T7 RNA polymerase variant and application thereof in RNA preparation

Abstract

According to the T7RNA polymerase variant and the preparation method thereof, the T7RNA polymerase variant is used for preparing RNA through in-vitro transcription, generation of dsRNA pollutants in RNA products can be remarkably reduced, and generation of dsRNA impurities is controlled from the source. In addition, the invention further provides a composition containing the T7RNA polymerase variant, a kit and a method for preparing RNA through in-vitro transcription.

Description

Field of the Invention

[0001] The present invention relates to the field of biotechnology, and particularly to RNA polymerase variants and their applications. Background of the Invention

[0003] The success of the mRNA vaccines of Pfizer and Moderna has sharply increased the attention of mRNA in the field of biopharmaceuticals. Compared with traditional subunit vaccines and inactivated vaccines, mRNA vaccines have a short production cycle and low cost, can quickly develop new candidate vaccines to cope with virus mutations, and have high immunogenicity as intracellular antigens, with excellent actual clinical effects. Therefore, they have irreplaceable advantages in the fields of cancer vaccines, influenza, HIV and other highly variable virus vaccines.

[0004] The generation and development of in vitro transcribed (IVT) mRNA technology have laid the foundation for the birth of mRNA vaccines. IVT mRNA technology refers to the process of transcribing and synthesizing mRNA in an acellular system in vitro, using DNA as a template and nucleotides as raw materials, and then using RNA polymerases derived from T3, T7 or SP6 phages. Among them, T7 RNA polymerase is the most widely used. In the process of preparing mRNA by IVT, T7 RNA polymerase only completes the conformational change from the initial promoter recognition, to the start of RNA synthesis, and then to the release and rotation of the RNA chain to the elongation state in a single subunit. In this process, the conformation of T7 RNA polymerase needs to undergo complex changes, and it is inevitable to produce impurities, including double-stranded RNA (dsRNA) impurities with high immunogenicity. DsRNA impurities can be recognized by RIG-1 and MDA5 in vivo, leading to adverse results such as protein synthesis inhibition and cell death. This makes the application of mRNA products very limited. Therefore, it is particularly important to reduce dsRNA in IVT mRNA products.

[0005] Traditional removal of dsRNA impurities is mainly achieved through downstream purification and additional additives, etc., but the purification steps are cumbersome and not economical. The 2023 Nobel laureates in Physiology or Medicine, Karikó et al, found that replacing uridine with m1ψ (N1-methylpseudouridine) in the synthesis of IVTmRNA can reduce the immunogenicity caused by dsRNA impurities and improve translation efficiency at the same time; however, Mulroney et al found that the introduction of modified nucleosides may lead to ribosome slippage and translate non-target proteins, thus bringing immunogenic impurities and causing unexpected consequences. Therefore, reducing the production of dsRNA from the source to reduce the immunogenicity of mRNA vaccines is a feasible method. Summary of the Invention

[0006] In a first aspect, the present invention provides a T7 RNA polymerase variant having an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or higher sequence identity compared to the wild-type T7 RNA polymerase (SEQ ID NO: 1), wherein said variant contains at least one amino acid mutation; wherein the type of mutation is insertion, substitution, deletion or any combination thereof; the amino acid positions of the variant sequence are referenced to SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides a class of biological materials selected from one or more of the following:

[0008] 1) A polynucleotide molecule encoding the above variant;

[0009] 2) An expression vector containing the polynucleotide molecule as described in 1);

[0010] 3) A host cell containing the polynucleotide molecule as described in 1), or a host cell containing the expression vector as described in 2).

[0011] In a third aspect, the present invention provides a method for preparing the above RNA polymerase variant.

[0012] In a fourth aspect, the present invention provides a composition comprising at least one RNA polymerase variant as described in the present invention.

[0013] In a fifth aspect, the present invention provides a kit comprising at least one RNA polymerase variant as described in the present invention.

[0014] In a sixth aspect, the present invention provides the use of the above RNA polymerase variant, composition or kit in the preparation of RNA by in vitro transcription.

[0015] In a seventh aspect, the present invention provides a method for reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription, and the present invention also provides a method for preparing RNA. DETAILED DESCRIPTION OF THE INVENTION

[0017] RNA POLYMERASE VARIANT

[0018] In a first aspect, the present invention provides an RNA polymerase variant having an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or higher sequence identity compared to the wild-type T7 RNA polymerase (SEQ ID NO: 1), wherein said variant contains at least one amino acid mutation; wherein the type of mutation is insertion, substitution, deletion or any combination thereof; the amino acid positions of the variant sequence are referenced to SEQ ID NO: 1.

[0019] In some embodiments, the RNA polymerase variant has an amino acid sequence that contains at least one mutation selected from the following amino acid sites compared to SEQ ID NO: 1: E167, V174, or G753, wherein the type of mutation is selected from substitution or deletion.

[0020] In some embodiments, the mutation at the E167 site is E167D.

[0021] In some embodiments, the mutations at the V174 site are selected from V174G, DEL174.

[0022] In some embodiments, the mutations at the G753 site are G753A, G753T, G753Q, G753N, or G753S.

[0023] In some embodiments, the amino acid sequence of the variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any of the amino acid sequences shown in SEQ ID NOs: 2-9.

[0024] In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NOs: 2-9.

[0025] Biological material

[0026] The present invention provides polynucleotides encoding any of the above T7 RNA polymerase variants. In some embodiments, in the polynucleotide sequences of the present invention, various modifications may be present in the coding region, as long as the variant amino acid sequence of the present invention does not change due to codon degeneracy or due to preferred codons in the organism expressing the variant. In some embodiments, the polynucleotide sequences encoding the RNA polymerase variants of the present invention may be selected from SEQ ID NOs: 11-18.

[0027] The expression vector provided by the present invention contains a polynucleotide molecule encoding the RNA polymerase variant of the present invention. In some embodiments, the expression vector further contains one or more regulatory sequences, such regulatory sequences including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, terminator sequences; wherein the regulatory sequences are operably linked to the polynucleotide molecule encoding the variant.

[0028] In some embodiments, the expression vector may be a linear or circular DNA molecule, usually containing elements such as multiple cloning sites, resistance genes, replication origins, etc. In some embodiments, the expression vector of the present invention is preferably pQE-80L.

[0029] The host cell provided by the present invention refers to any cell that is beneficial to the expression of the variants of the present invention, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vector described in the present invention, and encompasses any progeny cells that are different from the parental cells due to mutations occurring during replication.

[0030] In some embodiments, the host cell is a prokaryotic cell, and can be selected from Gram-positive bacteria or Gram-negative bacteria. In some embodiments, the host cell is a Gram-positive bacterium, 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 bacterium, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Pelobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21(DE3).

[0031] Method for preparing RNA polymerase variant

[0032] The method for preparing an RNA polymerase variant provided by the present invention includes: 1) culturing the host cell described in the present invention under conditions suitable for variant expression; and 2) recovering the variant.

[0033] In some embodiments, the method for recovering the variant can be a method well-known in the art, such as centrifugal separation, filtration, treatment with a crystalline protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations of the above methods.

[0034] In some embodiments, the preparation method further includes a step of purifying the variant, and the purification step can be a method well-known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), ammonium sulfate precipitation method, etc.

[0035] Composition

[0036] The composition provided by the present invention comprises at least one RNA polymerase variant described in the present invention.

[0037] The composition described in the present invention may be a composition for storing RNA polymerase variants. In some embodiments, in addition to the above-mentioned RNA polymerase variants, the composition of the present invention may optionally contain: 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 TritonX-100), stabilizers (such as glycerol), and other components. In some embodiments, the composition for storing RNA polymerase variants of the present invention contains: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, TritonX-100, and glycerol.

[0038] In some embodiments, the composition further contains template DNA. In some embodiments, the composition further contains at least one in vitro transcription component, and the in vitro transcription component may be selected from one or more buffer components, modified or unmodified ribonucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc.

[0039] Kit

[0040] The kit provided by the present invention contains at least one RNA polymerase variant as described in the present invention.

[0041] In some embodiments, the kit may further contain one or more in vitro transcription reaction reagents, such as: buffer components, modified or unmodified ribonucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the kit further contains a cap analog, and the cap analog may 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 contains at least one in vitro transcription component, and the in vitro transcription component may be selected from one or more buffer components, modified or unmodified ribonucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc. In one embodiment, the components of the in vitro transcription system may be selected from commercially available in vitro transcription reagents for RNA.

[0042] Application

[0043] The present invention provides the application of the above-mentioned RNA polymerase variants, compositions or kits in the preparation of RNA by in vitro transcription. The present invention also provides the application of at least one variant as described in the present invention in reducing the generation of dsRNA impurities in the preparation of RNA by in vitro transcription.

[0044] In some embodiments, the preparation of RNA by in vitro transcription includes contacting a DNA template with at least one RNA polymerase variant of the present invention and incubating in an in vitro transcription reaction system to obtain a target product. In some embodiments, the in vitro transcription system contains modified or unmodified ribonucleoside triphosphates.

[0045] Method

[0046] The present invention provides a method for reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription, including contacting a DNA template with one or more T7 RNA polymerase variants of the present invention and incubating in an in vitro transcription reaction system.

[0047] The present invention also provides a method for preparing RNA, including contacting a DNA template with at least one RNA polymerase variant of the present invention and incubating in an in vitro transcription reaction system to obtain a target product.

[0048] In some embodiments, the in vitro transcription system contains ribonucleoside triphosphates. In some embodiments, the ribonucleoside triphosphates can be selected from modified or unmodified ribonucleoside triphosphates (including their analogs). In some embodiments, the ribonucleoside triphosphates can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the ribonucleoside triphosphates can be selected from modified ribonucleoside triphosphates, including but not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), ribonucleoside triphosphates with a label (the label can be biotin, a fluorescent substance, digoxin, a radioactive element, etc.).

[0049] In vitro transcription reaction systems and incubation conditions suitable for generating RNA products are well known in the art. Those of ordinary skill in the art can determine appropriate reaction system pH values, reaction temperatures, reaction times, salt concentrations, or whether to add exogenous cofactors, etc., considering the optimal activity of the RNA polymerase. In some embodiments, the in vitro transcription reaction system of the present invention contains in vitro transcription reaction reagents: one or more buffer components, modified or unmodified ribonucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, in the incubation step of the present invention, the incubation temperature is 30-50 °C, preferably 37 °C. In some embodiments, in the incubation step of the present invention, the incubation time is 20-240 min, preferably 60 min.

[0050] In some embodiments, the RNA products prepared by the method of the present invention have higher yields, and / or higher integrity, and / or lower dsRNA impurity content, and / or more capped mRNA products, etc., compared to those prepared using wild-type RNA polymerase (SEQ ID NO: 1).

[0051] In some embodiments, compared to those prepared using wild-type RNA polymerase, the relative residual amount of dsRNA impurities (the ratio of the dsRNA residue in the variant group to the dsRNA residue in the WT group) in the RNA products prepared by the scheme of the present invention 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.

[0052] In some embodiments, the in vitro transcription reaction system contains one or more buffer components. In some embodiments, the buffer components can be selected from Tris-HCl, Hepes, citric acid, or commercially available buffer components. In some embodiments, the in vitro transcription buffer system further contains an RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system further contains 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 includes a cap analog, and the cap analog 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.

[0053] Other embodiments:

[0054] 1. An RNA polymerase variant, the amino acid sequence of which has at least 75% sequence identity compared to SEQ ID NO: 1 and contains at least one mutation selected from the following amino acid sites: E167, V174, or G753, wherein the mutation type is substitution or deletion; the amino acid positions of the variant sequence refer to SEQ ID NO: 1.

[0055] 2. The variant according to item 1, wherein:

[0056] (1) The mutation at the E167 site is E167D:

[0057] (2) The mutation at the V174 site is selected from V174G, DEL174;

[0058] (3) The mutation at the G753 site is selected from G753A, G753T, G753Q, G753N or G753S.

[0059] 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 higher sequence identity with any one of the sequences shown in SEQ ID NO: 2-9.

[0060] 4. A biological material, wherein the biological material is selected from one or more of the following:

[0061] 1) A polynucleotide molecule encoding any one of the variants described in items 1-3;

[0062] 2) An expression vector containing the polynucleotide molecule described in 1);

[0063] 3) A host cell containing the polynucleotide molecule described in 1), or a host cell containing the expression vector described in 2).

[0064] 5. A method for preparing any one of the variants described in items 1-3, which includes:

[0065] (1) Culturing the host cell described in item 4; and

[0066] (2) Recovering the variant.

[0067] 6. A composition, which contains any one of the variants described in items 1-3.

[0068] 7. A kit, which contains any one of the variants described in items 1-3.

[0069] 8. Use of any one of the variants described in items 1-3, the composition described in item 6 or the kit described in item 7 in in vitro transcription.

[0070] 9. Use of any one of the variants described in items 1-3, the composition described in item 6 or the kit described in item 7 in reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription.

[0071] 10. The use according to item 9, wherein the process of preparing RNA includes contacting a DNA template with any one of the variants described in items 1-3 and incubating in an in vitro transcription system.

[0072] 11. A method for reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription, which includes contacting a DNA template with at least one of the variants described in items 1-3 and incubating in an in vitro transcription reaction system.

[0073] 12. Use of the method described in item 11 in the preparation of RNA by in vitro transcription.

[0074] 13. A method for preparing RNA, which includes contacting a DNA template with at least one RNA polymerase variant described in items 1-3, and incubating in an in vitro transcription reaction system to obtain a target RNA product.

[0075] Beneficial effects

[0076] By modifying the wild-type T7 RNA polymerase and using it in the preparation of RNA by in vitro transcription, the present invention can significantly reduce the generation of dsRNA, achieving a reduction in dsRNA contamination from the source. All variants in Example 1 of the present invention can effectively reduce the production level of impurity dsRNA, and the residual amount of dsRNA impurities is reduced by at least 70%. Description of the drawings

[0077] Figure 1 It is a schematic diagram for the construction of a recombinant plasmid;

[0078] Figure 2 It is a schematic diagram for the difference in dsRNA residue values;

[0079] Among them, the corresponding relationship between the T7 wild-type polymerase and its variants and amino acids in the figure is shown in Table 1.

[0080] Table 1

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Detailed implementation manners

[0087] The following further illustrates the technical solutions of the present invention in combination with specific embodiments. However, the following embodiments are only examples of the present invention and do not represent or limit the protection scope of the present invention. The protection scope of the present invention is subject to the claims. In the following embodiments, unless otherwise specified, 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.

[0088] In the embodiments of the present invention, the definition of enzyme activity is that under the conditions of 37°C and pH 8.0, the amount of enzyme required to incorporate 1 nmol 3 of

[0089] Example 1 Preparation of RNA Polymerase Variants

[0090] The nucleotide sequences SEQ ID NO: 10 - 18 (the encoded amino acid sequences correspond to SEQ ID NO: 1 - 9) were synthesized by DNA sequencing, then amplified by PCR, and then introduced into the BseRI and HindIII restriction sites of the expression vector pQE - 80L to obtain a recombinant expression vector. The constructed vector was introduced into E. coli BL21(DE3) by transformation technology, coated on an LB plate containing ampicillin resistance, and cultured overnight in a 37°C incubator. The single colonies that grew out were subjected to plasmid extraction and sequencing, and finally a recombinant engineering bacterium containing the target gene was obtained. After the successfully sequenced E. coli recombinant strain was inoculated into LB medium for overnight activation culture, it was inoculated into the fermentation broth (LB medium) at 1 - 5% V / V, cultured until the OD600 value reached 0.6 - 0.8, IPTG with a final concentration of 0.5 mol / L was added and cultured for another 4 - 6 h, then the strain was collected by centrifugation at 12,000 rpm and 5°C, and the collected strain was washed with 0.2 M PBS buffer with a pH value of 7.0 to obtain the bacterial cells; after ultrasonic disruption, affinity chromatography purification was carried out to obtain the crude RNA polymerase solution.

[0091] Example 2 Verification of in vitro Transcription Reaction

[0092] The crude enzyme solution obtained in Example 1 was diluted with storage buffer to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 2 were loaded into an eight - row strip, mixed well, and centrifuged; the eight - row strip was placed on a PCR instrument and reacted at 37°C for 1 h, then 36 μL of magnetic beads (Vazyme, product number: N412) was added and mixed well, and incubated at room temperature for 2 - 5 min; the mixture was placed on a magnetic rack to purify the mRNA, and after purification, it was transferred to an RNase - free centrifuge tube to obtain the purified mRNA, and the dsRNA impurity content was tested with a dsRNA detection kit (Vazyme, product number: DD3509).

[0093] Table 2: Reaction System Ratio

[0094]

[0095] The dsRNA detection results are shown in Figure 2 , compared with the wild - type T7 RNA polymerase (WT), all variants in Example 1 can effectively reduce the level of impurity dsRNA production, and the residual amount of dsRNA impurities is reduced by at least 70%.

[0096] As described above, it is only the preferred embodiment of the present invention, and does not impose any formal restrictions on the technical solution of the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An RNA polymerase variant, characterized in that, The amino acid sequence of the variant has at least 75% sequence identity compared to SEQ ID NO: 1 and contains at least one mutation selected from the following amino acid positions: E167, V174, or G753, wherein the type of mutation is substitution or deletion; the amino acid positions of the variant sequence are referenced to SEQ ID NO:

1.

2. The variant according to claim 1, characterized in that: (1) The mutation at position E167 is E167D; (2) The mutation at position V174 is selected from V174G, DEL174; (3) The mutation at position G753 is selected from G753A, G753T, G753Q, G753N, or G753S.

3. An RNA polymerase variant, characterized in that, 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 higher sequence identity with any one of the sequences shown in SEQ ID NOs: 2-9.

4. A biomaterial, characterized in that, The biological material is selected from one or more of the following: 1) A polynucleotide molecule encoding the variant according to any one of claims 1-3; 2) An expression vector containing the polynucleotide molecule as described in 1); 3) A host cell containing the polynucleotide molecule as described in 1), or a host cell containing the expression vector as described in 2).

5. A method for preparing a variant according to any one of claims 1-3, characterized in that, Comprising: (1) Culturing the host cell according to claim 4; and (2) Recovering the variant.

6. A composition, characterized in that, Contains the variant according to any one of claims 1-3.

7. Kit, characterized in that, Contains the variant according to any one of claims 1-3.

8. Use of the variant according to any one of claims 1-3, the composition according to claim 6, or the kit according to claim 7 in in vitro transcription.

9. Use of the variant according to any one of claims 1-3, the composition according to claim 6, or the kit according to claim 7 in reducing the generation of dsRNA impurities during the preparation of RNA by in vitro transcription.

10. The application according to claim 9, characterized in that, The process of preparing RNA includes contacting a DNA template with the variant according to any one of claims 1-3 and incubating in an in vitro transcription system.

11. A method for reducing the generation of dsRNA impurities during in vitro transcription for preparing RNA, characterized in that, Includes contacting a DNA template with at least one variant as described in claims 1-3 and incubating in an in vitro transcription reaction system.

12. Use of the method according to claim 11 in the preparation of RNA by in vitro transcription.

13. A method for preparing RNA, characterized in that, Includes contacting a DNA template with at least one RNA polymerase variant as described in claims 1-3, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

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