T7 rna polymerase variants and their use in rna production

Abstract

The present application provides a T7 RNA polymerase variant and a preparation method thereof, and the T7 RNA polymerase variant of the present application is used for in vitro transcription to prepare RNA, which can significantly reduce the generation of dsRNA contaminants in the RNA product, and realizes the generation of dsRNA impurities from the source. In addition, the present application also provides a composition, a kit and a method for preparing RNA by in vitro transcription comprising a T7 RNA polymerase variant.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an RNA polymerase variant and its application. BACKGROUND

[0002] The success of mRNA vaccines of Pfizer and Moderna companies has sharply increased the attention of mRNA in the field of biological drugs. Compared with traditional subunit vaccines and inactivated vaccines, mRNA vaccines have short production cycle, low cost, can quickly develop new candidate vaccines to cope with viral mutations, and have high immunogenicity as intracellular antigens, and thus have excellent actual clinical effects. Therefore, they have irreplaceable advantages in the field of cancer vaccines, influenza, HIV and other highly variable virus vaccines.

[0003] The generation and development of in vitro transcription (IVT) mRNA technology lay the foundation for the emergence of mRNA vaccines. IVT mRNA technology refers to the use of DNA as a template, nucleotides as raw materials, and T3, T7 or SP6 bacteriophage-derived RNA polymerase to transcribe and synthesize mRNA in a cell-free system in vitro. Among them, T7 RNA polymerase is the most widely used. In the process of IVT mRNA preparation, T7 RNA polymerase only completes the conformational change from the initial promoter recognition to the start of RNA synthesis, and then to the release of the RNA chain to the elongation state. During this process, the conformation of T7 RNA polymerase needs to undergo complex changes, and impurities are inevitably produced, including double-stranded RNA (dsRNA) impurities with high immunogenicity. dsRNA impurities can be recognized by RIG-1 and MDA5 in vivo, leading to protein synthesis inhibition and cell death and other adverse results, which greatly limits the application of mRNA products. Therefore, it is particularly important to reduce dsRNA in IVT mRNA products.

[0004] Traditional dsRNA impurity removal is mainly achieved by downstream purification and additional additives, but the purification process is tedious and not cost-effective. Karikó et al. found that replacing uridine with m1ψ (N1-methylpseudouridine) in IVT mRNA synthesis can reduce the immunogenicity caused by dsRNA impurities and improve translation efficiency. However, Mulroney et al. found that the introduction of modified nucleosides can cause ribosome slippage, resulting in the translation of non-target proteins and unexpected consequences caused by immunogenic impurities. Therefore, reducing the production of dsRNA from the source to reduce the immunogenicity of mRNA vaccines is a feasible method. SUMMARY

[0005] In a first aspect, the present application provides a T7 RNA polymerase variant, which has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity to wild-type T7 RNA polymerase (SEQ ID NO: 1), wherein the variant comprises at least one mutation of an amino acid; wherein the type of mutation is insertion, substitution, deletion or any combination thereof; the amino acid position of the variant sequence is referred to SEQ ID NO: 1.

[0006] In a second aspect, the present application provides a biological material selected from one or more of the following:

[0007] 1) a polynucleotide molecule encoding the above-mentioned variant;

[0008] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0009] 3) a host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

[0010] In a third aspect, the present application provides a method for preparing the above-mentioned RNA polymerase variant.

[0011] In a fourth aspect, the present application provides a composition comprising at least one RNA polymerase variant as described in the present application.

[0012] In a fifth aspect, the present application provides a kit comprising at least one RNA polymerase variant as described in the present application.

[0013] In a sixth aspect, the present application provides the use of the above-mentioned RNA polymerase variant, composition or kit in the preparation of RNA by in vitro transcription.

[0014] In a seventh aspect, the present application provides a method for reducing the generation of dsRNA impurities in the preparation of RNA by in vitro transcription, and a method for preparing RNA. DETAILED DESCRIPTION

[0016] RNA polymerase variant

[0017] In a first aspect, the present application provides a T7 RNA polymerase variant, which has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more sequence identity to wild-type T7 RNA polymerase (SEQ ID NO: 1), wherein the variant comprises at least one mutation of an amino acid; wherein the type of mutation is insertion, substitution, deletion or any combination thereof; the amino acid position of the variant sequence is referred to SEQ ID NO: 1.

[0018] In some embodiments, the RNA polymerase variant comprises at least one mutation at an amino acid site selected from the group consisting of E167, V174, or G753, wherein the mutation type is selected from substitution or deletion, as compared to SEQ ID NO: 1.

[0019] In some embodiments, the mutation at the E167 site is E167D.

[0020] In some embodiments, the mutation at the V174 site is selected from V174G, DEL174.

[0021] In some embodiments, the mutation at the G753 site is G753A, G753T, G753Q, G753N, or G753S.

[0022] In some embodiments, the amino acid sequence of the variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2-9.

[0023] In some embodiments, the amino acid sequence of the variant is set forth in any one of SEQ ID NOs: 2-9.

[0024] Biological material

[0025] The present application provides a polynucleotide encoding any one of the above-mentioned T7 RNA polymerase variants. In some embodiments, various modifications can be present in the coding region of the polynucleotide sequence of the present application, as long as the variant amino acid sequence of the present application does not change with the degeneracy of the codon or the preferred codon in the organism expressing the variant. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of the present application can be selected from the group consisting of SEQ ID NOs: 11-18.

[0026] The expression vector provided by the present application comprises a polynucleotide molecule encoding the RNA polymerase variant of the present application. In some embodiments, the expression vector further comprises one or more regulatory sequences, such as but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, terminator sequences; wherein the regulatory sequence is operably linked to the polynucleotide molecule encoding the variant.

[0027] In some embodiments, the expression vector can be a linear or circular DNA molecule, which generally comprises elements such as a multiple cloning site, a resistance gene, a replication initiation site, etc. In some embodiments, the expression vector of the present application is preferably pQE-80L.

[0028] The host cell according to the present application refers to any cell that is favorable for the expression of the variant according to the present application, i.e. any cell that is susceptible to being transformed, transfected or transduced with the expression vector according to the present application, and encompasses any progeny cell that is not identical to the parent cell due to mutations that occur during replication.

[0029] In some embodiments, the host cell is a prokaryotic cell, which can be selected from gram-positive bacteria or gram-negative bacteria. In some embodiments, the host cell is a gram-positive bacteria, including but not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. In some embodiments, the host cell is a gram-negative bacteria, including but not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Limnobacter, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21 (DE3).

[0030] Method for preparing RNA polymerase variant

[0031] The method for preparing the RNA polymerase variant according to the present application comprises 1) culturing the host cell according to the present application under conditions suitable for the expression of the variant; and 2) recovering the variant.

[0032] In some embodiments, the method for recovering the variant can be any method known in the art, such as centrifugal separation, filtration, treatment with a protein precipitant (salting-out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0033] In some embodiments, the method for preparing further comprises a step of purifying the variant, which can be any method known in the art, such as chromatography (e.g. ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), ammonium sulfate precipitation, etc.

[0034] Composition

[0035] The composition according to the present application comprises at least one RNA polymerase variant according to the present application.

[0036] The composition described in the present application can be a composition for storing the RNA polymerase variant. In some embodiments, the composition described in the present application can optionally comprise, in addition to the RNA polymerase variant described above, buffer components (such as Tris base, Tris-HCl, HEPES, MOPS), salts (such as NaCl), enzyme inhibitors (such as EDTA), reducing agents (such as DTT), surfactants (such as Triton X-100), stabilizers (such as glycerol), and the like. In some embodiments, the composition for storing the RNA polymerase variant described in the present application comprises the RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0037] In some embodiments, the composition further comprises a template DNA. In some embodiments, the composition further comprises at least one in vitro transcription component, which can be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNAse inhibitors, inorganic pyrophosphatases, magnesium ions, and the like.

[0038] Kit

[0039] The kit provided in the present application comprises at least one RNA polymerase variant as described in the present application.

[0040] In some embodiments, the kit can further comprise one or more in vitro transcription reagents, such as buffer components, modified or unmodified nucleoside triphosphates, RNAse inhibitors, pyrophosphatases, magnesium ions, water, and the like. In some embodiments, the kit further comprises a cap analog, which can be selected from an unmethylated cap analog, a dimethylated cap analog, a trimethylated cap analog, a dimethylated symmetric cap analog, or an anti reverse cap analog. In some embodiments, the kit further comprises at least one in vitro transcription component, which can be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNAse inhibitors, inorganic pyrophosphatases, magnesium ions, and the like. In one embodiment, the in vitro transcription system component can be selected from commercially available RNA in vitro transcription reagents.

[0041] Applications

[0042] The present application provides the use of the above-described RNA polymerase variant, composition, or kit in the preparation of RNA by in vitro transcription. The present application also provides the use of at least one variant as described in the present application in the preparation of RNA by in vitro transcription to reduce the generation of dsRNA impurities.

[0043] In some embodiments, the in vitro transcription to prepare RNA comprises contacting a DNA template with at least one RNA polymerase variant described herein, incubating in an in vitro transcription reaction system, and obtaining a target product.

[0044] Methods

[0045] The present application provides a method for reducing the generation of dsRNA impurities in the process of in vitro transcription to prepare RNA, comprising contacting a DNA template with one or more T7 RNA polymerase variants described herein, incubating in an in vitro transcription reaction system.

[0046] The present application also provides a method for preparing RNA, comprising contacting a DNA template with at least one RNA polymerase variant described herein, incubating in an in vitro transcription reaction system, and obtaining a target product.

[0047] In some embodiments, the in vitro transcription system comprises nucleotide triphosphates. In some embodiments, the nucleotide triphosphates can be selected from modified or unmodified nucleotide triphosphates (including analogs thereof). In some embodiments, the nucleotide triphosphates can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the nucleotide triphosphates can be selected from modified nucleotide triphosphates, including but not limited to mlA (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), mlψ (N1-methyl-pseudouridine), nucleotide triphosphates with labels (the labels can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0048] The in vitro transcription reaction system and incubation conditions suitable for generating RNA products are well known in the art, and those of ordinary skill in the art can determine the appropriate reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, etc. in consideration of the optimal activity of the RNA polymerase. In some embodiments, the in vitro transcription reaction system described herein comprises in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleotide triphosphates, RNAase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the incubation temperature in the incubation step described herein is 30-50°C, preferably 37°C. In some embodiments, the incubation time in the incubation step described herein is 20-240 min, preferably 60 min.

[0049] In some embodiments, the RNA product prepared by the method of the present application has higher yield, and / or has higher integrity, and / or has less dsRNA impurity content, and / or more capped mRNA product, etc. compared to the use of wild type RNA polymerase (SEQ ID NO: 1).

[0050] In some embodiments, the relative residual amount of dsRNA impurity (ratio of dsRNA residual amount of variant group / dsRNA residual amount of WT group) of the RNA product prepared by the protocol of the present application is lower than 95%, lower than 90%, lower than 85%, lower than 80%, lower than 75%, lower than 70%, lower than 65%, lower than 60%, lower than 55%, lower than 50%, lower than 45%, lower than 40%, lower than 35%, lower than 30%, lower than 25%, lower than 20%, lower than 15%, lower than 10%, lower than 5%, lower than 2%, lower than 1% or lower compared to the use of wild type RNA polymerase.

[0051] In some embodiments, one or more buffer components are included in the in vitro transcription reaction system. In some embodiments, the buffer component can be selected from Tris-HCl, Hepes, citric acid or commercially available buffer components. In some embodiments, the in vitro transcription buffer system further comprises RNAse inhibitor, inorganic pyrophosphatase, magnesium ion. In some embodiments, the in vitro transcription buffer system further comprises water (such as: DEPC-water, RNase-free water, DNase-free water, sterilized purified water, deionized water, distilled water, etc.). In some embodiments, the in vitro transcription buffer system further comprises a cap analog, which can be selected from an unmethylated cap analog, a dimethylated cap analog, a trimethylated cap analog, a dimethylated symmetric cap analog, or an anti reverse cap analog.

[0052] Other embodiments:

[0053] 1. An RNA polymerase variant, which has at least 75% sequence identity to SEQ ID NO: 1 and comprises at least one mutation selected from the amino acid positions E167, V174 or G753, wherein the mutation type is substitution or deletion; the amino acid position of the variant sequence is referred to SEQ ID NO: 1.

[0054] 2. The variant of item 1, wherein:

[0055] (1) the mutation at E167 position is E167D:

[0056] (2) the mutation at V174 position is selected from V174G, DEL174;

[0057] (3) the mutation at position G753 is selected from G753A, G753T, G753Q, G753N, or G753S.

[0058] 3. An RNA polymerase variant, wherein the amino acid sequence of the variant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more sequence identity to any one of the sequences set forth in SEQ ID NOs: 2-9.

[0059] 4. A biological material, wherein the biological material is selected from one or more of the following:

[0060] 1) a polynucleotide molecule encoding a variant as claimed in any one of items 1-3;

[0061] 2) an expression vector comprising a polynucleotide molecule as claimed in 1);

[0062] 3) a host cell comprising a polynucleotide molecule as claimed in 1), or a host cell comprising an expression vector as claimed in 2).

[0063] 5. A method of producing a variant as claimed in any one of items 1-3, comprising:

[0064] (1) culturing a host cell as claimed in item 4; and

[0065] (2) recovering the variant.

[0066] 6. A composition comprising a variant as claimed in any one of items 1-3.

[0067] 7. A kit comprising a variant as claimed in any one of items 1-3.

[0068] 8. Use of a variant as claimed in any one of items 1-3, a composition as claimed in item 6, or a kit as claimed in item 7, in in vitro transcription.

[0069] 9. Use of a variant as claimed in any one of items 1-3, a composition as claimed in item 6, or a kit as claimed in item 7, to reduce the production of dsRNA impurities in a process for preparing RNA by in vitro transcription.

[0070] 10. Use as claimed in item 9, wherein the process for preparing RNA by in vitro transcription comprises contacting a DNA template with a variant as claimed in any one of items 1-3, and incubating in an in vitro transcription system.

[0071] 11. A method of reducing the production of dsRNA impurities in a process for preparing RNA by in vitro transcription, comprising contacting a DNA template with at least one variant as claimed in any one of items 1-3, and incubating in an in vitro transcription reaction system.

[0072] 12. Use of the method of item 11 for in vitro transcription of RNA.

[0073] 13. A method for preparing RNA, comprising contacting a DNA template with at least one RNA polymerase variant of items 1-3, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

[0074] Advantages

[0075] The present application can significantly reduce the generation of dsRNA by modifying the wild-type T7 RNA polymerase for use in the process of in vitro transcription to prepare RNA, thereby reducing dsRNA pollution from the source. All variants in Example 1 of the present application can effectively reduce the level of impurity dsRNA, and the residual amount of dsRNA impurities is reduced by at least 70%. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 Figure is a schematic diagram for construction of recombinant plasmid;

[0077] Figure 2 Figure is a schematic diagram for dsRNA residual value difference;

[0078] In the figure, the correspondence between T7 wild-type polymerase and its variants and amino acids is shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] DETAILED DESCRIPTION

[0086] The technical solutions of the present application will be further illustrated below in combination with specific examples. However, the following examples are only examples of the present application and do not represent or limit the protection scope of the present application. The protection scope of the present application is subject to the claims. In the following examples, if not specifically stated, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and technical means used are conventional methods and means in the art.

[0087] In the embodiments of the present application, the definition of enzyme activity is that 1 nmol of [H]ATP is incorporated into acid-insoluble precipitate per 1 unit of enzyme in 1 hour under the condition of 37°C and pH 8.0. 3 The amount of enzyme required for incorporating 1 nmol of [H]ATP into acid-insoluble precipitate per 1 unit of enzyme in 1 hour is defined as 1 unit of activity.

[0088] Example 1 Preparation of RNA polymerase variants

[0089] After the nucleotide sequences SEQ ID NO: 10-18 (the encoded amino acid sequences correspond to SEQ ID NO: 1-9) are synthesized by DNA sequence synthesis, PCR amplification is performed, and then the BseRI and HindIII enzyme digestion sites of the expression vector pQE-80L are introduced to obtain a recombinant expression vector. The constructed vector is introduced into E. coli BL21 (DE3) by transformation technology, coated on an LB plate containing ampicillin resistance, and cultured in a 37°C incubator overnight. The single colonies that grow out are subjected to plasmid extraction and sequencing, and finally a recombinant engineering bacterium containing the target gene is obtained. The E. coli recombinant strain that has passed sequencing is inoculated into LB medium for overnight activation culture, 1-5% V / V is inoculated into fermentation broth (LB medium), and the culture is continued until the OD 600 value is 0.6-0.8. Then, 0.5 mol / L IPTG is added to continue the culture for 4-6 h. The strain is collected by centrifugation at 12000 rpm and 5°C, and the collected strain is washed with 0.2M PBS buffer with a pH value of 7.0 to obtain the bacterial body. After ultrasonic crushing, affinity chromatography purification is performed to obtain the RNA polymerase stock solution.

[0090] Example 2 In vitro transcription reaction verification

[0091] The enzyme stock solution obtained in Example 1 is diluted with a storage buffer to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 2 are loaded into an eight-row array and mixed, and then centrifuged. The eight-row array is placed in a PCR instrument for 1 h of reaction at 37°C, and then 36 μL of magnetic beads (Vazyme, item number: N412) are added and mixed, and the mixture is incubated at room temperature for 2-5 min. The mixture is placed on a magnetic stand to purify the mRNA. After purification, the purified mRNA is transferred to an RNase-free centrifuge tube, and the dsRNA impurity content is tested by a dsRNA detection kit (Vazyme, item number: DD3509).

[0092] Table 2: Reaction system ratio

[0093]

[0094] The dsRNA detection results are shown in Table 3. Figure 2 Compared with the wild-type T7 RNA polymerase (WT), all the variants in Example 1 can effectively reduce the level of dsRNA impurities, and the residual amount of dsRNA impurities is reduced by at least 70%.

[0095] The above merely describes the preferred embodiments of the present application, and is not intended to limit the technical solutions of the present application in any form. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A variant of RNA polymerase characterized in that, The amino acid sequence of the variant comprises a mutation at position G753 relative to SEQ ID NO: 1, the amino acid sequence of the variant being as set forth in any one of SEQ ID NOs: 5-9.

2. Biomaterial, characterized in that, The biological material is selected from one or more of: 1) a polynucleotide molecule encoding a variant as claimed in claim 1 ; 2) an expression vector comprising a polynucleotide molecule as claimed in 1); 3) a host cell comprising a polynucleotide molecule as claimed in 1, or a host cell comprising an expression vector as claimed in 2).

3. A method of producing a variant according to claim 1, characterized in that, comprising: (1) culturing a host cell as claimed in claim 2; and (2) recovering the variant.

4. Composition, characterized in that, comprising a variant as claimed in claim 1.

5. A kit characterized in that, comprising a variant as claimed in claim 1.

6. Use of a variant as claimed in claim 1, a composition as claimed in claim 4, or a kit as claimed in claim 5, in the preparation of mRNA in vitro transcription.

7. Use of a variant as claimed in claim 1, a composition as claimed in claim 4, or a kit as claimed in claim 5, in reducing the generation of dsRNA impurities in the preparation of mRNA in vitro transcription.

8. Use according to claim 7, wherein the compound is ###0002### The process of preparing mRNA comprises contacting a DNA template with a variant as claimed in claim 1, incubating in an in vitro transcription system.

9. A method of reducing the formation of dsRNA impurities in an in vitro transcription process for preparing mRNA, characterized in that, comprising contacting a DNA template with at least one variant as claimed in claim 1, incubating in an in vitro transcription reaction system.

10. Use of a method as claimed in claim 9, in the preparation of mRNA in vitro transcription.

11. A method of preparing mRNA, characterized in that, comprising contacting a DNA template with at least one RNA polymerase variant as claimed in claim 1, incubating in an in vitro transcription reaction system, to obtain a target mRNA product. comprising contacting a DNA template with at least one RNA polymerase variant as claimed in claim 1, incubating in an in vitro transcription reaction system, to obtain a target mRNA product.

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