Enzyme-based system for producing messenger RNA with increased transfection efficiency

Abstract

To provide an optimized method for producing a clinical production grade enzyme for use in therapeutic mRNA synthesis.SOLUTION: A method of producing a protein for use in in vitro transcription (IVT) of messenger RNA (mRNA), wherein the protein is assessed for purity and effectiveness by the efficiency with which mRNA synthetically derived therefrom is subsequently transfected into cells and the encoded protein is produced.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 244,990, filed September 16, 2021. The entire disclosure of U.S. Provisional Patent Application No. 63 / 244,990 is incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as an ST.26XML file. The file entitled "PAT005268_Sequence_Listing.xml" has a size of 61,000 bytes and was created on September 12, 2022. The information contained in the ST.26XML file is incorporated herein by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

[0003] The present disclosure relates to the production of synthetic messenger RNA (mRNA) via in vitro transcription (IVT) using a novel and cost-effective method of enzyme production. [Background technology]

[0004] The recent success of messenger RNA-based vaccines in eradicating the COVID-19 pandemic has demonstrated the efficacy of RNA therapeutics and emphasized the need for efficient methods for mRNA production. Optimizing mRNA production conditions will aim to achieve better yields of the core proteins involved in mRNA synthesis, increased mRNA purity, and improved assays to assess mRNA quantity and quality. Optimized proteins for mRNA synthesis are considered beneficial due to increased mRNA production efficiency and the cost of the proteins themselves, which are commercially available. Key components of the strategy include optimized plasmid constructs for recombinant expression of core proteins used in IVT, optimized protein production conditions, and optimized protein purification protocols. Hornblower et al. (2015) "Minding your caps and tails—considerations for functional mRNA synthesis," a New England Biolabs White Paper, describes an exemplary in vitro mRNA synthesis scheme. Optimized methods for producing clinical-grade enzymes for use in therapeutic mRNA synthesis remain needed. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein are devices, systems, and methods for in vitro transcription of mRNA, a process optimized to produce effective and inexpensive batches of clinically relevant agents for human diseases. The methods described herein relate to improved production of core proteins involved in in vitro transcription (IVT). The improvements include optimization of expression vector sequences used in bacterial protein production of the protein. The methods further include optimizing production conditions, such as time and temperature. Finally, the methods include a means for assessing mRNA quality, whereby mRNA transfection and expression of the encoded protein are evaluated relative to the quality of the protein components used in the IVT process.

[0006] In one embodiment, the optimized bacterial expression vector contains nucleotide sequences encoding three IVT proteins that are individually cloned into the expression vector. In one aspect, a first DNA sequence or insert is cloned into a first bacterial expression vector, the first insert comprising a gene sequence for T7 RNA polymerase. The insert further comprises an arabinose promoter sequence upstream of the polymerase sequence. In one aspect, the insert consists of SEQ ID NO: 19. In one aspect, the codon-optimized gene sequence for T7 RNA polymerase is SEQ ID NO: 22.

[0007] In another embodiment, a second gene sequence is cloned into a second bacterial expression vector, wherein the insert comprises gene sequences for the D1 and D12 subunits of vaccinia virus capping enzyme (VVCE). The insert further comprises an arabinose promoter sequence upstream of the D1 and D12 subunits. The insert further comprises a ribosome binding site (RBS) immediately downstream of the arabinose promoter. In one embodiment, the insert consists of SEQ ID NO: 20. In one embodiment, the codon-optimized gene sequence for the D1 subunit of VVCE is SEQ ID NO: 23. In one embodiment, the codon-optimized gene sequence for the D12 subunit of VVCE is SEQ ID NO: 24.

[0008] In another embodiment, a third insert is cloned into a third bacterial expression vector, the insert comprising a gene sequence for poly(A) polymerase. The insert further comprises an arabinose promoter sequence upstream of the poly(A) polymerase sequence. In one embodiment, the insert consists of SEQ ID NO: 21. In one embodiment, the gene sequence for poly(A) polymerase is SEQ ID NO: 25.

[0009] In another embodiment, the bacterium contains an expression vector for protein expression and subsequent purification. In some cases, conditions for protein growth and purification are enabled by modifying the insert cloned into the expression plasmid. Such modifications include placing an arabinose promoter upstream of the protein-encoding nucleotide sequence. Another modification includes placing two arabinose promoters upstream of VVCE, one upstream of the D1 subunit, and a second upstream of the D12 subunit. A further modification includes the addition of a nucleic acid sequence encoding a His tag, where a polymeric histidine is encoded in-frame with the protein sequence and placed at either the N- or C-terminus, allowing the protein to be column purified after fermentation. A further modification includes the addition of a Tobacco Etch Virus protease sequence, allowing the His tag sequence to be proteolytically removed following protein purification.

[0010] In one embodiment, purified proteins are added sequentially to an in vitro transcription reaction. First, purified T7 RNA polymerase is added to a reaction containing linearized plasmid DNA encoding an mRNA transcript of interest. RNA is then transcribed from the linearized DNA through the action of T7 RNA polymerase. The second reaction contains the RNA transcript from the first reaction, S-adenosylmethionine, and purified VVCE. The RNA transcript is then N-terminally capped to produce an uncapped mRNA. The third reaction contains the capped mRNA from the VVCE reaction, purified poly(A) polymerase, and ATP, which adds a polyadenylated tail to the 3' end of the capped mRNA transcript.

[0011] Various objects, features, aspects and advantages will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings, in which like numerals represent like elements. [Brief explanation of the drawings]

[0012] [Figure 1] Structural features of mRNA are shown. Diagram from Vaccines (2020) 5:11. Critical quality traits that dictate mRNA expression performance are shown. Five critical quality traits are identified that indicate efficient expression of a gene of interest. Three of these traits require the use of enzymes: transcription (T7 RNA polymerase); capping (guanylyl transferase); and poly(A) tailing (poly(A) polymerase). Currently, these three core enzymes must be purchased for IVT. [Figure 2] The T7 polymerase expression construct is shown. Leaky expression from the T7-promoted system could result in deleterious protein effects and mutations in the gene sequence. Switching to a tightly regulated promoter enabled the proposed cloning of the gene. [Figure 3A] Replacing the T7 promoter with an arabinose promoter enabled growth and protein expression in BL21 DE3 Clear Coli. Optimized growth conditions are shown. Figure 3A shows the expression construct used. Leaky expression from the T7-driven system could result in adverse protein reactions and mutations in the gene sequence. Switching to a tightly regulated promoter enabled the proposed cloning of the gene. Figure 3B shows the protein gel results from a 4-hour fermentation. Figure 3C shows the protein gel results from an overnight (O / N) fermentation. In each Coomassie-stained protein gel: Lane 0, protein ladder; Lane 1, cell pellet; Lane 2, supernatant; Lane 3, flow-through; Lane 4, wash; Lane 5, wash 2; Lane 6, imidazole wash; Lane 7, elution 1; Lane 8, elution 2; Lane 9, elution 3; Lane 10, elution 4; Lane 11, elution 5; Lane 12, elution 6; Lane 13, elution 7. [Figure 3B] This is a continuation of Figure 3A. [Figure 3C] This is a continuation of Figure 3B. [Figure 4A]Demonstration of poor VVCE solubility when expressed behind the T7 promoter. Figure 4A shows the expression construct used. Protein expression in BL21 DE3 Clear Coli. Results shown on a protein gel (Figure 4B). In the Coomassie-stained protein gel: Lane 0 protein ladder; Lane 1 cell pellet; Lane 2 supernatant; Lane 3 flow-through; Lane 4 wash; Lane 5 imidazole wash; Lane 6 elution 1; Lane 7 elution 2; Lane 8 elution 3; Lane 9 elution 4; Lane 10 elution 5. Enzyme solubility was hindered when expressed behind the T7 promoter. [Figure 4B] This is a continuation of Figure 4A. [Figure 5A] Figure 5A shows the improved solubility of VVCE D1 and D12 subunits when the T7 promoter was replaced with an arabinose promoter. Figure 5A shows the expression construct used. Protein expression in BL21 DE3 Clear Coli (Figure 5C). Coomassie-stained protein gel: Lane 0, protein ladder; Lane 1, cell pellet; Lane 2, supernatant; Lane 3, flow-through; Lane 4, wash 1; Lane 5, wash 2; Lane 6, imidazole wash; Lane 7, elution 1; Lane 8, elution 2; Lane 9, elution 3; Lane 10, elution 4; Lane 11, elution 5. Enzyme solubility was hindered when expressed behind the T7 promoter. Switching to a tightly regulated promoter and slowing expression (18°C) improved solubility. [Figure 5B] This is a continuation of Figure 5A. [Figure 6A]The addition of an arabinose promoter allows for the growth and expression of insoluble poly(A) polymerase in BL21 DE3 Clear Coli. Figure 6A shows the expression construct used. In a Coomassie-stained protein gel (Figure 6B): Lane 0, protein ladder; Lane 1, cell pellet; Lane 2, supernatant; Lane 3, flow-through; Lane 4, wash 1; Lane 5, wash 2; Lane 6, imidazole wash; Lane 7, elution 1; Lane 8, elution 2; Lane 9, elution 3; Lane 10, elution 4; Lane 11, elution 5. The overexpressed protein is approximately 43 kDa. Protein expression resulted in an insoluble cleaved enzyme. [Figure 6B] This is a continuation of Figure 6A. [Figure 7A] The poly(A) polymerase gene with a UUG start codon allowed for the expression of a protein of appropriate size and good solubility. Expression in BL21 DE3 Clear Coli. Figure 7A shows the expression construct used. Figure 7B shows a Western blot using anti-His. Lane 0: protein ladder; Lane 1: cell pellet; Lane 2: supernatant; Lane 3: flow-through; Lane 4: wash 1; Lane 5: wash 2; Lane 6: imidazole wash; Lane 7: elution 1; Lane 8: elution 2; Lane 9: elution 3. Protein expression resulted in an insoluble cleaved enzyme. Addition of an unusual start codon and transcriptional regulatory sequences allowed the enzyme to be expressed properly. [Figure 7B] This is a continuation of Figure 7A. [Figure 8] Transfection-compatible mRNA production workflow. [Figure 9-1] Flow cytometry histogram analysis is shown. Performance comparison of enhanced Clear Coli expression to commercial in vitro transcriptases. Plots show detection of GFP expression in 293T cells transfected with IVT GFP-mRNA. Plots shown represent triplicates. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 10A]Performance comparison of improved Clear Coli expression to commercially available in vitro transcriptases. IVT GFP-mRNA transfection results obtained using different enzymes. Figure 10A shows the average percentage of GFP positives. Figure 10B shows the average geometric mean fluorescence intensity. In each graph, RNA samples are as follows: (1) untransfected 293t; (2) in vitro transcripts without modifications; (3) in vitro transcripts with 5' caps; (4) uncapped HiScribe transcripts with tails; (5) ARCA-capped HiScribe transcripts; (6) NEB VVCE-capped HiScribe transcripts; and (7) mRNA transcripts produced with in-house enzymes (arrows). [Figure 10B] This is a continuation of Figure 10A. DETAILED DESCRIPTION OF THE INVENTION

[0013] The disclosed components, compositions, systems, kits, and methods can be used to perform cell-free messenger RNA (mRNA) synthesis. Cell-free mRNA synthesis using in vitro transcription (IVT) utilizes an ensemble of catalytic proteins produced from the lysate of transfected bacterial cells. The purified proteins contain the essential components of the IVT reaction. Various methods exist for producing protein components for cell-free mRNA synthesis, including those described herein.

[0014] All patents and published applications identified in this specification are incorporated by reference to the same extent as if each individual patent or application was specifically and individually indicated to be incorporated by reference. In the event that a definition or use of a term in an incorporated reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0015] definition In some embodiments, numbers expressing quantities of ingredients, properties, e.g., concentrations, reaction conditions, and the like, used to describe and claim particular embodiments are understood to be modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written specification and attached claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments are approximations, the numerical values ​​set forth in the specific examples are reported precisely as feasible. The numerical values ​​set forth in some embodiments may necessarily contain certain errors resulting from the standard deviation found in their respective testing measurements.

[0016] Unless the context dictates otherwise, all ranges set forth herein should be interpreted as inclusive of their endpoints, and open-ended ranges should be interpreted as including only practicable values. Similarly, all lists of values ​​should be interpreted as including intermediate values ​​unless the context dictates otherwise.

[0017] As used herein below and throughout the claims, the meanings of "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Also, as used herein, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0018] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value within the range. Unless otherwise specified herein, each individual value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to specific embodiments herein is intended merely for clarity and does not impose a limitation on the scope of the claimed invention. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the claimed invention.

[0019] Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the modified group and the written description of all Markush groups used in the appended claims is accordingly fulfilled.

[0020] Expression vector Expression vectors containing nucleic acids encoding one or more mRNAs described herein are provided. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0021] messenger RNA When the purchased enzymes T7 RNA polymerase, VVCE, and poly(A) polymerase are replaced with in-house produced enzymes in the mRNA production process, a surprising increase in mRNA quality occurs, as determined by the expression level of the protein expressed in mRNA-transfected cells. Enzyme production involves transfection of a plasmid expression vector into bacteria, in which a gene encoding the protein of interest is operably linked to a promoter sequence that drives expression of the transfected gene. Figure 1 shows the key characteristics of mRNA.

[0022] The promoter sequence can be any promoter suitable for driving protein expression in bacteria. In a preferred embodiment, the promoter is an arabinose promoter. Further engineering of gene sequences inserted into plasmid expression vectors includes the use of tags by which proteins can be identified and / or purified. In a preferred embodiment, a gene encoding a protein of interest is linked to a sequence encoding a polymeric histidine (His) tag in frame at either the 5' or 3' end of the protein. The His tag can be separated from the gene of interest by a tobacco etch virus (TEV) protease recognition sequence, whereby TEV can be added to the protein and the His tag cleaved off.

[0023] Replacement of the T7 promoter with the arabinose promoter driving expression of T7 RNA polymerase (Figure 2 and Figures 3A-3C) was shown to allow growth of BL21 DE3 Clear Coli cells and drive heterologous protein expression therein. Increasing the fermentation time from 4 hours to overnight (18 hours) also increased protein expression. Similar replacement of the T7 promoter driving expression of the D1 and D12 subunits of VVCE with the arabinose promoter enabled expression of the subunits (Figures 4A-4B and Figures 5A-5B). Performing protein expression at 18°C ​​also improved protein expression.

[0024] The sequence for producing an IVT protein can be further modified by adding an alternative start codon to the protein-coding sequence. In a preferred embodiment, the alternative start codon is linked to the end of poly(A) polymerase. This unusual UUG start codon of poly(A) polymerase was first postulated by Cao & Sarkar (Proc. Natl. Acad. Sci. USA. Vol. 89, pp. 10380-10384, November 1992). The N-terminal sequence containing the UUG start codon (SEQ ID NO: 21) is encoded in frame immediately upstream of the lysine at amino acid position 11 with respect to the wild-type protein sequence of SEQ ID NO: 25.

[0025] The expression plasmid insert sequence for production of IVT proteins can be further modified by the use of novel promoter sequences. Surprisingly, the arabinose promoter significantly increases the solubility of VVCE, thus increasing the yield of VVCE expressed in E. coli. Also unexpectedly, because the use of a T7 promoter upstream of T7 RNA polymerase and poly(A) polymerase resulted in the inability to produce cloned expression plasmids that could be successfully propagated in bacteria using standard techniques, cloning the T7 RNA polymerase and poly(A) polymerase genes into the expression vector itself became possible by placing the arabinose promoter upstream of the start codons of both proteins.

[0026] Disclosed herein is a method for optimizing the production of T7 RNA polymerase for protein yield. After transfecting BL21 DE3 Clear Coli with the expression plasmid, the fermentation temperature was lowered to 18°C ​​and the luminescence time was extended to 18 hours. Figure 7 shows the increase in the yield of soluble T7 RNA polymerase when these fermentation conditions were applied.

[0027] Expression vectors containing nucleic acids encoding one or more mRNAs described herein are provided. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably. However, the disclosed methods and compositions are intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0028] After optimizing bacterial production of T7 RNA polymerase, VVCE, and poly(A) polymerase, and purifying and characterizing the proteins by standard methods, the proteins are applied to a series of reactions whereby linearized plasmid DNA containing a gene of interest is transcribed, capped, and poly-adenosine-tailed. The plasmid with the inserted DNA can be suitable for propagation in viruses, prokaryotes, or eukaryotes.

[0029] Template DNA is typically propagated and stored as supercoiled plasmid DNA. Plasmid DNA is linearized by the action of one or more restriction enzymes, thereby removing or excising the gene of interest from the circular plasmid DNA. Generally, unique restriction sites occur at the 5' and 3' ends of the gene of interest. The restriction sites may be identical or different. Any restriction enzyme can be used, provided it does not recognize a restriction site within the gene of interest. The linearized DNA is then isolated by column purification, although other standard methods, such as ethanol or isopropanol precipitation, may also be used. The purified linearized DNA containing the gene sequence is then isolated by size determination and quantified spectrophotometrically. 1 μg template DNA is then added to a reaction containing nucleotide triphosphates (ATP, CTP, GTP, and UTP) and purified T7 RNA polymerase. The reaction proceeds overnight at 37°C in a thermal cycler. The RNA transcripts are then column purified and quantified spectrophotometrically.

[0030] Conversion of RNA transcripts to uncapped RNA requires three sequential enzymatic steps: removal of the 5'-terminal gamma-phosphate by RNA triphosphatase activity (TPase), transfer of a GMP group to the resulting diphosphate 5'-terminus by RNA guanylyltransferase activity (GTase), and modification of the N7 amine of the guanosine cap by guanine-N7 methyltransferase activity (MTase). Vaccinia virus capping enzyme consists of D1 and D12 subunits, with all three enzymatic steps carried out by the D1 subunit. Purified and quantified RNA transcripts (10 μg) are then added to a reaction containing GTP, s-adenosylmethionine, and purified VVCE. The reaction is then allowed to proceed for 1 hour at 37°C. The capped RNA is then column purified and quantified spectrophotometrically.

[0031] Polyadenylation is the addition of a poly(A) tail to an mRNA transcript. The poly(A) tail is important for the nuclear transport, translation, and stability of mRNA. The final step in in vitro mRNA production is the addition of a poly(A) tail to the capped transcript. Capped mRNA (10 μg) transcripts are added to a reaction containing ATP and purified poly(A) polymerase, and the reaction proceeds for 1 hour at 37°C. The capped, polyadenylated mRNA transcripts are then column-purified and quantified spectrophotometrically. The purified mRNA should be used immediately or frozen at -80°C or colder.

[0032] An overview of the mRNA workflow is shown in Figure 8. The resulting mRNA can be formulated for delivery to cells (in vitro transfection) or tissues (in vivo transfection). Any suitable formulation can be used. Exemplary nanoparticle formulations are described in U.S. Patent Application No. 16 / 622,908, the disclosure of which is incorporated herein by reference. Evaluation of mRNA-encoded protein delivery and expression using standard methods is shown in Figures 9A-9F, where HEK293 cells are transfected and assessed by flow cytometry. Figures 10A and 10B show improved protein yields with mRNA produced by the methods described herein compared to commercially available RNA transcripts capped with VVCE protein from NEW ENGLAND BIOLABS®.

[0033] It will be appreciated that the disclosed technology provides many advantageous technical effects, such as increased production of IVT core proteins T7 RNA polymerase, vaccinia virus capping enzyme, and poly(A) polymerase, and increased yields of protein products derived from the mRNA transcripts produced thereby. [Example]

[0034] Preparation of the arabinose promoter insert. The pBAD-DEST49 DNA plasmid was used as a template for PCR used to amplify the arabinose promoter. The arabinose promoter was amplified using primers SEQ ID NO:1 (forward) and SEQ ID NO:2 (reverse). The amplification mixture contained (per reaction): 10 μL 5× PRIMESTAR® GXL polymerase buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng pBAD-DEST49; 1 μL PRIMESTAR® GXL polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used to amplify the arabinose insert: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 55°C for 15 seconds (×30 cycles); 68°C for 90 seconds (×30 cycles); and 68°C for 1 minute. PCR products were purified using QIAQUICK® PCR spin columns.

[0035] The purified PCR product was then used in another PCR using primers SEQ ID NOs: 3 and 4. The amplification mixture contained (per reaction): 10 μL 5×PRIMESTAR® GXL Polymerase Buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng pBAD-DEST49; 1 μL PRIMESTAR® GXL Polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used for amplification of the arabinose insert: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 60°C for 15 seconds (×30 cycles); 68°C for 90 seconds (×30 cycles); and 68°C for 1 minute. The arabinose promoter insert with Gibson Assembly handle was then gel extracted and purified using a QIAGEN® Gel Extraction Kit.

[0036] BglIII restriction digestion protocol. The pET22b lactose-inducible expression system was digested using BglIII restriction enzyme purchased from NEW ENGLAND BIOLABS®. Two separate reactions were completed for pET22b and BglIII restriction digestion as follows (per reaction): 10 μL Buffer 3.1; 3 μL BglIII restriction enzyme; 46 μL pET22b DNA; 41 μL MILLI-Q® water. Each reaction was then incubated for 2 hours at 37°C in a thermal cycler. Following incubation, 500 μL Buffer PB was added to each reaction. Buffer PB and the restriction digest mixture were added to two separate miniprep columns purchased from QIAGEN®. The columns were centrifuged at 15,000 × g for 1 minute, and the flow-through was discarded. 1 mL of Buffer PE containing ethanol was added to each column. The columns were centrifuged again at 15,000 x g for 1 minute, and the flow-through was discarded. The columns were then spun down at 15,000 x g for 2 minutes. The columns were then transferred to new microcentrifuge tubes. 50 μL of MILLI-Q® water was added to each column, and the columns were centrifuged at 15,000 x g for 2 minutes.

[0037] Xba1 restriction digestion protocol. 50 μL of purified and digested material was used in a digestion with the following protocol (per reaction): 9 μL CUTSMART® buffer; 3 μL Xba1; 50 μL BglIII digested material; and 28 μL MILLI-Q® water. The reaction was incubated for 2 hours at 37°C in a thermal cycler. Antarctic phosphatase was then added (10 μL / reaction of Antarctic phosphatase buffer & 2 μL / reaction of Antarctic phosphatase). The reaction mixture was then incubated for 1 hour at 37°C. The digested vector was gel extracted and purified using a QIAGEN® Gel Extraction and Purification Kit.

[0038] GIBSON ASSEMBLY® Protocol. 100 ng of digested and purified vector was prepared for the reaction. 70 ng of amplified and purified promoter was also prepared for the reaction. Each reaction contained only 0.1 pmol of total DNA used in GIBSON ASSEMBLY®. Vector DNA and insert DNA were mixed with MILLI-Q® water to a final volume of 10 μL. 10 μL of 2x NEB HIFI ASSEMBLY Master mix (obtained from NEW ENGLAND BIOLABS®) was then added to the reaction mixture. The prepared GIBSON ASSEMBLY® reaction was then incubated at 50°C for 15 minutes in a thermal cycler. Following the incubation period, 8 μL of the reaction mixture was then transformed using NEB5α ultracompetent cells. Transformants with the arabinose promoter cloned into the pET22b expression plasmid were used to amplify the plasmid DNA, which was then harvested using the QIAGEN® Maxi Prep purification system.

[0039] Cloning of pBM100 to generate a T7 RNA polymerase expression system. The RNA polymerase with a Gibson handle and an integrated 6xHis tag was PCR amplified using SEQ ID NOs: 5 and 6 as primers. The PCR reaction mixture contained (per reaction): 10 μL 5x PRIMESTAR® GXL Polymerase Buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng RNA polymerase DNA; and 1 μL PRIMESTAR® GXL Polymerase. PCR to amplify the RNA polymerase insert was completed as follows: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 60°C for 15 seconds (×30 cycles); 68°C for 120 seconds (×30 cycles); and 68°C for 1 minute. The completed PCR was then purified using QIAQUICK® PCR spin columns. 20 μg of pBM98 was used in a double restriction digest using the enzymes Nde1 and Nco1-HF with the following reaction recipe (per reaction): 90 μL CUTSMART® buffer; 1 μL Nco1-HF; 1 μL Nde1; 20 μg RNA polymerase DNA; and water to a final volume of 90 μL. The restriction digest was then incubated for 2 hours at 37°C in a thermal cycler. Antarctic phosphatase was then added (10 μL / reaction of Antarctic phosphatase buffer and 2 μL / reaction of Antarctic phosphatase). The reaction mixture was then incubated for 1 hour at 37°C. The digested vector was gel extracted and purified using a QIAGEN® Gel Extraction and Purification Kit.

[0040] 100 ng of digested and purified vector was prepared for the GIBSON ASSEMBLY® reaction. 126 ng of amplified and purified RNA polymerase insert was also prepared for the reaction. Less than 0.1 pmol of total DNA was used in each GIBSON ASSEMBLY® reaction. The vector DNA and insert DNA were mixed with MILLI-Q® water to a final volume of 10 μL. 10 μL of 2x NEB HIFI ASSEMBLY Master mix (obtained from NEW ENGLAND BIOLABS®) was then added to the reaction mixture. The prepared GIBSON ASSEMBLY® reaction was then incubated at 50°C for 15 minutes in a thermal cycler. Following the incubation period, 8 μL of the reaction mixture was then transformed using NEB5α ultracompetent cells. Transformants with T7 RNA polymerase cloned into an arabinose-driven expression plasmid were used to amplify the plasmid DNA, which was then harvested using the QIAGEN® Maxi Prep purification system.

[0041] Construction of a Dual Arabinose-Driven Expression Plasmid. A dual-expressing arabinose-driven system was constructed for cloning the vaccinia virus capping enzyme D1 and D12 subunits. The pET-Duet purified plasmid was digested with Xba1 using the following protocol (per reaction): 9 μL CUTSMART® buffer; 3 μL Xba1; 10 μg pET-Duet plasmid; and MILLI-Q® water to a final volume of 90 μL. The reaction was incubated at 37°C for 2 hours in a thermal cycler. Antarctic phosphatase was then added (10 μL / reaction of Antarctic phosphatase buffer & 2 μL / reaction of Antarctic phosphatase). The reaction mixture was then incubated at 37°C for 1 hour.

[0042] The digested vector was then gel extracted and purified using a QIAGEN® Gel Extraction and Purification Kit. The arabinose promoter insert was then amplified from pBM98 using SEQ ID NO:1 and SEQ ID NO:2 as primers with the following reaction mixture (per reaction): 10 μL 5×PRIMESTAR® GXL Polymerase Buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng pBM98; 1 μL PRIMESTAR® GXL Polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used for amplifying the arabinose insert: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 55°C for 15 seconds (×30 cycles); 68°C for 90 seconds (×30 cycles); and 68°C for 1 minute. The PCR product was purified using QIAQUICK® PCR spin columns.

[0043] The purified PCR product was then used in another PCR using SEQ ID NOs: 7 and 8 as primers. This PCR product was used in GIBSON ASSEMBLY® to create multiple cloning site #1 with an arabinose-promoted system. 100 ng of digested and purified vector was prepared for the GIBSON ASSEMBLY® reaction. 70 ng of amplified and purified promoter was also prepared for the reaction. Less than 0.1 pmol of total DNA was used in the GIBSON ASSEMBLY®. The vector DNA and insert DNA were mixed with MILLI-Q® water to a final volume of 10 μL. 10 μL of 2x NEB HIFI ASSEMBLY Master mix (obtained from NEW ENGLAND BIOLABS®) was then added to the reaction mixture. The prepared GIBSON ASSEMBLY® reaction was then incubated at 50°C for 15 minutes in a thermal cycler. Following an incubation period, 8 μL of the reaction mixture was then transformed using NEB5α ultracompetent cells. The transformed cells with the arabinose promoter cloned into the pET-Duet dual expression plasmid were used to amplify the plasmid DNA, which was then collected using a QIAGEN® MaxiPrep purification system. The resulting plasmid was labeled pBM122 to indicate the presence of the arabinose promoter in MCS1 and the T7 promoter in MCS2.

[0044] The newly constructed plasmid was then used to replace the downstream T7 promoter with another plasmid-driven insert. A double restriction digest was completed on the prepared plasmid using the enzymes Bsrg1-HF and Nde1 with the following reaction mixture (per reaction): 9 μL CUTSMART® buffer; 1.5 μL Bsrg1-HF; 1.5 μL Nde1; 30 μg pBM122; and MILLI-Q® water to a final volume of 90 μL.

[0045] The restriction digest was incubated at 37°C for 1 hour. Antarctic phosphatase was then added (10 μL / reaction of Antarctic phosphatase buffer & 2 μL / reaction of Antarctic phosphatase). The reaction mixture was then incubated at 37°C for 1 hour. The digested vector was then gel extracted and purified using a QIAGEN® Gel Extraction and Purification Kit. The arabinose promoter was prepared using SEQ ID NO: 1 and SEQ ID NO: 2 as primers. PCR was completed using the following protocol (per reaction): 10 μL 5x PRIMESTAR® GXL Polymerase Buffer; 4 μL DNTPs; 1 μL 10 μM Forward Primer; 1 μL 10 μM Reverse Primer; 50 ng pBM98; 1 μL PRIMESTAR® GXL Polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used for amplification of the arabinose insert: 98°C for 2 minutes; 98°C for 20 seconds (x30 cycles); 55°C for 15 seconds (x30 cycles); 68°C for 90 seconds (x30 cycles); and 68°C for 1 minute. PCR products were purified using QIAQUICK® PCR spin columns.

[0046] The purified PCR product was then used in another PCR amplification using primers SEQ ID NOs: 9 and 10. Each PCR reaction contained: 10 μL of 5×PRIMESTAR® GXL Polymerase Buffer; 4 μL of DNTPs; 1 μL of 10 μM forward primer; 1 μL of 10 μM reverse primer; 50 ng of generated PCR product; 1 μL of PRIMESTAR® GXL Polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used for amplification of the arabinose insert: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 60°C for 15 seconds (×30 cycles); 68°C for 90 seconds (×30 cycles); and 68°C for 1 minute. The PCR product was purified using QIAQUICK® PCR spin columns.

[0047] The purified PCR product was then used in another PCR using SEQ ID NOs: 9 and 10 as primers in a reaction mixture containing the following (per reaction): 10 μL 5×PRIMESTAR® GXL Polymerase Buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng of generated PCR product; 1 μL PRIMESTAR® GXL Polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used for amplification of the arabinose insert: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 60°C for 15 seconds (×30 cycles); 68°C for 90 seconds (×30 cycles); and 68°C for 1 minute. The PCR product was purified using QIAQUICK® PCR spin columns.

[0048] 100 ng of the digested and purified vector was prepared for the GIBSON ASSEMBLY® reaction. 58 ng of the amplified and purified promoter was also prepared for the reaction. Less than 0.1 pmol of total DNA was used in the GIBSON ASSEMBLY®. The vector DNA and insert DNA were mixed with MILLI-Q® water to a final volume of 10 μL. 10 μL of 2x NEB HIFI ASSEMBLY Master mix (obtained from NEW ENGLAND BIOLABS®) was then added to the reaction mixture. The prepared GIBSON ASSEMBLY® reaction was then incubated at 50°C for 1 hour in a thermal cycler. Following the incubation period, 8 μL of the reaction mixture was then transformed using NEB5α ultracompetent cells.

[0049] Transformants with the arabinose promoter cloned into the pET-Duet dual expression plasmid were used to amplify the plasmid DNA, which was then collected using the QIAGEN® MaxiPrep purification system. The resulting plasmid was labeled pBM123 for use in cloning.

[0050] Cloning of pBM127, Vaccinia Virus Capping Enzyme, in a Dual Arabinose-Facilitated Expression System. The D12 subunit was amplified using primers SEQ ID NOs: 11 and 12 for cloning into the second multiple cloning site of the arabinose-facilitated system in the following reaction mixture (per reaction): 10 μL 5×PRIMESTAR® GXL polymerase buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng D12 subunit template; 1 μL PRIMESTAR® GXL polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used for amplification of the D12 GIBSON ASSEMBLY® insert: 98°C for 2 minutes; 98°C for 20 seconds (x30 cycles); 55°C for 15 seconds (x30 cycles); 68°C for 90 seconds (x30 cycles); 55°C for 15 seconds (x30 cycles); 68°C for 120 seconds (x30 cycles); and 68°C for 1 minute. PCR products were purified using QIAQUICK® PCR spin columns.

[0051] 10 μg of pBM125 plasmid was digested using Nco1-HF restriction enzyme using the following reaction mixture (per reaction): 9 μL CUTSMART® buffer; 2 μL Nco1-HF; 10 μg pBM125; and MILLI-Q® water to a final volume of 90 μL. Incubation was completed at 37°C for 1 hour in a thermal cycler. Antarctic phosphatase was then added (10 μL / reaction of Antarctic phosphatase buffer & 2 μL / reaction of Antarctic phosphatase). The reaction mixture was then incubated at 37°C for 1 hour. The digested vector was then gel extracted and purified using a QIAGEN® Gel Extraction and Purification Kit.

[0052] 100 ng of digested and purified vector was prepared for the GIBSON ASSEMBLY® reaction. 125 ng of amplified and purified subunit was also prepared for the reaction. Less than 0.1 pmol of total DNA was used in the GIBSON ASSEMBLY®. The vector and insert DNA were mixed with MILLI-Q® water to a final volume of 10 μL. 10 μL of 2× NEB HIFI ASSEMBLY Master mix (obtained from NEW ENGLAND BIOLABS®) was then added to the reaction mixture. The prepared GIBSON ASSEMBLY® reaction was then incubated at 50°C for 15 minutes in a thermal cycler. Following the incubation period, 8 μL of the reaction mixture was then transformed using NEB5α ultracompetent cells. The transformed cells with the cloned D12 subunit were used to amplify the plasmid DNA, which was then collected using a QIAGEN® MaxiPrep purification system. The resulting DNA was labeled as pBM127 and used to transform CLEARCOLI™ competent cells for protein expression.

[0053] Cloning of pBM135 to generate an E. coli poly(A) polymerase expression system. Using SEQ ID NOs: 15 and 16 as primers, a PCR reaction mixture was prepared as follows (volume per reaction): 10 μL of 5×PRIMESTAR® GXL polymerase buffer; 4 μL of DNTPs; 1 μL of 10 μM forward primer; 1 μL of 10 μM reverse primer; 50 ng of poly(A) polymerase DNA; and 1 μL of PRIMESTAR® GXL polymerase. PCR to amplify the poly(A) polymerase insert was completed as follows: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 55°C for 15 seconds (×30 cycles); 68°C for 90 seconds (×30 cycles); and 68°C for 1 minute. The completed PCR product was then purified using a QIAQUICK® PCR spin column.

[0054] The purified PCR product was then used in another PCR using SEQ ID NOs: 17 and 18 as primers. PCR reactions were prepared as follows (per reaction): 10 μL 5×PRIMESTAR® GXL Polymerase Buffer; 4 μL DNTPs; 1 μL 10 μM forward primer; 1 μL 10 μM reverse primer; 50 ng of PCR product; 1 μL PRIMESTAR® GXL Polymerase; and MILLI-Q® water to a final volume of 50 μL. The following conditions were used to amplify the poly(A) polymerase insert: 98°C for 2 minutes; 98°C for 20 seconds (×30 cycles); 55°C for 15 seconds (×30 cycles); 68°C for 60 seconds (×30 cycles); 68°C for 1 minute. The PCR product was purified using QIAQUICK® PCR spin columns.

[0055] 20 μg of pBM98 was used in a double restriction digest using the enzymes Nde1 and Nco1-HF with the following reaction recipe (per reaction): 90 μL CUTSMART® buffer; 1 μL Nco1-HF; 1 μL Nde1; 20 μg of pBM98; and water to a final volume of 90 μL. The restriction digest was incubated for 2 hours at 37°C in a thermal cycler. Antarctic phosphatase was then added (10 μL / reaction of Antarctic phosphatase buffer & 2 μL / reaction of Antarctic phosphatase). The reaction mixture was then incubated for 1 hour at 37°C. The digested vector was gel extracted and purified using a QIAGEN® Gel Extraction and Purification Kit.

[0056] 100 ng of digested and purified vector was prepared for the GIBSON ASSEMBLY® reaction. 63 ng of amplified and purified poly(A) polymerase insert was also prepared for the reaction. Less than 0.1 pmol of total DNA was used in the GIBSON ASSEMBLY®. The vector DNA and insert DNA were mixed with MILLI-Q® water to a final volume of 10 μL. 10 μL of 2× NEB HIFI ASSEMBLY Master mix (obtained from NEW ENGLAND BIOLABS®) was then added to the reaction mixture. The prepared GIBSON ASSEMBLY® reaction was then incubated at 50°C for 15 minutes in a thermal cycler. Following the incubation period, 8 μL of the reaction mixture was then transformed using NEB5α ultracompetent cells.

[0057] Transformants with E. coli poly(A) polymerase cloned into an arabinose-driven expression plasmid were used to amplify the plasmid DNA, which was then harvested using the QIAGEN® Mini Prep purification system.

[0058] Production of RNA transcripts from linearized DNA. This procedure is used to prepare reactions for the production of in vitro RNA transcripts encoding desired genes of interest for further downstream modification. The list of equipment used in the process includes: a 96-well thermal cycler; 10x transcription buffer; micropipettes; NTPs (100 mM); 8-strip PCR tubes; inorganic pyrophosphatase; RNAse inhibitor (mouse); Not1-HF enzyme; in-house purified T7 RNA polymerase; and a QIAGEN® PCR purification kit.

[0059] To generate the in vitro transcription (IVT) template, obtain an aliquot of pRNI-GFP in preparation for restriction digestion. For all three IVT reactions, prepare 10 μg of digested template DNA using the following recipe (volumes per reaction): 5 μL CUTSMART® Buffer; 2 μL Not1-HF Restriction Enzyme; 10 μg pRNI-GFP; and nuclease-free water to a final volume of 100 μL. Add the contents to a 1.5 mL microcentrifuge tube labeled "pRNI-GFP Digest." Incubate for 2 hours in a 37°C incubator. Following incubation, remove the digested DNA and add 500 μL of Buffer PB. Transfer the mixture of Buffer PB and digested DNA to a purple QIAGEN® PCR Purification column. Centrifuge the column at maximum speed for 1 minute in a tabletop centrifuge and discard the flow-through. 750 μL of Buffer PE was applied directly to the column, followed by centrifugation at maximum speed for 1 minute. Again, the flow-through was discarded. The column was then spun at maximum speed for 3 minutes. A 1.5 mL microcentrifuge tube was labeled "digested pRNI-GFP." The column was placed in a newly labeled microcentrifuge tube. 40 μL of nuclease-free water was placed directly on the membrane. After incubation at 37°C for 2 minutes, the column was spun at maximum speed for 2 minutes. The elution fractions were analyzed using a spectrophotometer according to the manufacturer's instructions.

[0060] To prepare the IVT reaction, label a PCR tube with the following name: "IVT Reaction." Obtain the following tubes: a vial of in-house purified T7 RNA polymerase; a vial of inorganic pyrophosphatase; a vial of RNAse inhibitor (mouse); and a tube of "pRNI-GFP digest" and keep them on ice at all times. Thaw the following reagents at room temperature: 10x reaction buffer; ATP; CTP; UTP; and GTP. Add the following reagents in the following order to the labeled PCR tube: RNAse-free water (≤20 μL); 10x reaction buffer (2 μL); 100 mM ATP (2 μL); 100 mM CTP (2 μL); 100 mM GTP (2 μL); 100 mM UTP (2 μL); template DNA (1 μg); T7 RNA polymerase mix (2 μL); RNAse inhibitor (1 μL); and inorganic pyrophosphatase (2 μL). The final volume of one reaction will be 20 μL. The prepared PCR tubes were centrifuged briefly and then incubated overnight at 37°C in a thermal cycler.

[0061] Purification of IVT RNA transcripts. After overnight incubation, the tubes were removed from the thermal cycler and 100 μL of Buffer PB was added to the reaction using a filtered p200 micropipette. It is common for the reaction vessel to become cloudy after overnight incubation at this point in the process. Upon addition of Buffer PB, a translucent solid will be present, which should be mixed gently using a pipette.

[0062] The mixture was added directly to the membrane of a purple QIAGEN® PCR Purification column. The columns were centrifuged at top speed for 1 minute, and the flow-through was discarded. 750 μL of Buffer PE was added to each column and centrifuged at top speed for 1 minute. The flow-through was discarded. The columns were spun at top speed for 3 minutes. A new 1.5 mL microcentrifuge tube was labeled "In Vitro Transcripts." The columns were transferred to the labeled microcentrifuge tube, and 35 μL of RNAse-free water was added directly to the membrane. The columns were centrifuged at top speed for 2 minutes. The eluted transcripts were then analyzed in a spectrophotometer according to the manufacturer's instructions. After spectrophotometric analysis, the purified RNA transcripts should be kept on ice.

[0063] If the capping reaction cannot be completed immediately after purification, the RNA transcripts should be stored at -80 °C. For best results, frozen RNA should not be stored for more than one week.

[0064] Production of capped RNA transcripts. The following components were stored at -20°C prior to starting the procedure: 10x capping buffer, 32 mM S-adenosylmethionine (SAM), 10 mM guanosine triphosphate (GTP), RNAse inhibitor (mouse), and in-house purified vaccinia virus capping enzyme (VVCE). These were kept on ice during reaction assembly. A microcentrifuge tube was labeled "2 mM SAM." SAM was diluted 1:16 by adding 30 μL of RNAse-free water and 2 μL of the 32 mM stock to the labeled microcentrifuge tube. For highest capping efficiency, it is best to dilute SAM immediately before the reaction. Two new PCR strip tubes were labeled "VVCE Capping Reaction." Using the collected purified RNA, 10 μg of RNA was added to each labeled tube. RNAse-free water was added to each tube to a final volume of 14 μL. The RNA and water mixture was denatured in a thermal cycler at 65°C for 5 minutes. Immediately after denaturation, the RNA was placed on ice for 5 minutes. Reagents were added to each reaction in the following order (volumes per reaction): 2µL 10x capping buffer; 1µL 10mM GTP; 1µL 2mM SAM; 1µL VVCE; and 1µL RNAse inhibitor. The contents were gently mixed using a pipette and spun down. The reactions were incubated in a thermal cycler at 37°C for 1 hour.

[0065] After incubation at 37°C, the tubes were removed from the thermal cycler, and 100 μL of Buffer PB was added to each reaction using a filtered p200 micropipette. The contents of each reaction tube were carefully transferred onto the membrane of a purple QIAGEN® PCR Purification column. The columns were centrifuged at top speed for 1 minute, and the flow-through was discarded. 750 μL of Buffer PE was added to each column and centrifuged at top speed for 1 minute. After discarding the flow-through, the columns were dehydrated by centrifuging at top speed for 3 minutes. Two new 1.5 mL microcentrifuge tubes were labeled "Capped RNA Transcripts." The columns were placed in the labeled microcentrifuge tubes, and 35 μL of RNAse-free water was added directly to the membrane and incubated for 2 minutes. The products were collected by centrifugation at top speed for 2 minutes. The prepared RNA transcripts were analyzed using a spectrophotometer according to the manufacturer's instructions. Following SPEC, the purified RNA transcripts were kept on ice and transferred directly to the tailing reaction. If the tailing reaction cannot be completed immediately after purification, the RNA transcripts should be stored at -80°C for a period of time (for best results, no more than one week).

[0066] Production of polyadenylated mRNA. The following components: 10x tailing buffer; 10 mM adenosine triphosphate (ATP); RNAse inhibitor (mouse); and in-house purified poly(A) polymerase were stored at 20°C prior to starting and kept on ice during reaction assembly. Two new PCR strip tubes were labeled "Poly(A) Polymerase Tail Reaction." Using the collected purified RNA, 10 μg of RNA was added to each labeled tube. RNAse-free water was added to each tube to a final volume of 14 μL. Reagents were added to each reaction in the following order: 2 μL 10x tailing buffer; 2 μL 10 mM ATP; 1 μL poly(A) polymerase; and 1 μL RNAse inhibitor. The contents were gently mixed using a pipette and spun down. The reactions were incubated at 37°C for 1 hour in a thermal cycler.

[0067] After the incubation period, the tubes were removed from the thermal cycler, and 100 μL of Buffer PB was added to each reaction using a filtered p200 micropipette. The mixture was added directly onto the membrane of a purple QIAGEN® PCR Purification column. The columns were centrifuged at top speed for 1 minute, and the flow-through was discarded. 750 μL of Buffer PE was added to each column, followed by centrifugation at top speed for 1 minute. The flow-through was discarded, and the columns were then spun at top speed for 3 minutes. The columns were placed in new 1.5 mL microcentrifuge tubes, and 35 μL of RNAse-free water was added directly to the membrane. The columns were allowed to sit for 2 minutes, then centrifuged at top speed for 2 minutes. The prepared RNA transcripts were analyzed using a spectrophotometer according to the manufacturer's instructions. Following SPEC, purified RNA transcripts should be kept on ice and proceed directly to the transfection reaction. If transfection cannot be completed immediately after purification, RNA transcripts should be stored at -80°C.

[0068] The following discussion provides many example embodiments. Each embodiment represents one combination of the elements of the invention, but the inventive subject matter is considered to encompass all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the inventive subject matter is also considered to encompass any other combination of the remaining A, B, C, or D, even if not explicitly disclosed.

[0069] As used herein, and unless the context indicates otherwise, the term "coupled to" is intended to encompass both direct coupling (where the two elements coupled to each other touch each other) and indirect coupling (where at least one additional element is located between the two elements). Thus, the terms "coupled to" and "coupled with" are used interchangeably.

[0070] In addition to the modifications already described, it will be apparent to those skilled in the art that many more modifications are possible without departing from the spirit of the present disclosure. Accordingly, the present disclosure is not limited except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprise" and "comprising" should be interpreted in a non-exclusive manner to refer to elements, components, or steps, indicating that a referenced element, component, or step may be present, used, or combined with other elements, components, or steps not specifically referenced. When the specification or claims refer to at least one of something selected from the group consisting of A, B, C, ..., and N, the written text should be interpreted as requiring only one element from that group, rather than A+N, or B+N, etc.

Claims

1. transfecting a first plasmid vector comprising an arabinose promoter and a nucleic acid sequence encoding T7 RNA polymerase into a first plurality of competent bacterial cells; transfecting a second plasmid vector comprising at least one arabinose promoter and a nucleic acid sequence encoding vaccinia virus capping enzyme (VVCE) into a second plurality of competent bacterial cells; transfecting a third plasmid vector comprising an arabinose promoter, a UUG start codon, and a nucleic acid sequence encoding polyadenosine (poly(A)) polymerase into a third plurality of competent bacterial cells; expressing and purifying T7 RNA polymerase from said first plurality; expressing and purifying VVCE from said second plurality; expressing and purifying poly(A) polymerase from said third plurality; adding linearized plasmid DNA to a first composition comprising the purified T7 RNA polymerase to produce RNA transcripts; adding the RNA transcript to a second composition comprising the purified VVCE to produce a capped RNA transcript; adding the capped RNA transcripts to a third composition comprising the purified poly(A) polymerase to produce polyadenylated capped mRNA transcripts; and purifying mRNA from said third composition; A method for in vitro synthesis of mRNA, comprising:

2. 2. The method of claim 1, wherein the first plasmid vector comprises a nucleotide sequence at least 85% identical to SEQ ID NO:

19.

3. 3. The method of claim 2, wherein the first plasmid vector comprises a nucleotide sequence at least 90% identical to SEQ ID NO:

19.

4. 4. The method of claim 3, wherein the first plasmid vector comprises a nucleotide sequence at least 95% identical to SEQ ID NO:

19.

5. 5. The method of claim 4, wherein the first plasmid vector comprises a nucleotide sequence comprising SEQ ID NO:

19.

6. 2. The method of claim 1, wherein the second plasmid vector comprises a nucleotide sequence at least 85% identical to SEQ ID NO:

20.

7. 7. The method of claim 6, wherein the second plasmid vector comprises a nucleotide sequence at least 90% identical to SEQ ID NO:

20.

8. 8. The method of claim 7, wherein the second plasmid vector comprises a nucleotide sequence at least 95% identical to SEQ ID NO:

20.

9. 9. The method of claim 8, wherein the second plasmid vector comprises a nucleotide sequence comprising SEQ ID NO:

20.

10. 2. The method of claim 1, wherein the third plasmid vector comprises a nucleotide sequence at least 85% identical to SEQ ID NO:

21.

11. 11. The method of claim 10, wherein the third plasmid vector comprises a nucleotide sequence at least 90% identical to SEQ ID NO:

21.

12. 12. The method of claim 11, wherein the third plasmid vector comprises a nucleotide sequence at least 95% identical to SEQ ID NO:

21.

13. 13. The method of claim 12, wherein the third plasmid vector comprises a nucleotide sequence comprising SEQ ID NO:

21.

14. 2. The method of claim 1, wherein the bacterial competent cells are Clear Coli BL21 (DE3) competent cells.

15. The method of claim 1, wherein VVCE is expressed at 18°C.

16. 7. The method of claim 6, wherein the VVCE is expressed over a fermentation period of 18 hours.

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