Composition for in vitro transcription reaction and in vitro transcription reaction method

Abstract

This composition comprises: at least one buffer selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-morpholinoethanesulfonic acid; and a capping reagent containing a capping compound capable of bonding to a terminal of a transcription reaction product, wherein the buffer concentration is 5-35 mM, and the pH is 5.0-7.5.

Description

Composition for in vitro transcription reaction and method for in vitro transcription reaction

[0001] The present disclosure relates to a composition for an in vitro transcription reaction and an in vitro transcription reaction method.

[0002] In vitro transcription (IVT) refers to an RNA synthesis reaction carried out in a test tube (in vitro) using RNA (ribonucleic acid) polymerase and DNA (deoxyribonucleic acid) as a template. IVT can be used for research and development of mRNA (messenger RNA) drugs. Research and development of mRNA drugs is progressing rapidly in various fields, including not only vaccines for infectious diseases but also cancer vaccines and therapeutic drugs for genetic diseases. IVT enables the synthesis of long-chain RNA such as mRNA drugs with high purity and high yield.

[0003] Various conditions for IVT have been investigated, and for example, trishydroxymethylaminomethane (Tris) buffer and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer are known as compositions for IVT reactions (e.g., Karnyart Samnuan et al., "Design-of-experiments in vitro transcription yield optimization of self-amplifying RNA", F1000 Research 2022, 11:333, First published: 18 Mar 2022). , investigated the IVT reaction conditions to maximize the yield of approximately 9.4 kb long-chain RNA. According to Non-Patent Document 1, magnesium has the greatest effect on the RNA yield.

[0004] In IVT, RNA having a capping compound at the 5' end is synthesized for the purpose of improving the stability of the synthesized RNA, etc., and there is a demand for high-yield synthesis of RNA of a desired length that has a capping compound. Therefore, an object of the present disclosure is to provide a composition for in vitro transcription reaction and an in vitro transcription reaction method that can synthesize RNA of a desired length that has a capping compound in an IVT reaction in high yield.

[0005] In order to achieve the above-mentioned object, the present inventors have conducted extensive research and found that by selecting an appropriate range for the type, concentration, and pH of a buffer used in an in vitro transcription reaction, it is possible to synthesize RNA of a desired length having a capping compound in high yield, and have completed the present disclosure.

[0006] <1> A composition for in vitro transcription reactions, comprising at least one buffer selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-morpholinoethanesulfonic acid, and a capping reagent containing a capping compound capable of binding to the terminus of a transcription reaction product, wherein the buffer concentration is 5 mM (mmol / L; the same applies hereinafter) to 35 mM, and the buffer has a pH in the range of 5.0 to 7.5. <2> The composition for in vitro transcription reactions according to <1>, further comprising at least one compound selected from the group consisting of spermidine and dithiothreitol. <3> The composition for in vitro transcription reactions according to <1> or <2>, further comprising an RNA polymerase and a nucleotide reagent that serves as a substrate for the RNA polymerase. <4> An in vitro transcription reaction method comprising the steps of preparing a reaction solution containing the composition for in vitro transcription reaction according to any one of <1> to <3> and template DNA, and synthesizing RNA having a capping compound by transcribing the template DNA in the reaction solution. <5> The in vitro transcription reaction method according to <4>, further comprising the step of preparing template DNA. <6> The in vitro transcription reaction method according to <4> or <5>, further comprising the step of recovering the RNA having the capping compound.

[0007] According to the composition for in vitro transcription reaction and the in vitro transcription reaction method of the present disclosure, RNA having a capping compound and of a desired length can be synthesized in high yield by an in vitro transcription reaction.

[0008] Hereinafter, embodiments of the present disclosure will be described. The description is for illustrating the embodiments and is not intended to limit the scope of the present disclosure.

[0009] In the following embodiments, the components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the present disclosure. For example, the present disclosure allows addition, omission, substitution, modification, etc. of the number, amount, position, ratio, material, configuration, type, order, etc., within the scope of the spirit of the present disclosure.

[0010] In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in the present disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another staged numerical range. Furthermore, in numerical ranges described in the present disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples. In the present disclosure, each component may contain multiple corresponding substances. When multiple substances corresponding to each component are present in a composition, etc., the content or amount of each component means the total content or amount of the multiple substances present in the composition, etc., unless otherwise specified. In this specification, the term "process" does not only refer to an independent process, but also includes processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved.

[0011] In this disclosure, the term "gene" is not limited to a region encoding a protein, but also includes, for example, an expression control region, an intron region, and non-coding RNA (miRNA) of unknown function. In addition, in this disclosure, "DNA" and "RNA" are abbreviations for deoxyribonucleic acid and ribonucleic acid, respectively. In this disclosure, "DNA" and "RNA" refer to molecules having multiple nucleotides in any form, including single-stranded, double-stranded, oligonucleotides, or polynucleotides. "Base sequence" and "nucleotide sequence" refer to the order of nucleotides in single-stranded "DNA" and "RNA."

[0012] In this disclosure, "in vitro" means an artificial environment other than within a living organism (e.g., an animal, plant, or microorganism), such as a test tube, a reaction vessel, a cell culture vessel, or the like.

[0013] In the present disclosure, "pH" is a value measured with a pH meter at a temperature adjusted to 25±0.2° C. As the pH meter, for example, a HORIBA F-72 desktop pH meter can be used.

[0014] <Composition for In Vitro Transcription Reactions> The composition for in vitro transcription reactions of the present disclosure comprises at least one buffer selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-morpholinoethanesulfonic acid, and a capping reagent containing a capping compound capable of binding to the terminus of a transcription reaction product, wherein the buffer concentration is 5 mM to 35 mM and the buffer pH is in the range of 5.0 to 7.5. Hereinafter, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid will be referred to as HEPES, and 2-morpholinoethanesulfonic acid will be referred to as MES. The composition for in vitro transcription reactions of the present disclosure enables high-yield synthesis of RNA of a desired length that contains a capping compound in an in vitro transcription reaction.

[0015] Here, whether RNA having a capping compound and of a desired length can be synthesized in high yield by an in vitro transcription reaction can be comprehensively determined based on the yield, capping rate, and purity (referred to as FA purity in the examples described below). Here, the yield is calculated as the ratio of the actual yield contained in the reaction solution after completion of the reaction to the theoretical yield. The capping rate is calculated as the proportion of RNA having a capping compound among the synthesized RNA after completion of the reaction. The purity is calculated as the proportion of RNA having the desired length among the synthesized RNA.

[0016] In the present disclosure, the yield is preferably 80% or higher, more preferably 85% or higher, even more preferably 90% or higher, and even more preferably 95% or higher. Furthermore, in the present disclosure, the capping rate is preferably 96% or higher, more preferably 97% or higher, even more preferably 98% or higher, and even more preferably 99% or higher. Furthermore, in the present disclosure, the purity is preferably 75% or higher, more preferably 80% or higher, even more preferably 85% or higher, even more preferably 90% or higher, and even more preferably 95% or higher. In particular, the composition for in vitro transcription reactions of the present disclosure can achieve the above-mentioned ranges for all of the yield, capping rate, and purity, allowing RNA having a capping compound and of a desired length to be synthesized in high yield by in vitro transcription reactions.

[0017] <Buffer> HEPES and MES contained in the composition for in vitro transcription reaction of the present disclosure are one of the so-called Good's buffers, and are buffers that exhibit a buffering effect around neutral. As the HEPES and MES of the present disclosure, commercially available products can be used as appropriate.

[0018] In the in vitro transcription reaction composition of the present disclosure, at least one buffer selected from the group consisting of HEPES and MES is contained in the composition at 5 mM to 35 mM, preferably 5 mM to 30 mM, more preferably 10 mM to 30 mM, even more preferably 10 mM to 20 mM, and most preferably 20 mM. By maintaining the buffer concentration in the composition within this range, the capping rate and yield can be increased, for example, within the ranges described above. The concentration of the buffer in the composition can be measured by proton NMR analysis using an internal standard.

[0019] The buffer contained in the composition for in vitro transcription reaction of the present disclosure has a pH in the range of 5.0 to 7.5, preferably 5.3 to 7.2, more preferably 5.7 to 6.9, even more preferably 6.0 to 6.5, even more preferably 6.2 to 6.4, and most preferably 6.3. By setting the pH of the buffer in this range, it is possible to achieve a high purity, for example, within the range described above.

[0020] <Capping Compound> In the composition for in vitro transcription reaction of the present disclosure, a capping compound generally refers to a compound that is added to the 5' end of mature mRNA to form a so-called 5' cap structure. In the present disclosure, a capping reagent refers to a reagent containing at least a capping compound, and can be a solution containing a capping compound and a solvent. The 5' cap structure can be formed by a modified nucleotide, particularly a guanine nucleotide derivative. The capping compound is not particularly limited as long as it is a compound that can form such a 5' cap structure, and conventionally known compounds can be used. Examples of capping compounds include m ３’，７ G (Am) G, m ７ GpppA, m ７ GpppG, m ３’，７ GpppG, m ７ G(Am)U,m ７G(Am)G. As the capping compound, commercially available CleanCap (registered trademark, the same applies hereinafter) Reagent AG, CleanCap Reagent AG(3'OMe), CleanCap Reagent M6, CleanCap Reagent AU, etc. can also be used.

[0021] <Other Components> Preferably, the in vitro transcription reaction composition of the present disclosure further contains at least one compound selected from the group consisting of spermidine and dithiothreitol (DTT). Among these, spermidine can contribute to the stabilization or activation of the enzyme (RNA polymerase) that catalyzes the transcription reaction. Among these, dithiothreitol (DTT) can contribute to the activation of RNase inhibitors that inhibit the activity of RNases. Therefore, by further containing at least one compound selected from the group consisting of spermidine and dithiothreitol (DTT), these in vitro transcription reaction compositions can synthesize RNA with a high yield. The in vitro transcription reaction composition may also contain a polyamine other than spermidine that contributes to the stabilization or activation of RNA polymerase. The in vitro transcription reaction composition may also contain a reducing agent other than dithiothreitol (DTT) that contributes to the activation of RNase inhibitors.

[0022] Furthermore, the in vitro transcription reaction composition of the present disclosure may contain other components necessary for the in vitro transcription reaction. For example, the in vitro transcription reaction composition of the present disclosure may further contain an RNA polymerase and a nucleotide reagent that serves as a substrate for the RNA polymerase. The nucleotide reagent may include ribonucleotides such as ATP (adenosine triphosphate), CTP (cytidine triphosphate), GPT (guanosine triphosphate), and UTP (uridine triphosphate), or may include analogs of these ribonucleotides (also referred to as analogs). Note that "nucleotide" generally refers to a molecule in which a phosphate group is covalently bonded to the sugar moiety of a nucleoside. "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar is typically, but not limited to, a pentofuranosyl sugar. Examples of pentofuranosyl sugars include ribose and deoxyribose. Bases (nucleobases) generally include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Nucleotides containing adenine (A), guanine (G), cytosine (C), and uracil (U) are used in the in vitro transcription reaction composition of the present disclosure. The bases may be either modified or unmodified.

[0023] The UTP analog may include a uridine analog listed below. That is, the uridine analog may be selected from the group consisting of 1-methylpseudouridine, 1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In particular, the uracil analog is preferably one containing 1-methyl-pseudouridine.

[0024] CTP analogs may include the following cytidine analogs: 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 5-formylcytidine, 4-methylcytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zeb and cytidine analogs selected from the group consisting of lysidine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, α-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, 4-acetyl-2'-O-methyl-cytidine, 4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine (f5Cm), 4,4,2'-O-trimethyl-cytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0025] ATP analogs may include the adenine analogs or adenosine analogs listed below. Thus, adenine analogs and adenosine analogs include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, 2-methyl-adenine, 6-methyladenosine, 2-methylthio-6-methyl-adenosine, 6-isopentenyladenosine, 2-methylthio 6-Isopentenyl-adenosine, 6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-6-(cis-hydroxyisopentenyl)adenosine, 6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-6-threonylcarbamoyl-adenosine, 6,6-dimethyl-adenosine, 6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-6-hydroxynorvalylcarbamoyl-adenosine, 6-acetyl-adenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine , 6,2'-O-dimethyl-adenosine, 6,6,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine (phosphate)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and 6-(19-amino-pentaoxanonadecyl)-adenosine.

[0026] GTP analogs may include the guanine or guanosine analogs listed below. Guanine and guanosine analogs include inosine, 2-aminopurine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, unmodified hydroxywybutosine, 7-deaza-guanosine, queosine, epoxyqueosine, galactosyl-queosine, mannosyl-queosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methyl- Guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methylguanosine, 2-methyl-guanosine, 2,2-dimethyl-guanosine, 2,7-dimethyl-guanosine, 2,2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, 2-methyl-6-thio-guanosine, 2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine, 2-methyl-2'-O-methyl-guanosine, 2,2-dimethyl-2'-O-methyl-guanosine, 1-methyl-2'-O-methyl-guanosine, 2,7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl-inosine, 1,2' and guanine or guanosine analogs selected from the group consisting of -O-dimethyl-inosine, 2'-O-ribosylguanosine (phosphate), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0027] The RNA polymerase contained in the in vitro transcription reaction composition of the present disclosure is not particularly limited, and any conventionally known RNA polymerase can be used. For example, commercially available RNA polymerases can be used. Examples of commercially available RNA polymerases include CUGA (registered trademark, the same applies hereinafter) 3 RNA polymerase, CUGA 7 RNA polymerase, CUGA 6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and Codex HiCap RNA polymerase.

[0028] The composition for in vitro transcription reactions of the present disclosure may also contain pyrophosphatase. Pyrophosphatase, also known as inorganic pyrophosphatase, hydrolyzes inorganic pyrophosphate generated during transcription reactions catalyzed by RNA polymerase. By including pyrophosphatase, the composition for in vitro transcription reactions of the present disclosure can increase the yield of RNA.

[0029] Furthermore, the in vitro transcription reaction composition of the present disclosure may contain an RNase inhibitor that inhibits the RNA degradation activity of ribonuclease (RNase). The RNase inhibitor is not particularly limited, and any RNase inhibitor known in the art can be used. Specific examples of RNase inhibitors include DEPC (Diethylpyrocarbonate), vanadyl ribonucleotides, inhibitory proteins against ribonuclease A, ribonuclease B, and ribonuclease C, and anti-ribonuclease antibodies. Commercially available RNase inhibitors include RNaseOUT (Life Technologies), RNasin® (Promega), and Ribonuclease Inhibitor (derived from porcine liver) (Takara Bio).

[0030] Furthermore, the in vitro transcription reaction composition of the present disclosure may contain deoxyribonuclease (DNase) and a buffer solution used in the reaction solution for the DNase-mediated DNA degradation reaction. After synthesizing the target RNA using the in vitro transcription reaction composition, highly pure RNA can be obtained by degrading DNA, such as template DNA, contained in the reaction system with DNase. The DNase is not particularly limited, and any DNase known in the art can be used. An example of a commercially available DNase is DNase I derived from bovine pancreas, which is an RNase-free product. The buffer solution used for the DNase may be, for example, Tris-HCl.

[0031] <In vitro transcription reaction method> The in vitro transcription reaction method of the present disclosure includes the steps of preparing a reaction solution containing the in vitro transcription reaction composition of the present disclosure and template DNA, and synthesizing RNA containing a capping compound by transcribing the template DNA in the reaction solution. According to the in vitro transcription reaction method of the present disclosure, RNA having a capping compound and a desired length can be synthesized in high yield by an in vitro transcription reaction. In other words, by using the in vitro transcription reaction method of the present disclosure, RNA having a capping compound and a desired length can be synthesized in high yield with excellent yield, capping rate, and purity.

[0032] <Template DNA> The in vitro transcription reaction method of the present disclosure can include a step of preparing template DNA. In the present disclosure, template DNA includes a promoter recognizable by RNA polymerase and a region transcribed by RNA polymerase. Examples of promoters include, but are not limited to, the SP6 promoter and the T7 promoter. The region transcribed by RNA polymerase may encode a protein or a portion thereof, or may not encode a protein. The template DNA may be a reaction product obtained by amplifying and synthesizing a transcription region linked to a promoter by PCR (Polymerase Chain Reaction), or a reaction product obtained by linearizing plasmid DNA, into which a transcription region linked to a promoter sequence has been inserted, with a restriction enzyme.

[0033] The region transcribed by RNA polymerase preferably contains an open reading frame encoding a pharmaceutically useful polypeptide or a pharmaceutically useful fragment thereof. The pharmaceutically useful polypeptide or a pharmaceutically useful fragment thereof is not particularly limited as long as it is a polypeptide or fragment used for treating or preventing a disease. The pharmaceutically useful polypeptide or fragment thereof may be a polypeptide or fragment thereof useful for preventing an infectious disease, treating cancer, or treating a disease. The polypeptide or fragment thereof for preventing an infectious disease may be an antigenic polypeptide or an immunogenic fragment used to prevent infection with or the onset of a viral infection caused by COVID-19 (Corona Virus Infectious Disease, emerged in 2019) virus, cytomegalovirus, respiratory syncytial virus, influenza A virus, influenza B virus, human metapneumovirus, parainfluenza virus, West Nile virus, Zika virus, rabies virus, Epstein-Barr virus, human immunodeficiency virus, Nipah virus, or dengue virus.

[0034] Examples of such antigen polypeptides or fragments thereof include SARS-CoV-2 spike protein, SARS-CoV-2 protein, influenza HA protein, influenza HA / NA protein, West Nile virus NS1 protein, Zika virus structural protein, and Dengue virus particle surface protein.

[0035] The cancer therapeutic polypeptide or fragment thereof may be a tumor-associated antigen protein, tumor-associated antigen polypeptide or tumor-associated immunogenic fragment targeted to solid cancer / melanoma, colon cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, HPV16-positive head and neck cancer, triple-negative breast cancer, ovarian cancer.

[0036] The polypeptide or fragment thereof useful for treating a disease may be a polypeptide or fragment thereof useful for treating cardiovascular disease, ischemic vascular disease, ornithine transcarbamylase deficiency, transthyretin amyloidosis (ATTR), chikungunya, propionic acidemia, cystic fibrosis, autoimmune disease, subsequent methylmalonic acidemia, glycogen storage disease type I, or glycogen storage disease type III.

[0037] Furthermore, the region transcribed by RNA polymerase may contain one or more open reading frames encoding a pharmaceutically useful polypeptide or a pharmaceutically useful fragment thereof. Furthermore, the template DNA is not limited to a configuration containing a region transcribed by one RNA polymerase, but may contain a plurality of regions transcribed by RNA polymerase.

[0038] The RNA transcribed from the template DNA can have a 3'-terminal polyadenylated (poly(A)) tail. Having a 3'-terminal polyadenylated (poly(A)) tail on the transcribed RNA can increase the stability of the RNA. A 3'-terminal polyadenylated (poly(A)) tail refers to a homopolymer containing an adenine nucleoside attached to the 3'-end of the RNA, and can be of any length. The 3'-terminal polyadenylated (poly(A)) tail may contain one or more guanine nucleosides, uracil nucleosides, or cytosine nucleosides between the homopolymer containing the adenine nucleoside. The 3'-terminal polyadenylated (poly(A)) tail may be, for example, a homopolymer containing a total of 100 adenine nucleoside residues at the 3'-end, divided into 30 and 70 residues.

[0039] The RNA transcribed from the template DNA may further contain a 5' untranslated region (5'UTR) between the 5' cap structure and the open reading frame, and a 3' untranslated region (3'UTR) between the open reading frame and the 3' poly(A) tail, respectively.

[0040] <RNA Recovery> The in vitro transcription reaction method of the present disclosure can include a step of recovering RNA. According to the in vitro transcription reaction method of the present disclosure, the reaction solution contains various impurities in addition to the synthesized RNA. The step of recovering RNA refers to a step of separating the RNA (target product) contained in the reaction solution from the impurities. In general, impurities include, for example, various components and by-products used in RNA production, undesired RNA species, proteins, DNA or fragments thereof, pyrophosphates, and free nucleotides.

[0041] The RNA recovery step can be performed using RNA purification methods known in the art. For example, the RNA purification method can include DNase treatment followed by precipitation of the synthesized RNA using lithium chloride. In addition, the RNA purification method can also include ion-pair reversed-phase HPLC (High Performance Liquid Chromatography) using a porous reversed stationary phase or affinity chromatography using Poly(T) resin.

[0042] The present disclosure will be described in more detail below using examples, but the technical scope of the present disclosure is not limited to the following examples.

[0043] Example 1 (1) In vitro transcription (IVT) reaction mRNA was synthesized by in vitro transcription (IVT) reaction. The reaction solution contained 20 mM HEPES (pH 6.3) as a buffer, 10 mM dithiothreitol, 2 mM spermidine, 15.5 mM magnesium acetate, 4 mM CleanCap AG (3'OMe), 5 mM ATP, 5 mM GTP, 5 mM CTP, 5 mM N1-methylpseudo-UTP, 0.002 U / μL pyrophosphatase, 1.25 U / μL RNase inhibitor, 8 U / μL T7 RNA polymerase, and 10 ng / μL template DNA. The reaction solution was reacted at 37°C for 2.5 hours, and then 20.0 μL of the IVT reaction solution was collected in a 1.5 mL tube. The pH values ​​of the buffers were measured using a HORIBA F-72 benchtop pH meter at a temperature of 25±0.2°C when prepared as 1 M buffers. The 1 M buffers of various pH values ​​used in the examples and comparative examples other than Example 1 were prepared using HEPES (Sigma-Aldrich), Tris-HCl (Nippon Gene or Invitrogen), MES (Dojindo Chemical Industries), TES (Dojindo Chemical Industries), or BES (Sigma-Aldrich), water for injection (Otsuka Pharmaceutical), hydrochloric acid, or aqueous sodium hydroxide solution.

[0044] <DNA Degradation Reaction and Preparation of mRNA Aqueous Solution> To degrade the template DNA contained in the IVT reaction solution, DNase I, Tris-HCl (pH 7.5), and calcium chloride were added to 20.0 μL of the collected IVT reaction solution and heated at 37°C for 15 minutes. Subsequently, to prepare mRNA precipitates, 60.0 μL of 3.75 M LiCl aqueous solution was added to the reaction solution, followed by stirring and spinning down, and then allowed to stand overnight at -20°C. Next, the centrifuge was set to 4°C and 15,000 rpm, and the mixture was spun down for 15 minutes, after which the supernatant was removed. 500 μL of 70% ethanol was added, and the centrifuge was set to 4°C and 15,000 rpm, and the mixture was spun down for 5 minutes, after which the supernatant was removed, yielding a white pellet-like mRNA solid. The mRNA solid was mixed with 40.0 μL of water for injection, and the mixture was stirred and spun down multiple times to completely dissolve the mRNA solid. The obtained aqueous mRNA solution was used as a sample for various evaluation measurements.

[0045] (2) Calculation of yield The RNA concentration of the mRNA aqueous solution was measured using NanoDrop (Thermo Fisher Scientific), and the average of two measurements was used as the RNA concentration of the solution. Since the mRNA contained in 40.0 μL of IVT quality evaluation sample solution was derived from 20.0 μL of IVT reaction solution, the reaction yield was calculated using the following formula (1). Formula (1): Reaction yield (mg / mL) = crude RNA weight (mg) ÷ IVT reaction solution volume (mL) = RNA concentration of mRNA aqueous solution [mg / mL] × 0.040 [mL] ÷ 0.020 [mL] Since the theoretical yield was 5.27 mg / mL, the yield was calculated using the following formula (2). Formula (2): Yield (%) = [Reaction yield (mg / mL) ÷ 5.27 (mg / mL)] × 100

[0046] (3) Measurement of Capping Rate The capping rate was measured according to the method described in a non-patent document (Katalin Kariko et al., “Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA”, Pharmaceutics 2022, 14:328, First published: 29 January 2022).

[0047] (4) Measurement of Fragment Analyzer (FA) Purity Measurement of FA purity was performed using a 5200 Fragment Analyzer (Agilent) according to the standard protocol provided with the instrument. For calibration, the analysis results of ssRNA Ladder solution (New England Labs) were used as a calibration curve between migration time and base length. For the eight peaks identified, the base lengths were designated as 15, 500, 1000, 2000, 3000, 5000, 7000, and 9000 nt, in order of shortest migration time. For the analysis results of the mRNA sample, the peak area percentage of the peak corresponding to the target length (approximately 4300 nt) was analyzed and used as the FA purity of the mRNA.

[0048] (5) Evaluation: After the in vitro transcription reaction, if the yield, capping rate, and FA purity all exceeded predetermined levels, the result was judged to be "pass," indicating that RNA of the desired length containing a capping compound could be synthesized in high yield. Furthermore, if any one of the yield, capping rate, and FA purity was below a predetermined level after the in vitro transcription reaction, the result was judged to be "fail," indicating that RNA of the desired length containing a capping compound could not be synthesized in high yield. Specifically, the yield level was set to 80%, the capping rate level to 96%, and the FA purity level to 75%.

[0049] [Example 2] In vitro transcription reaction was performed in the same manner as in Example 1, except that 5 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured. [Example 3] In vitro transcription reaction was performed in the same manner as in Example 1, except that 10 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured. [Example 4] In vitro transcription reaction was performed in the same manner as in Example 1, except that 30 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured. [Example 5] In vitro transcription reaction was performed in the same manner as in Example 1, except that the buffer pH was 5.3, and the yield, capping rate, and FA purity were measured. [Example 6] In vitro transcription reaction was performed in the same manner as in Example 1, except that the buffer pH was 5.7, and the yield, capping rate, and FA purity were measured. [Example 7] In vitro transcription reaction was performed in the same manner as in Example 1, except that the buffer pH was 6.9, and the yield, capping rate, and FA purity were measured. [Example 8] In vitro transcription reaction was carried out in the same manner as in Example 1, except that the pH of the buffer was changed to 7.2, and the yield, capping rate, and FA purity were measured. [Example 9] In vitro transcription reaction was carried out in the same manner as in Example 1, except that 20 mM MES was used as the buffer, and the yield, capping rate, and FA purity were measured.

[0050] [Comparative Example 1] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 20 mM Tris-HCl was used as the buffer, and the yield, capping rate, and FA purity were measured. [Comparative Example 2] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 40 mM Tris-HCl was used as the buffer, and the yield, capping rate, and FA purity were measured. [Comparative Example 3] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 40 mM Tris-HCl was used as the buffer and the pH of the reaction solution was set to 7.0, and the yield, capping rate, and FA purity were measured. [Comparative Example 4] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 40 mM Tris-HCl was used as the buffer and the pH of the reaction solution was set to 7.5, and the yield, capping rate, and FA purity were measured. Comparative Example 5 An in vitro transcription reaction was carried out in the same manner as in Example 1, except that 40 mM Tris-HCl was used as the buffer and the pH of the reaction solution was set to 8.0, and the yield, capping rate, and FA purity were measured.

[0051] [Comparative Example 6] An in vitro transcription reaction was performed in the same manner as in Example 1, except that no buffer was used, and the yield, capping rate, and FA purity were measured. [Comparative Example 7] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 40 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured. [Comparative Example 8] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 50 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured. [Comparative Example 9] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 60 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured. [Comparative Example 10] An in vitro transcription reaction was performed in the same manner as in Example 1, except that 70 mM HEPES was used as the buffer, and the yield, capping rate, and FA purity were measured.

[0052] [Comparative Example 11] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that the buffer pH was 4.1, and the yield, capping rate, and FA purity were measured. [Comparative Example 12] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that the buffer pH was 4.4, and the yield, capping rate, and FA purity were measured. [Comparative Example 13] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that the buffer pH was 4.7, and the yield, capping rate, and FA purity were measured. [Comparative Example 14] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that the buffer pH was 4.9, and the yield, capping rate, and FA purity were measured. [Comparative Example 15] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that the buffer pH was 8.0, and the yield, capping rate, and FA purity were measured.

[0053] [Comparative Example 16] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that 20 mM TES (2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid) was used as the buffer, and the yield, capping rate, and FA purity were measured. [Comparative Example 17] An in vitro transcription reaction was carried out in the same manner as in Example 1, except that 20 mM BES (2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid) was used as the buffer, and the yield, capping rate, and FA purity were measured.

[0054] [Results 1] Table 1 shows the results of comparing Example 1 with Comparative Examples 1 to 5.

[0055]

[0056] As shown in Table 1, when Tris-HCl was used as a buffer for the in vitro transcription reaction, the capping rate was low and the FA purity was also not at a practical level. In contrast, by using HEPES as a buffer for the in vitro transcription reaction at a predetermined concentration and under predetermined pH conditions as in Example 1, the FA purity, yield, and capping rate were all at satisfactory levels, demonstrating that RNA having a capping compound and of the desired length could be synthesized in high yield.

[0057] [Results 2] Table 2 shows the results of comparing Examples 1 to 4 with Comparative Examples 6 to 10.

[0058]

[0059] As shown in Table 2, when HEPES is used as a buffer for in vitro transcription reactions, by setting the HEPES concentration in the range of 5 mM to 35 mM, the FA purity, yield, and capping rate are all at satisfactory levels, and RNA having a capping compound and of the desired length can be synthesized in high yield.

[0060] [Result 3] Table 3 shows the results of comparing Examples 1 and 5 to 8 with Comparative Examples 11 to 15.

[0061]

[0062] As shown in Table 3, when HEPES is used as a buffer for the in vitro transcription reaction, by setting the pH of the reaction solution in the range of 5.0 to 7.5, the FA purity, yield, and capping rate are all at satisfactory levels, and RNA having a capping compound and of the desired length can be synthesized in high yield.

[0063] [Results 4] Table 4 shows the results of comparing Example 1, Example 9, Comparative Example 1, Comparative Example 16, and Comparative Example 17.

[0064]

[0065] As shown in Table 4, when MES, which is structurally similar to HEPES, was used at a concentration of 20 mM and the reaction solution pH was 6.3, the FA purity, yield, and capping rate were all at satisfactory levels, indicating that RNA of the desired length having a capping compound could be synthesized in high yield. In contrast, when TES and BES, which have structures different from HEPES and similar to Tris, were used, the capping rate and yield were low.

[0066] The results in Tables 1 to 4 show that by performing an in vitro transcription reaction at a concentration of at least one buffer selected from the group consisting of HEPES and MES of 5 mM to 35 mM and at a pH in the range of 5.0 to 7.5, the FA purity, yield, and capping rate are all at satisfactory levels, and RNA of the desired length having a capping compound can be synthesized in high yield.

[0067] The disclosure of Japanese Patent Application No. 2024-109241, filed on July 5, 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A composition for in vitro transcription reactions, comprising at least one buffer selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2-morpholinoethanesulfonic acid, and a capping reagent containing a capping compound capable of binding to the end of a transcription reaction product, wherein the concentration of the buffer is 5 mM to 35 mM and the pH of the buffer is in the range of 5.0 to 7.

5.

2. The composition for an in vitro transcription reaction according to claim 1, further comprising at least one compound selected from the group consisting of spermidine and dithiothreitol.

3. The composition for an in vitro transcription reaction according to claim 1, further comprising an RNA polymerase and a nucleotide reagent that serves as a substrate for the RNA polymerase.

4. An in vitro transcription reaction method comprising the steps of: preparing a reaction solution containing the composition for in vitro transcription reactions described in claim 1 and template DNA; and synthesizing RNA having a capping compound by transcribing the template DNA in the reaction solution.

5. The in vitro transcription reaction method according to claim 4, further comprising the step of preparing the template DNA.

6. The in vitro transcription reaction method according to claim 4, further comprising the step of recovering the RNA having the capping compound.

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