CN116265581A - In vitro preparation method of transcript mRNA - Google Patents
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Abstract
The invention provides an in vitro preparation method of transcript mRNA. In order to solve the problems that the existing preparation method cannot simultaneously consider the yield, the impurity dsRNA and the mRNA expression quantity of transcripts, which results in high preparation cost or large influence on immunogenicity, and the like, the invention adjusts and optimizes the formula of a transcription system, reduces the quantity of the impurity dsRNA in the transcripts, balances the yield, the impurity content and the expression quantity of the transcripts, realizes the purposes of high yield, high expression quantity and low impurity content, and the obtained transcripts can be directly used for mRNA drugs, mRNA vaccines, polypeptides or protein drugs and the like through conventional lithium chloride purification, has high in-vitro translation efficiency and small influence on immunogenicity, omits the steps of transferring the transcripts, purifying and removing the dsRNA through RP-HPLC which is complicated and difficult to produce in a large scale under GMP conditions, and obviously reduces the preparation difficulty and cost.
Description
Technical Field
The invention relates to an in vitro preparation method of transcript mRNA.
Background
mRNA therapy includes 2 major factors: mRNA encoding a particular protein and delivery system. Designing an mRNA drug requires achieving a certain level of efficacy at a certain dosage. One of the technical difficulties of mRNA therapy is that the immune system must avoid "abnormal" recognition of mRNA, and thus immune response.
In humans, ssRNA (single-stranded RNA) recognizes TLR7 and TLR8, thereby activating MyD88, whereas dsRNA recognizes TLR3, activating IFN beta, thereby causing the production of a number of inflammatory factors, including IFN alpha.
RNA of different lengths can be prepared enzymatically in vitro using phage RNA polymerase, such as T7 RNA polymerase. Advantages of T7 RNA polymerase include its strong activity and ease of protein production. However, T7 RNA polymerase mediated RNA synthesis produces a wide variety of abnormal byproducts, resulting in transcripts that typically stimulate the innate immune system of vertebrates. Among these, dsRNA is a key by-product formed from the target transcript and the fully reverse complementary transcript.
At present, the transcription product of T7 RNA polymerase must be subjected to RP-HPLC purification operation to reduce the content of dsRNA in transcript mRNA, but the method using RP-HPLC is complex, the development difficulty is high, the loading capacity is low, the loss of the product is great, and because most of the organic phase intervened by the method is a toxic reagent, the amplified production under the GMP condition is very difficult. Thus, there is a need for a process for preparing transcript mRNA from T7 RNA polymerase transcription to reduce dsRNA production. However, the decrease of dsRNA content in an in vitro transcription system is accompanied by a rapid decrease in the yield of transcript mRNA, and the expression level of the transcript mRNA is related to the variation of the proportion of components of the transcription system. How to balance the relation among the yield of the transcript mRNA, the dsRNA content and the expression level of the transcript mRNA, reduces the cost of preparing the transcript mRNA, and becomes the research focus of the in-vitro industrialization and standardization preparation method of the transcript mRNA.
Disclosure of Invention
The object of the present invention is to provide an in vitro method for preparing transcript mRNA having high transcript mRNA yield, low dsRNA content and high transcript mRNA expression level, which can reduce the mass percentage of double-stranded RNA (dsRNA) in the transcript mRNA prepared in vitro, thereby eliminating the RP-HPLC purification operation.
In order to solve the technical problems, the invention adopts the following technical scheme:
an in vitro preparation method of transcript mRNA, which is obtained by in vitro transcription of a DNA template and precipitation of lithium chloride,
the transcription system of the in vitro transcription comprises a DNA template, tris-HCl, DTT, inorganic pyrophosphatase, naCl, T7 RNA polymerase, ATP, GTP, CTP, UTP, spermidine and Mg 2+ Controlling the transcription system to be in an initial state, wherein the concentration of the T7 RNA polymerase in the transcription system is 2-5U/. Mu.L, and the Mg 2+ 25-27 mM, the molar concentration of said NTP in the transcription system being in relation to said Mg 2+ The molar ratio in the transcription system is 1:0.5 to 0.7.
The mass percentage of double-stranded RNA in the transcript mRNA obtained by the in vitro preparation method is less than 0.7 percent.
In the transcription system of the present invention, if Mg 2+ If the concentration of (2) is too low, the yield of the transcript will be significantly reduced; as the concentration of magnesium ions increases, the impurity content and yield rise substantially synchronously, the rate of change slightly differentiates, but the in vitro expression level of mRNA shows a decreasing trend. If Mg is 2+ If the concentration of (2) is too high, the dsRNA content in the transcript is significantly increased, so that the transcript needs to be purified by RP-HPLC which has high requirements on production plants, high cost and complex operation. Therefore, we need to find the balance among the yield, the impurity and the expression level to obtain the optimal reaction system with high yield, high expression and low impurity.
Preferably, the transcription system is controlled such that in the initial state, the molar concentrations of ATP, GTP, CTP and UTP are equal.
Preferably, the transcription system is controlled to have a molar concentration of ATP, GTP, CTP and UTP of 8-12 mM in the initial state.
Further, the transcription system is controlled to have the molar concentration of the ATP, the GTP, the CTP and the UTP of 8-10 mM in the initial state.
Preferably, the molar concentration of said ATP, said GTP, said CTP and said UTP is 10mM.
Preferably, the concentration of the T7 RNA polymerase in the transcription system is controlled to be 2-3U/. Mu.L in the initial state of the transcription system.
Preferably, the concentration of the DNA template in the transcription system is 30-60 ng/. Mu.L.
Preferably, the concentration of DTT in the transcription system is 8-12 mM. For example, the concentration of DTT in the transcription system is 8mM, 9mM, 10mM, 11mM, 12mM.
Preferably, the concentration of NaCl in the transcription system is 3-6 mM. For example, the concentration of NaCl in the transcription system is 3mM, 4mM, 5mM, 6mM.
Specifically, adding a DNA template, tris-HCl, DTT, inorganic pyrophosphatase, naCl, T7 RNA polymerase, ATP, GTP, CTP, UTP, spermidine and magnesium chloride into a transcription reaction liquid, then incubating for 1-3 h at 35-38 ℃, and finally adding DNase I to digest and remove the DNA template, thus obtaining a reaction liquid containing RNA.
Preferably, the DNase I is added in an amount of 0.02 to 0.08U/. Mu.L, more preferably 0.04 to 0.06U/. Mu.L.
Preferably, the temperature of the digestion reaction is 35 to 38 ℃, and the time of the digestion reaction is 10 to 30min, more preferably 10 to 20min.
Preferably, the transcription system comprises 30-60 ng/. Mu.L of DNA template, 35-45 mM Tris-HCl, 8-12 mM DTT, 2-6U/mL inorganic pyrophosphatase, 3-6 mM NaCl, 2-5U/. Mu.L T7 RNA polymerase, 8-12 mM ATP, 8-12 mM GTP, 8-12 mM CTP, 8-12 mM UTP, 1-3 mM spermidine, 25-27 mM MgCl 2 。
According to some specific and preferred embodiments, the transcription system specifically comprises 50 ng/. Mu.L of DNA template, 40mM Tris-HCl (pH 7.9), 10mM DTT,4U/mL inorganic pyrophosphatase, 5mM NaCl, 2.5U/. Mu.LT 7 RNA polymerase, 10mM ATP,10mM GTP,10mM CTP,10mM UTP,2mM spermidine, 25-27 mM MgCl 2 。
Further preferably, the transcription system comprises 50 ng/. Mu.L of DNA template, 40mM Tris-HCl,10mM DTT,4U/mL inorganic pyrophosphatase, 5mM NaCl, 2.5U/. Mu. L T7 RNA polymerase, 10mM ATP,10mM GTP,10mM CTP,10mM UTP,2mM spermidine, 26mM MgCl 2 。
The second aspect of the present invention also provides a kit for preparing transcript mRNA, said kit comprising reagents for preparing a transcription system, and optionally lithium chloride, said reagents comprising n X (35-45) mM Tris-HCl, n X (8-12) mM DTT, n X (2-6) U/mLN× (3-6) mM NaCl, n× (2-5) U/. Mu. L T7 RNA polymerase, n× (8-12) mM ATP, n× (8-12) mM GTP, n× (8-12) mM CTP, n× (8-12) mM UTP, n× (1-3) mM spermidine, n× (25-27) mM MgCl 2 Wherein n is more than or equal to 1 and less than or equal to 20.
The components of the kit can be packaged independently, or part of the components can be mixed and packaged, for example, tris-HCl, DTT, inorganic pyrophosphatase, naCl, spermidine and MgCl 2 Package in combination, and/or package ATP, GTP, CTP and UTP in combination.
According to some specific and preferred embodiments, the reagent comprises n.times.40 mM Tris-HCl, n.times.10 mM DTT, n.times.04U/mL inorganic pyrophosphatase, n.times.5 mM NaCl, n.times.2.5U/. Mu. L T7 RNA polymerase, n.times.10 mM ATP, n.times.10 mM GTP, n.times.10 mM CTP, n.times.10 mM UTP, n.times.2 mM spermidine, n.times.25-27 mM MgCl 2 Wherein n is more than or equal to 1 and less than or equal to 20.
In a third aspect, the invention provides an in vitro method of preparing a transcript mRNA using said method or use of a transcript mRNA prepared using said kit in medicine.
Specifically, the drug is an mRNA drug, an mRNA vaccine, a polypeptide or a protein drug.
By optimizing the reaction system and method, the yield and the expression quantity of the in-vitro prepared transcript mRNA can be obviously improved without complex purification methods such as RP-HPLC (high-performance liquid chromatography) and the in-vitro translation efficiency of the prepared transcript mRNA is high while the dsRNA content percentage in the in-vitro prepared transcript mRNA is reduced to below 0.7 percent.
For the preparation method, a purification method which is high in toxicity and difficult to expand production is omitted, so that industrialization is facilitated, and the production cost of transcript mRNA is reduced.
For disease treatment, such as tumor immunotherapy by using mRNA-encoded antibody, the translation efficiency of mRNA is improved, the toxic and side effects are reduced, and the dosage of mRNA medicine and the toxic and side effects of the medicine can be greatly reduced, so that the method has great positive significance in terms of medicine formation and curative effect.
Compared with the prior art, the invention has the following advantages:
the inventor can obviously inhibit the formation of dsRNA in a transcription product by adjusting the formula of a transcription system for preparing the transcript mRNA through in vitro transcription, so that the yield, the dsRNA content and the expression quantity of the transcript mRNA are balanced, the cost of industrially and standardizing in vitro production of the transcript mRNA is reduced, the transcript mRNA has higher in vitro translation efficiency and small influence on immunogenicity, can be directly used for mRNA medicaments, mRNA vaccines, polypeptides or protein medicaments and the like, avoids the step of removing the dsRNA by transferring the transcription product through complicated RP-HPLC purification which is difficult to produce in an amplifying way under the GMP condition, and has great significance for development and application of the mRNA medicaments, the mRNA vaccines, the polypeptides or the protein medicaments and the like.
Drawings
FIG. 1 shows different Mg 2+ Yield of mRNA transcribed in vitro at concentration;
FIG. 2 shows different Mg 2+ dsRNA content in mRNA transcribed in vitro at concentration;
FIG. 3 shows different Mg 2+ In vitro expression levels of transcripts at concentrations.
Detailed Description
The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other. The materials, reagents, etc. used in the examples of the present invention are commercially available.
Because the existing transcript mRNA preparation method cannot simultaneously give consideration to the yield, the impurity dsRNA and the expression quantity of the transcript mRNA, the preparation cost is high or the influence on immunogenicity is large, and the like, the large-scale production cost of the transcript mRNA is too high, the large-scale production of the transcript mRNA is difficult under the GMP condition, and the development of mRNA drugs, mRNA vaccines, polypeptides or protein drugs is limited. The inventors have made extensive studies to propose an in vitro preparation method of transcript mRNA by in vitro transcription using DNA templates and chlorinePrecipitation of lithium-oxide to obtain the transcript mRNA, the in vitro transcribed transcription system comprising a DNA template, tris-HCl, DTT, inorganic pyrophosphatase, naCl, T7 RNA polymerase, ATP, GTP, CTP, UTP, spermidine, and Mg 2+ Controlling the transcription system to be in an initial state, wherein the concentration of the T7 RNA polymerase in the transcription system is 2-5U/. Mu.L, and the Mg 2+ The molar concentration of ATP, GTP, CTP or UTP in the transcription system is 25-27 mM, and the molar concentration of Mg 2+ The molar ratios in the transcription system were 1:2 to 3.
The technical scheme and effect of the present invention will be further described with reference to specific examples and comparative examples.
In the examples and comparative examples of the present invention:
inorganic pyrophosphatase is available from offshore protein biotechnology Co., ltd (cat# GMP-M036);
t7 RNA polymerase was purchased from offshore protein Biotechnology Co., ltd (cat# GMP-E121);
NTP is purchased from Shanghai megadimension technology development Co., ltd (ATP product number: R1331; CTP product number: R3331; GTP product number: R2331; UTP product number: R5331);
DNase I was purchased from offshore protein Biotechnology Co., ltd (cat# GMP-E127).
DNA linearization
IVT plasmid pJ241 (autonomously developed, comprising kanamycin resistance gene, T7 promoter sequence, 5 '-and 3' -UTR, poly (A) and type-IIS restriction site sequences), comprises the target sequence of the Receptor Binding Domain (RBD) of the Spike (S) protein encoding the novel coronavirus SARS-CoV-2, 5'-UTR, 3' -UTR and polyA signal. pJ241 was digested with type-IIS restriction enzymes. Each 10. Mu.g of plasmid was mixed with 10U of Esp I/BsmBI and incubated at 37℃for 4 hours to ensure complete linearization. The reaction was quenched by the addition of 1/10 volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, thoroughly mixed and cooled at-20℃for 1 hour. The linearized DNA was precipitated by centrifugation at 13800g for 15min at 4℃and washed twice with 70% ethanol and resuspended in nuclease-free water.
In vitro transcription of mRNA
The transcription systems of examples and comparative examples are essentially identical, except for MgCl 2 Final concentration of (2).
Transcription reaction system: 50 ng/. Mu.L of linearized plasmid DNA template, 40mM Tris-HCl,5mM NaCl,10mM DTT,2mM spermidine, 4U/mL inorganic pyrophosphatase, 2.5U/. Mu. L T7 RNA polymerase, 10mM ATP,10mM GTP,10mM CTP,10mM UTP, an appropriate amount of MgCl 2 。
Wherein, in different reaction systems, mgCl 2 The final concentration of (C) was 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 26mM, 36mM, 46mM, respectively.
After the reaction systems are respectively and uniformly mixed, incubating for 2 hours at 37 ℃, adding DNase I with the adding amount of 0.05U/mu L, and digesting for 15 minutes at 37 ℃ to obtain a reaction solution containing RNA.
After DNAse I digestion, the mRNA was purified using a Monorch RNA purification kit (#T2057L, NEB), dissolved using DEPC-Treated water, and the concentration was measured using an ultraviolet spectrophotometer (NanoDrop 2000, thermo) to calculate the mRNA yield m 1 。
The mRNA yields at various magnesium ion concentrations are shown in FIG. 1 and Table 1.
Table 1: mg of + Concentration and mRNA yield
Overall, with increasing magnesium ion concentration, the in vitro transcribed mRNA yield gradually increased, but the stepwise changes differentiated. When the magnesium ion concentration reached 26mM, the yield reached the plateau. Wherein the yield per reaction increases more slowly during the magnesium ion concentration range from 16mM to 21 mM; the yield increase per reaction was more pronounced during the magnesium ion concentration range from 21mM to 26 mM; there was no significant change in the yield per reaction during the increase in magnesium ion concentration from 26mM to 46mM.
In vitro transcription typically incorporates dsRNA (double stranded RNA) in an amount detected by the following method: direction 96Adding standard substance or sample into the pore plate, adding antibody of anti-dsRNA receptor and antibody of anti-dsRNA donor, incubating at 4deg.C for 24 hr, detecting dsRNA concentration on HTRF equipment by using visual double-stranded RNA detection kit (Cat#64RNAPEG, cisbio), and calculating dsRNA mass m in the reaction solution according to the volume of RNA suspension 2 . mass percent of dsRNA in mRNA = mass m of dsRNA 2 Yield of mRNA m 1 ×100％，
The dsRNA content at various magnesium ion concentrations is shown in FIG. 2 and Table 2.
Table 2: mg of + Concentration and dsRNA content
| Mg + Concentration (mM) | Percentage by mass of dsRNA | Mg + Concentration (mM) | Percentage by mass of |
| 16 | 0.060％ | 22 | 0.160％ |
| 17 | 0.045％ | 23 | 0.280％ |
| 18 | 0.033％ | 24 | 0.660％ |
| 19 | 0.040％ | 26 | 0.690％ |
| 20 | 0.035％ | 36 | 0.900％ |
| 21 | 0.037％ | 46 | 1.290％ |
Overall, as the magnesium ion concentration increases, the dsRNA content in the in vitro transcript increases, but the stepwise changes differentiate. The dsRNA content was somewhat reduced, but the trend was not apparent, when the magnesium ion concentration was increased from 16mM to 21 mM; the increase in magnesium ion concentration from 21mM to 46mM showed a substantially linear increase in dsRNA content, but a plateau occurred in the 24-26mM segment.
Referring to FIGS. 1 and 2, the effect of magnesium ion concentration adjustment on yield and dsRNA impurity content was not completely synchronized with other conditions of the transcription system.
RNA capping
Each 10. Mu.g of uncapped mRNA was heated at 65℃for 10 minutes, placed on ice for 5 minutes, and then incubated with 10U of vaccinia virus capping enzyme, 50U of mRNA Cap 2' -O-methyltransferase, 0.2mM SAM, 0.5mM GTP and 1U of RNase inhibitor at 37℃for 60 minutes to produce Cap1 modified structure.
RNA purification
The modified mRNA was purified using a Monarch RNA purification kit (#T2057L, NEB), and the RNA was resuspended in nuclease-free water and stored at-20 ℃.
Mrna expression in vitro:
the purified transcription product is transfected into human embryo kidney cells to detect the expression level of RBD protein. Specifically, human embryonic cells (HEK 293T, ATCC) were selected and cultured using DMEM (# 11965092, thermosusher) medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco-FBS, life Technologies). Cells were seeded in 6-well flat bottom cell culture plates (Corning) at 80,000 cells per well, transfected with Lipofectamine 2000 (# 11668019,Thermo Fisher) after 24 hours, 3. Mu.g mRNA per well, and cell culture supernatants were collected after 18-24 hours of transfection, and analyzed for RBD protein expression using SARS-CoV-2 (2019-nCoV) Spike RBD ELISA KIT (KIT 40592, yinPaoshan).
The protein expression levels of transcripts obtained at different magnesium ion concentrations are shown in FIG. 3 and Table 3.
Table 3: mg of + Concentration and in vitro protein expression level of the transcripts obtained
Overall, the increase in magnesium ion concentration inhibited the translation activity of mRNA under the other conditions of the transcription system, but the translation activity was increased when the magnesium ion concentration was 26 mM.
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. An in vitro preparation method of transcript mRNA is characterized in that the transcript mRNA is obtained by in vitro transcription and lithium chloride precipitation of a DNA template,
the transcription system of the in vitro transcription comprises a DNA template, tris-HCl, DTT, inorganic pyrophosphatase, naCl and T7 RNA polymerase, NTP, spermidine, and Mg 2+ ，
Controlling the transcription system to be in an initial state, wherein the concentration of the T7 RNA polymerase in the transcription system is 2-5U/. Mu.L, and the Mg 2+ 25-27 mM, the molar concentration of said NTP in the transcription system being in relation to said Mg 2+ The molar ratio in the transcription system is 1:0.5 to 0.7.
2. The method for preparing transcript mRNA according to claim 1, wherein the mass percentage of double-stranded RNA in the transcript mRNA is less than 0.7%.
3. The method for preparing transcript mRNA in vitro according to claim 1, wherein the molar concentration of ATP, GTP, CTP and UTP in the transcription system is controlled to be 8 to 12mM, respectively, in the initial state.
4. The method for preparing transcript mRNA in vitro according to claim 1, wherein the concentration of said T7 RNA polymerase in the transcription system is 2 to 3U/. Mu.L in the initial state of the transcription system.
5. The method for preparing transcript mRNA in vitro according to claim 1, 3 or 4, wherein said DNA template has a concentration of 30 to 60 ng/. Mu.L in the transcription system.
6. The method for preparing transcript mRNA in vitro according to claim 5, wherein said DTT has a concentration of 8mM to 12mM in the transcription system.
7. The method for preparing transcript mRNA in vitro according to claim 1, wherein the concentration of said NaCl in the transcription system is 3 to 6mM.
8. The method for preparing transcript mRNA in vitro according to claim 1, wherein said transcription system in the initial stateComprises 30 to 60 ng/. Mu.L of DNA template, 35 to 45mM of Tris-HCl,8 to 12mM of DTT,2 to 6U/mL of inorganic pyrophosphatase, 3 to 6mM of NaCl,2 to 5U/. Mu.L of T7 RNA polymerase, 8 to 12mM of ATP,8 to 12mM of GTP,8 to 12mM of CTP,8 to 12mM of UTP,1 to 3mM of spermidine and 25 to 27mM of MgCl 2 。
9. A kit for preparing transcript mRNA, comprising a reagent for preparing a transcription system, and optionally lithium chloride, wherein the reagent comprises n X (35-45) mM Tris-HCl, n X (8-12) mM DTT, n X0 (2-6) U/mL inorganic pyrophosphatase, n X (3-6) mM NaCl, n X (2-5) U/. Mu. L T7 RNA polymerase, n X (8-12) mM ATP, n X (8-12) mM GTP, n X (8-12) mM CTP, n X (8-12) mM UTP, n X (1-3) mM spermidine, n X (25-27) mM MgCl 2 Wherein n is more than or equal to 1 and less than or equal to 20.
10. Use of the transcript mRNA according to any one of claims 1 to 8 for in vitro preparation or the transcript mRNA prepared using the kit according to claim 9 in medicine.
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