of: a. obtaining a template DNA for mRNA transcription; b. contacting the template DNA of step a. with a first mixture, said first mixture comprising:

[0016] - a buffer consisting of Tris-HCI, MgCh, DTT and KCI;

[0017] - oligonucleotide triphosphates (ATP, CTP. GTP and ipUTP); and

[0018] - water; P024156WO-01 Notarbartolo & Gervasi S.p.A. c. contacting the resulting mixture of step b. with a second mixture, said second mixture comprising:

[0019] - a T7 RNA polymerase;

[0020] - an RNAse Inhibitor; and

[0021] - a Pyrophosphatase; d. contacting the resulting mixture of step c. with a third mixture, said third mixture comprising:

[0022] - guanosine triphosphate (GTP);

[0023] - a capping enzyme, preferably Vaccinia or Faustovirus;

[0024] - a mRNA Cap 2'-O-Methyltransferase; and

[0025] - S-adenosylmethionine (SAM); e. performing in vitro transcription and capping reaction to obtain a capped mRNA.

[0026] In a second aspect the invention herein described relates to a kit for a single-pot in vitro transcription of synthetic capped mRNA reaction comprising:

[0027] - a buffer consisting of Tris-HCI, MgCh, DTT and KCI;

[0028] - oligonucleotide triphosphates (ATP, CTP. GTP and ipUTP);

[0029] - a capping enzyme, preferably Vaccinia or Faustovirus;

[0030] - a T7 RNA polymerase;

[0031] - a mRNA Cap 2'-O-Methyltransferase;

[0032] - S-adenosylmethionine (SAM);

[0033] - an RNAse Inhibitor;

[0034] - a Pyrophosphatase;

[0035] - water; and

[0036] - instructions for use in the method as herein disclosed.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The invention is described in detail below with reference to the Figures, specifically: Figure 1 : Schematization of the classical mRNA manufacturing process.

[0039] Figure 2: Pareto Chart reporting the effects of different amounts of chemicals in the Reaction Buffer. A Represents the concentration of Tris-HCI (mM); B represents the P024156WO-01 Notarbartolo & Gervasi S.p.A. concentration of MgCL (mM); C represents the concentration of DTT (mM) and D represents the concentrations of KCI (mM). AA represents the contribution of the quadratic term A and AB, AD and AC represent contribution of the relevant first order interactions between variables AB, AD and AC represent the combination of the aforementioned chemicals.

[0040] Figure 3 (A-D) Graphic representation of the results: counter and surface plots for the buffer component optimization E: Graphic representation of the mathematical model generated using design of experiments.

[0041] Figure 4: Single pot “Optimum” reaction delivers high yield of 5’-capped EGFP mRNA

[0042] (A). EGFP mRNA yield after in-vitro transcription performed in “Optimum” single pot reaction with Vaccinia Capping Enzyme (“Optimum”), II step reaction buffer using Vaccinia Capping Enzyme (Standard II Step) and reaction without capping (No CAP).

[0043] (B) EGFP expression in Hek293 cells transfected with EGFP mRNA obtained from “Optimum” single pot reaction with Vaccinia Capping Enzyme, II step reaction buffer (Standard II Step) using Vaccinia Capping Enzyme and without capping. (C) percentage of Hek293 EGFP-positive cells transfected with mRNA obtained from “Optimum” single pot reaction with Vaccinia Capping Enzyme, II step reaction buffer (Standard II Step) and without capping. Data represents mean of three independent experiments.

[0044] Figure 5: mRNA synthesized using “Optimum” single step reaction is 5’ capped. mRNA obtained using single pot reaction showed higher capping efficiency than the II step reaction (Standard II step). Low capping efficiency was observed in material obtained performing reactions without capping (negative control). Data represents mean of three different experiments.

[0045] Figure 6: Single pot “Optimum” reaction delivers high yield of 5’capped spCas9 mRNA. (A) spCas9 mRNA yield after in vitro transcription performed in “Optimum” single pot reaction with Vaccinia Capping Enzyme (“Optimum”), II step reaction buffer using Vaccinia Capping Enzyme (Standard II step) and without capping (no CAP). (B) spCas9 mRNA obtained using single pot reaction (“Optimum”) showed higher capping efficiency than the Standard II step reaction (Standard II Step). Low capping efficiency P024156WO-01 Notarbartolo & Gervasi S.p.A. was observed in material obtained performing reactions without capping (No CAP, negative control). Data represents mean of three different experiments.

[0046] Figure 7 Single pot “Optimum” reaction delivers high yield of 5’ -capped EGFP mRNA using Faustovirus capping Enzyme (FCE). (A) EGFP mRNA yield after in-vitro transcription performed in “Optimum” single pot reaction with Faustovirus Capping Enzyme and reaction without capping (No CAP). (B) EGFP expression in Hek293 cells transfected with EGFP mRNA obtained from “Optimum” single pot reaction with Faustovirus Capping Enzyme and without capping (no CAP). (C) Percentage of Hek293 EGFP-positive cells transfected with mRNA obtained from “Optimum” single pot reaction with Faustovirus Capping Enzyme and without capping (No CAP). (D) EGFP mRNA obtained using single pot reaction and Faustovirus capping enzyme showed high capping efficiency. Low capping efficiency was observed in material obtained performing reactions without capping (No CAP, negative control). Data represents mean of three different experiments.

[0047] The characteristics and advantages of the invention will be clear from the detailed description which follows and from the Experimental section which shows the results which have led to its definition.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] The growing popularity of mRNA as a therapeutic candidate has sustained the great hope to combat a wide range of incurable diseases.

[0050] In the last few years, rapid advances in biotechnology and molecular medicine have enabled the production of almost any functional protein / peptide in the human body by introducing mRNA as a vaccine or therapeutic agent.

[0051] The production process of mRNA therapeutics begins with the design of a DNA template for subsequent in vitro transcription (IVT).

[0052] Usually, DNA template is in the form of plasmid DNA (pDNA), however PCR products or synthetic double-stranded oligonucleotides could be also considered as valid alternatives.

[0053] Typically, the DNA template should include the following elements: a promoter P024156WO-01 Notarbartolo & Gervasi S.p.A. sequence, the gene of interest (GOI), 5' and 3' untranslated regions (UTRs), a poly(A) tail. After DNA template preparation and linearization, mRNA manufacturing process can start by using the steps represented in Figure 1 .

[0054] There is a pressing need to optimize mRNA synthesis processes by increasing efficiency and yield while minimizing operational complexity. Furthermore, reducing overall manufacturing costs and the expense of consumables is essential to ensure the scalability and accessibility of mRNA therapeutics for broader clinical application. In a first aspect the invention describes a method for single-pot in vitro transcription of synthetic capped mRNA, also referred to herein as “Optimum”, said method comprising the steps of: a. obtaining a template DNA for mRNA transcription; b. contacting the template DNA of step a. with a first mixture, said first mixture comprising:

[0055] - a buffer consisting of Tris-HCI, MgCh, DTT and KCI;

[0056] - oligonucleotide triphosphates (ATP, CTP. GTP and iplITP); and

[0057] - water; c. contacting the resulting mixture of step b. with a second mixture, said second mixture comprising:

[0058] - a T7 RNA polymerase;

[0059] - an RNAse Inhibitor; and

[0060] - a Pyrophosphatase; d. contacting the resulting mixture of step c. with a third mixture, said third mixture comprising:

[0061] - guanosine triphosphate (GTP);

[0062] - a capping enzyme, preferably Vaccinia (VCE) or Faustovirus (FCE);

[0063] - a mRNA Cap 2'-O-Methyltransferase; and

[0064] - S-adenosylmethionine (SAM); e. performing in vitro transcription and capping reaction to obtain a capped mRNA. P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0065] The invention also describes a method for single-pot in vitro transcription of synthetic capped mRNA, said method consisting of the steps of: a. obtaining a template DNA for mRNA transcription; b. contacting the template DNA of step a. with a first mixture, said first mixture comprising:

[0066] - a buffer consisting of Tris-HCI, MgCh, DTT and KCI;

[0067] - oligonucleotide triphosphates (ATP, CTP, GTP and iplITP); and

[0068] - water; c. contacting the resulting mixture of step b. with a second mixture, said second mixture comprising:

[0069] - a T7 RNA polymerase;

[0070] - an RNAse Inhibitor; and

[0071] - a Pyrophosphatase; d. contacting the resulting mixture of step c. with a third mixture, said third mixture comprising:

[0072] - guanosine triphosphate (GTP);

[0073] - a capping enzyme, preferably Vaccinia or Faustovirus;

[0074] - a mRNA Cap 2'-O-Methyltransferase; and

[0075] - S-adenosylmethionine (SAM); e. performing in vitro transcription and capping reaction to obtain a capped mRNA.

[0076] In a preferred embodiment, in the method for single-pot in vitro transcription of synthetic capped mRNA according to the invention, said Tris-HCI is in the range from 35 to 75 mM, said MgCh is in the range from 25 to 45 mM, said DTT is in the range from 0.5 to 2.5 mM and said KCI is in the range from 0.01 to 1 mM, more preferably said Tris-HCI is in the amount of 53 mM, said MgCh is in the amount of 32 mM, said DTT is in the amount of 1 mM and said KCI is in the amount of 0.01 mM.

[0077] In a further preferred embodiment, in the method for single-pot in vitro transcription of synthetic capped mRNA according to the invention, the oligonucleotides in the first P024156WO-01 Notarbartolo & Gervasi S.p.A. mixture are in the range from 2.5 to 20 mM, preferably ATP, CTP, GTP and iplITP are in the amount of 5 mM.

[0078] In a further preferred embodiment, in the method for single-pot in vitro transcription of synthetic capped mRNA according to the invention, the Vaccinia capping enzyme is in the range from 500 to 2000 U / mL, the T7 RNA polymerase is in the range from 5000 and 15000 U / mL, the mRNA Cap 2'-O-Methyltransferase is in the range from 2500 to 7500 U / mL, the S-adenosylmethionine (SAM) is in the range from 0.1 to 0.5 mM, the RNAse Inhibitor is in the range from 500 to 2000 U / mL and the Pyrophosphatase is in the range from 10 to 50 U / mL; more preferably the Vaccinia capping enzyme is in the amount of 1000 U / mL, the T7 RNA polymerase is in the amount of 8000 U / mL, the mRNA Cap 2'-O-Methyltransferase is in the amount of 5000 U / mL, the S- adenosylmethionine (SAM) is in the amount of 0.2 mM, the RNAse Inhibitor is in the amount of 1000 U / mL and the Pyrophosphatase is in the amount of 20 U / mL.

[0079] In a still further preferred embodiment, in the method for single-pot in vitro transcription of synthetic capped mRNA of the present invention:

[0080] - step b. is performed at 37°C in a range from 15 minutes to 1 hour, preferably for 30 minutes;

[0081] - step c. is performed at 37°C in a range from 2 to 4 hours, preferably for 3 hours; and

[0082] - step d. is performed at 37°C in a range from 1 to 3 hours, preferably for 2 hours.

[0083] Data obtained clearly revealed that, using the single pot RNA manufacturing platform, the present invention allows to synthetize large amounts of mRNA with higher capping efficiency as compared with standard two step enzymatic reaction, thus paving the way towards a more sustainable, simple and streamlined mRNA manufacturing process.

[0084] In a second aspect the invention herein described relates to a kit for a single-pot in vitro transcription of synthetic capped mRNA reaction comprising:

[0085] - a buffer consisting of Tris-HCI, MgCL, DTT and KCI;

[0086] - oligonucleotide triphosphates (ATP, CTP, GTP and ipUTP);

[0087] - a capping enzyme, preferably Vaccinia or Faustovirus enzyme;

[0088] - a T7 RNA polymerase;

[0089] - a mRNA Cap 2'-O-Methyltransferase; P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0090] - S-adenosylmethionine (SAM);

[0091] - an RNAse Inhibitor;

[0092] - a Pyrophosphatase;

[0093] - water; and

[0094] - instructions for use in the method as herein disclosed.

[0095] One or more components of a kit may be included in one container for a single step reaction, or one or more components may be contained in one container, but separated from other components for sequential use or parallel use.

[0096] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0097] EXPERIMENTAL SECTION

[0098] Reference is now made to the following experimental section, which together with the above description illustrates some embodiments of the invention.

[0099] In order to optimize the reaction buffer compositions for the one-step in vitro transcription and enzyme mediated capping of the method and kit of the present invention, various components and concentrations were assessed using a Design of Experiment approach.

[0100] In particular, the contribution of four chemical compounds were analyzed in a Screening Experiment: tris-hydrochloride (Tris-HCI), magnesium chloride (MgCL), DL- dithiothreitol (DTT), and potassium chloride (KCI).

[0101] In addition, for each of these, various concentrations were tested in order to identify the minimum and the maximum concentrations of each for obtaining an adequate reaction yield, capping reaction yield and protein expression.

[0102] To start the investigation, mRNA encoding for Emerald Green Fluorescent Protein (EGFP) was used for the preliminary tests and a scale of 50 pL was set as a starting point.

[0103] In particular, 9 different conditions were tested in triplicate carrying out 27 reactions. In P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0104] Figure 2 it can be appreciated that only two out of the four tested chemical compounds have an impact on the in vitro transcription (IVT) reaction yield and final protein expression.

[0105] In particular, MgCh concentration appeared to be important for T7 polymerase activity, whereas Tris-HCI content was significant for the vaccinia capping system performance. The contribution of the remaining buffer components tested in these first experiments was negligible (Figure 2).

[0106] Having identified the two significant factors in the reaction buffer composition, it was decided to use the Surface Response Methodology in order to study different concentrations of MgCh and Tris-HCI.

[0107] Results obtained from the experiment are summarized in Figure 3.

[0108] It is worth noting that a mathematical model was generated using this strategy and it was possible to estimate the ideal reaction conditions to optimize both mRNA yield and capping efficiency in a single pot reaction.

[0109] Moreover, the results indicated that it is also possible to adjust the concentration of MgCh and Tris-HCI in the buffer in order to obtain more specific results in terms of reaction yield or capping efficiency.

[0110] The “Optimum” buffer formulation was calculated (53 mM Tris-HCI and 32 mM MgCh), and it was decided to scale the reaction up.

[0111] In particular, a new experiment moving from a reaction scale of 50 pL to 200 pL was designed. In this new experiment, only the optimal condition (53 mM Tris-HCI and 32 mM MgCh) was tested to produce EGFP mRNA.

[0112] In addition, this experiment was performed in triplicate and both reaction yield and fluorescence expression in cells transfected with the aforementioned mRNA were measured.

[0113] As positive and negative controls in this new set of tests a reaction performed without capping, a two-step capping reaction and a reaction carried out using EZ-CAP as a co- transcriptional capping reagent were included.

[0114] Results obtained showed that the EGFP mRNA synthetized using the Optimum reaction was in the expected range of concentration (from 3 to 6 mg / mL) and P024156WO-01 Notarbartolo & Gervasi S.p.A. comparable with the control reactions (Figure 4A).

[0115] Afterwards, the obtained mRNA was used to transfect Hek293 cells and EGFP expression was evaluated 72 hours post-transfection using flow cytometry.

[0116] As shown in Figure 4B EGFP expression (measured as mean of fluorescence intensity) in Hek293 cells transfected with the “Optimum” mRNA of the invention was higher than what was observed transfecting cells with mRNA obtained performing 2 reactions. mRNA without capping worked and negative control respectively.

[0117] To strengthen the data, the percentage of EGFP-positive cells was also measured. As highlighted in Figure 4C, all the conditions tested, except the negative control, showed a percentage of positive cells close to 90% thus indicating a high transfection efficiency.

[0118] Overall, data obtained indicated that using the optimized single pot reaction RNA production can be scaled up without losing performance.

[0119] Since the assessment of protein expression is only an indirect measurement of capping efficiency, a technique based on two-step enzymatic degradation of uncapped mRNA to measure the percentage of residual capped material was used. In brief, RNA 5' Polyphosphatase was used for the removal of phosphates from 5’ -triphosphorylated RNA (uncapped mRNA). Then, 5’ monophosphorylated RNA was digested with Terminator™ 5’-Phosphate-Dependent Exonuclease.

[0120] In these conditions uncapped RNA will be degraded, while capped RNA will persist. After the double enzymatic digestion, RNA was loaded on an agarose gel of electrophoresis and bands were quantified densitometrically. Capping efficiency was calculated as quantity(digested) / quantity(control) x 100%.

[0121] Results obtained clearly showed a higher capping efficiency (>50%) in mRNA produced with the optimized condition as well as using co-transcriptional capping. Significant reduction on capping efficiency was observed in samples obtained from VCE II step reaction or without capping (Figure 5). These results further corroborated the observations on EGFP protein expression.

[0122] To strengthen the results, a second mRNA using a different template was produced. In particular, spCas9 was selected. This second set of experiments, using Cas9, P024156WO-01 Notarbartolo & Gervasi S.p.A. allowed to test longer constructs assessing the possibility to apply the single pot reaction to mRNA that could be more challenging to be produced.

[0123] By testing Cas9 production the same concentration of Tris-HCI and MgCh derived from EGFP experiments (as indicated above) were used and IVT reactions at 200 pL scale were performed.

[0124] Results obtained from preliminary experiments showed that, using the optimized reaction, a quantity of mRNA similar to that obtained in standard control conditions was obtained (Figure 6A).

[0125] Furthermore, some assessments of the capping efficiency on Cas9 mRNA were performed. Data obtained showed a higher percentage of capped mRNA derived from “Optimum” reaction than in standard two step conditions (Figure 6B).

[0126] The results indicate that the optimized reaction of the invention (Optimum) could be universal and the “Optimum” reaction conditions can be applied independently from mRNA lengths.

[0127] MATERIALS AND METHODS

[0128] Buffer preparation

[0129] The optimal buffer components and concentrations were designed by carrying out experiments in triplicate in the same plate the same day.

[0130] A series of 27 in vitro transcription reactions were carried out synthesizing EGFP mRNA at microscale (50 pL / reaction).

[0131] Table 1 below shows the optimal concentrations of the four components of the Optimum buffer.

[0132] Table 1 : P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0133] Reactions were prepared modifying the concentrations of Tris-HCI, MgCh, DTT and KCI, while maintaining the concentration of the remaining components of the reaction as described below in Table 2.

[0134] Table 2: mRNA generated from all the IVT reactions was quantified using Qubit Fluorimeter (Thermofisher Scientific) with Qubit RNA BR assay Kit (Thermofisher Scientific) and purified using Monarch RNA Cleanup kit 500 pg (New England Biolabs).

[0135] Purified mRNA was transfected in Hek293 cells using jet messenger reagent and following manufacturer’s instructions.

[0136] Fluorescence was recorded using Sony SH800 cytofluorimeter (Sony Biotechnology). mRNA yield, Percentage of EGFP-positive cells and mean of fluorescence intensity were the variables considered.

[0137] A second study was then designed to optimize the concentration of T ris-HCI and MgCh in the buffer. Therefore, the Response Surfaces Methodology was used. A Full Factorial Central Composite Design was generated based on the results of the first Screening study.

[0138] Following this model, 27 in vitro transcription reactions were carried out synthesizing EGFP mRNA at microscale (50 pL). Reactions were prepared changing the concentrations of Tris-HCI and MgCh, while maintaining the concentration of the remaining components as described in the Table 2 above. mRNA generated from all P024156WO-01 Notarbartolo & Gervasi S.p.A. the IVT reactions was quantified using Qubit Fluorimeter (Thermofisher Scientific) with Qubit RNA BR assay Kit (Thermofisher scientific) and purified using Monarch RNA Cleanup kit 500 pg (New England Biolabs). Purified mRNA was transfected in HEK293 cells using jet messenger reagent and following manufacturer’s instructions. Fluorescence was recorded using Sony SH800 cytofluorimeter (Sony Biotechnology). mRNA yield, Percentage of EGFP-positive cells and mean of fluorescence intensity were the variables considered.

[0139] In Vitro transcription Reaction.

[0140] Plasmid DNA (pDNA) containing a T7 promoter, a segmented polyA tail and a cutting site for the enzyme BspQI (New England Biolabs, NEB) was used as template for mRNA in-vitro transcription. Prior to RNA in vitro transcription, pDNAs encoding Emerald Green Fluorescent Protein (pmEGFP) and CRISPR-Associated Protein 9 (pmCAS9), were linearized using enzyme, according to the manufacturer’s instructions.

[0141] Cleanup of the linearized pDNAs was carried out using Wizard DNA Clean-up system (Promega). 50 pg / mL (Range 25-100 pg / mL) of linearized and purified pDNAs were used to perform IVT reactions.

[0142] In particular, three different IVT reactions were prepared as detailed below:

[0143] Reaction 1 Optimum (invention):

[0144] Tris(hydroxymethyl)aminomethane chlorhydrate (Tris-HCI) (Sigma Aldrich) 53 mM;

[0145] Magnesium Chloride (MgCL) (Sigma Aldrich) 32 mM;

[0146] Dithiothreitol (DTT) (Sigma Aldrich) 1 mM;

[0147] Adenosine Triphosphate (ATP) (Thermo Fisher Scientific) 5 mM;

[0148] Guanosine Triphosphate (GTP) (Thermo Fisher Scientific) 10 mM;

[0149] Cytidine triphosphate (CTP) (Thermo Fisher Scientific) 5 mM;

[0150] N1 -Methylpseudo-Uridine, (ipUTP) (Thermo Fisher Scientific) 5 mM;

[0151] T7 RNA polymerase (New England Biolabs) 8000 U / mL;

[0152] Murine RNAse Inhibitor (New England Biolabs) 1000 U / mL;

[0153] Inorganic Pyrophosphatase (New England Biolabs) 20 U / mL;

[0154] Vaccinia Capping Enzyme (VCE) (New England Biolabs) 1000 U / mL; P024156WO-01 Notarbartolo & Gervasi S.p.A. mRNA Cap 2'-O-Methyltransferase (New England Biolabs) 5000 U / mL;

[0155] S-adenosylmethionine (SAM) (New England Biolabs) 0.2 mM.

[0156] Reaction 2 Standard 2 Step (control)

[0157] RNA polymerase Buffer 1X

[0158] Magnesium Chloride (MgCL) (Sigma Aldrich) 40 mM;

[0159] Dithiothreitol (DTT) 10 mM;

[0160] Adenosine Triphosphate (ATP) (Thermo Fisher Scientific) 5 mM;

[0161] Guanosine Triphosphate (GTP) (Thermo Fisher Scientific) 10 mM;

[0162] Cytidine triphosphate (CTP) (Thermo Fisher Scientific) 5 mM;

[0163] N1 -Methylpseudo-Uridine, (ipUTP) (Thermo Fisher Scientific) 5 mM;

[0164] T7 RNA polymerase (New England Biolabs) 8000 U / mL;

[0165] Murine RNAse Inhibithor (New England Biolabs) 1000 U / mL;

[0166] Inorganic Pyrophosphatase (New England Biolabs) 20 U / mL;

[0167] Vaccinia Capping Enzyme (VCE) (New England Biolabs) 1000 U / mL; mRNA Cap 2'-O-Methyltransferase (New England Biolabs) 5000 U / mL;

[0168] S-adenosylmethionine (SAM) (New England Biolabs) 0.2 mM.

[0169] Reaction 3 No Cap (negative control)

[0170] RNA polymerase Buffer 1X

[0171] Magnesium Chloride (MgCL) (Sigma Aldrich) 40 mM;

[0172] Ditiothreitol (DTT) 10 mM;

[0173] Adenosine Triphosphate (ATP) (Thermo Fisher Scientific) 5 mM;

[0174] Guonosine Triphosphate (GTP) (Thermo Fisher Scientific) 5 mM;

[0175] Cytidine triphosphate (CTP) (Thermo Fisher Scientific) 5 mM;

[0176] N1 -Methylpseudo-Uridine, (ipUTP) (Thermo Fisher Scientific) 5 mM;

[0177] T7 RNA polymerase (New England Biolabs) 8000 U / mL;

[0178] Murine RNAse Inhibithor (New England Biolabs) 1000 U / mL;

[0179] Inorganic Pyrophosphatase (New England Biolabs) 20 U / mL.

[0180] To prepare the Optimum reaction non-enzymatic components were mixed and preheated at +37 °C for 30 minutes. P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0181] Subsequently, T7 RNA polymerase, Murine RNAse Inhibitor and Inorganic Pyrophosphatase were added to each reaction and tubes were incubated at +37 °C for 3 hours using a pre-heated thermoblock.

[0182] After three hours 5 mM GTP, Vaccinia Capping Enzyme (VCE) mRNA Cap 2'-O- Methyltransferase (New England Biolabs) and S-adenosylmethionine (SAM) (New England Biolabs) were added to Reaction 1 (Optimum) and tubes were incubated at +37 °C for 2 hours.

[0183] Conversely Reaction 2 (standard 2 step) was subjected to a buffer exchange using Monarch RNA Cleanup kit 500 pg (New England Biolabs) following manufacturer’s instructions. Cleaned mRNA from Reaction 2 was then used as substrate for enzymatic capping reaction adding 5 mM GTP, Vaccinia Capping Enzyme (VCE) mRNA Cap 2'- O-Methyltransferase (New England Biolabs) and S-adenosylmethionine (SAM) (New England Biolabs). Reaction 2 was incubated for 2 hours at +37°C.

[0184] Reaction 3 was left at +37°C for 2 hours without any further enzymatic addition.

[0185] After 2 hours RNA concentration in each reaction was determined using Qubit fluorometer (Thermofisher Scientific) and Qubit RNA BR Assay kit (Thermofisher scientific).

[0186] Same procedure was used to prepare both EGFP and Cas9 mRNAs up to 200 pL scale.

[0187] Reaction scale up

[0188] Reaction scale up was performed up to 5 mL using same procedure described above except that reaction mixture was prepared in 5 mL bags (FlexBoy 2D bags) (Sartorius) and incubated at +37°C in rocking motion with an angle of 7 ° (Range 6-8°) and speed set at 40 rpm (Range 35-50 rpm).

[0189] The same Optimum reaction was performed using 1000 U / mL (Range 550-2000) of Faustovirus capping Enzyme S17 (FCE) (Takara).

[0190] Deccaping procedure:

[0191] For determination of capping efficiency of IVT reactions, a method based on enzymatic degradation of uncapped mRNA was used. P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0192] 2 pg of mRNA from aforementioned IVT reactions, for both EGFP and Cas9, were purified using Monarch RNA Cleaup kit 500 pg (New England Biolabs). Purified mRNAs were incubated with RNA 5' Polyphosphatase in order to remove phosphates from 5’ -triphosphorylated RNA (uncapped mRNA).

[0193] Mixture of 1x RNA 5' Polyphosphatase Reaction Buffer (LGC Biosearch), 1 ll / pl RNase Inhibitor (New England Biolabs), 2 pg of mRNA, 0.5 U / pL of RNA 5' Polyphosphatase (LGC Biosearch) and nuclease-free water added up to 20 pl was incubated at +37°C for 60 min.

[0194] After first enzymatic reaction, a buffer exchange was performed using Monarch RNA Cleanup Kit 500 pg. 5’ monophosphorylated RNA was digested with Terminator™ 5’- Phosphate-Dependent Exonuclease. 1 x Terminator Reaction Buffer A (LGC Biosearch) 1 U / pL RNase Inhibitor (New England Biolabs), 0.05 U / pL of Terminator™ 5’-Phosphate- Dependent Exonuclease (LGC Biosearch) and nuclease free water was added to reaction mixture from first step up to 40 pL reaction volume.

[0195] Reaction was incubated at +30°C for 1 hour and quenched with 5mM EDTA pH 8. mRNA concentration in reaction was determined loading samples on 1 % agarose gel stained with Gel Red (Biotum). Residual capped mRNA was determined by densitometric analysis of the bands obtained. mRNA Transfection

[0196] HEK293 cells were seeded 24 hours before transfection in 24-well plates at a cell density of 100.000 cells / well in 0.5 mL of Iscove's Modified Dulbecco's Medium (IMDM) (ThermoFisher Scientific) supplemented with 10% Foetal Bovine serum (FBS) (Thermofisher Scientific).

[0197] After 24 hours of incubation at +37 °C and 5% CO2, cells were transfected with 0.5 pg / well (Range 0.4-0.6 pg / well) of purified EGFP mRNA using Jetmessenger (Polyplus) following manufacturer instructions. Transfected cells were incubated for 72 hours at +37 °C and 5% CO2 and then analyzed by flow cytometry using Sony SH 800 (Sony Biotechnology). Both percentage of EGFP-positive cells as well as mean of fluorescence intensity were recorded on samples ranging from 20000 to 50000 cells for each sample. P024156WO-01 Notarbartolo & Gervasi S.p.A.

[0198] From the above description and the above-noted examples, the advantage attained by the method and kit herein described and obtained according to the present invention are apparent.

Claims

P024156WO-01 Notarbartolo & Gervasi S.p.A.CLAIMS1 . A method for single-pot in vitro transcription of synthetic capped mRNA, said method comprising the steps of: a. obtaining a template DNA for mRNA transcription; b. contacting the template DNA of step a. with a first mixture, said first mixture comprising:- a buffer consisting of Tris-HCI, MgCL, DTT and KCI;- oligonucleotide triphosphates (ATP, CTP, GTP and iplITP); and- water; c. contacting the resulting mixture of step b. with a second mixture, said second mixture comprising:- a T7 RNA polymerase;- an RNAse Inhibitor; and- a Pyrophosphatase; d. contacting the resulting mixture of step c. with a third mixture, said third mixture comprising:- guanosine triphosphate (GTP);- a Vaccinia capping enzyme;- a mRNA Cap 2'-O-Methyltransferase; and- S-adenosylmethionine (SAM); e. performing in vitro transcription and capping reaction to obtain a capped mRNA.

2. The method for single-pot in vitro transcription of synthetic capped mRNA according to claim 1 , wherein said Tris-HCI is in the range from 35 to 75 mM, said MgCL is in the range from 25 to 45 mM, said DTT is in the range from 0.5 to 2.5 mM and said KCI is in the range from 0.01 to 1 mM.

3. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 or 2, wherein said Tris-HCI is in the amount of 53 mM, saidP024156WO-01 Notarbartolo & Gervasi S.p.A.MgCh is in the amount of 32 mM, said DTT is in the amount of 1 mM and said KCI is in the amount of 0.01 mM.

4. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 to 3, wherein the oligonucleotides in said first mixture are in the range from 2.5 to 20 mM, preferably ATP, CTP, GTP and iplITP are in the amount of 5 mM.

5. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 to 4, wherein the Vaccinia capping enzyme is in the range from 500 to 2000 U / mL, the T7 RNA polymerase is in the range from 5000 and 15000 U / mL, the mRNA Cap 2'-O-Methyltransferase is in the range from 2500 to 7500 U / mL, the S- adenosylmethionine (SAM) is in the range from 0.1 to 0.5 mM, the RNAse Inhibitor is in the range from 500 to 2000 U / mL and the Pyrophosphatase is in the range from 10 to 50 U / mL.

6. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 to 5, wherein the Vaccinia capping enzyme is in the amount of 1000 U / mL, the T7 RNA polymerase is in the amount of 8000 U / mL, the mRNA Cap 2'-O-Methyltransferase is in the amount of 5000 U / mL, the S-adenosylmethionine (SAM) is in the amount of 0.2 mM, the RNAse Inhibitor is in the amount of 1000 U / mL and the Pyrophosphatase is in the amount of 20 U / mL.

7. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 to 6, wherein step b. is performed at 37°C for 30 minutes.

8. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 to 7, wherein step c. is performed at 37°C for 3 hours.P024156WO-01 Notarbartolo & Gervasi S.p.A.

9. The method for single-pot in vitro transcription of synthetic capped mRNA according to any one of claims 1 to 8, wherein step d. is performed at 37°C for 2 hours.

10. A kit for a single-pot in vitro transcription of synthetic capped mRNA reaction comprising:- a buffer consisting of Tris-HCI, MgCh, DTT and KCI;- oligonucleotide triphosphates (ATP, CTP, GTP and ipUTP);- a Vaccinia capping enzyme;- a T7 RNA polymerase;- a mRNA Cap 2'-O-Methyltransferase;- S-adenosylmethionine (SAM);- an RNAse Inhibitor;- a Pyrophosphatase;- water; and- instructions for use in the method according to claims 1 to 9.

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