RNA polymerase variants

Abstract

The invention provides an RNA polymerase variant, and relates to the technical field of biology. The RNA polymerase variant provided by the invention can be used for remarkably reducing the generation of dsRNA impurities in an in-vitro transcription process and remarkably improving the integrity of an RNA transcription product, especially in transcription of long-fragment RNA. In addition, the invention provides an application of the RNA polymerase variant in in-vitro transcription, and also provides a method for preparing ultra-long saRNA (greater than 10000 nt).

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to RNA polymerase variants and applications thereof. Background of the Invention

[0003] The development of mRNA-based vaccines and treatments has become the hottest research direction recently. Two forms of vaccines have been developed: one is traditional mRNA, which consists of a 5' cap, a 5'-untranslated region (UTR), an open reading frame (ORF) encoding the vaccine antigen, a 3'-untranslated region (UTR), and a poly (A) tail. Traditional mRNA has poor stability and will be quickly degraded in cells, resulting in unsustainable protein expression levels. If used for long-term treatment, patients may need to be injected multiple times and in large quantities, which will increase the toxic side effects of the therapy. The other is self-amplifying mRNA (saRNA) derived from the positive-strand RNA viral genome. The biggest feature of saRNA is that it not only maintains the self-replication activity derived from the RNA viral vector, but also carries a sequence that can express RNA polymerase (RNA-dependent RNApolymerase, RdRP). When the enzyme is generated, it can generate more saRNA using saRNA as a template, that is, only a low dose of saRNA is needed to obtain a high level of protein expression. And the self-replication of saRNA in the body can also lead to longer expression of antigenic proteins, thereby triggering a relatively more lasting immune response. In addition, saRNA produces double-stranded RNA structures during replication, which can be recognized by pattern recognition receptors (PRRs) in cells and activate the innate immune response of cells, further improving the immune response effect of saRNA vaccines.

[0004] Existing studies have shown that compared with traditional mRNA vaccines, saRNA can still produce the same immune response when the injection dose is hundreds or even thousands of times smaller. For therapeutic RNA, the possible toxic side effects of mRNA delivery vectors can be reduced with a smaller dosage and administration frequency; for the production of RNA vaccines, a lower dosage can reduce the production cost of saRNA. However, due to the complexity of the encoding gene, the molecular weight of saRNA is much larger than that of traditional mRNA. During the synthesis of saRNA, the integrity of the product has always been a difficult problem to solve. This problem is particularly important for ultra-long fragments of >10000nt, and it is the primary problem in the optimization of saRNA synthesis process. At present, most in vitro transcription (IVT) schemes are aimed at small RNA fragments, while saRNA fragments are longer (>8000nt) and have more secondary structures, so conventional IVT schemes are not suitable for the synthesis of saRNA. Therefore, there is a need for an RNA preparation method that is suitable for IVT preparation of saRNA and can ensure the integrity of saRNA products. Summary of the invention

[0005] In a first aspect, the present disclosure provides a class of RNA polymerase variants, whose amino acid sequence comprises at least one mutation selected from the following amino acid positions compared with SEQ ID NO: 1: H300, H176.

[0006] In a second aspect, the present disclosure provides a type of biomaterial, which is selected from one or more of the following:

[0007] 1) A polynucleotide molecule encoding the above variant;

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

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

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

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

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

[0013] In a sixth aspect, the present disclosure provides the use of the above RNA polymerase variants in preparing RNA by in vitro transcription.

[0014] In a seventh aspect, the present disclosure also provides a method for preparing RNA. DETAILED DESCRIPTION OF THE INVENTION

[0016] RNA polymerase variants

[0017] The RNA polymerase variant provided by the present disclosure comprises, compared with the amino acid sequence of wild-type T7 RNA polymerase SEQ ID NO: 1, at least one mutation selected from the following amino acid sites: H176, H300; the mutation type can be selected from substitution or deletion.

[0018] In some embodiments, the amino acid sequence of the variant comprises any one mutation selected from the following relative to SEQ ID NO: 1: H176D, H300D.

[0019] In some embodiments, the amino acid sequence of the variant is as shown in any one of SEQ ID NOs: 2-3.

[0020] Biomaterials

[0021] The present disclosure provides polynucleotides encoding RNA polymerase variants. Due to the degeneracy of codons or the preference of codons of host cells expressing polypeptides, the polynucleotide sequence is any polynucleotide sequence encoding a variant without changing the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of the present disclosure can be selected from SEQ ID NO: 5-6.

[0022] The disclosure provides an expression vector comprising a polynucleotide molecule encoding an RNA polymerase variant of the disclosure. In some embodiments, the expression vector further comprises one or more regulatory sequences, such regulatory sequences including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequence is operably linked to the polynucleotide molecule encoding the variant.

[0023] In some embodiments, the expression vector may be a linear or circular DNA molecule, which generally comprises elements such as a multiple cloning site, a resistance gene, a replication origin site, etc. In some embodiments, the expression vector described in the present disclosure is preferably pQE-80L.

[0024] The host cells provided by the present disclosure refer to any cells that are favorable for the expression of the variants of the present disclosure, that is, any cells that are susceptible to transformation, transfection or transduction with the expression vectors described in the present disclosure, including any progeny cells that are different from the parent cells due to mutations that occur during replication.

[0025] 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, Oceanic Bacillus, 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, Silvae, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21.

[0026] Method for preparing RNA polymerase variants

[0027] The present disclosure provides a method for preparing an RNA polymerase variant, comprising 1) culturing the host cell described in the present disclosure under conditions suitable for variant expression; and 2) recovering the variant.

[0028] In some embodiments, the method for recovering the variant can be a method well known in the art, such as centrifugation, filtration, treatment with a crystal protein precipitant (salting out), extraction, ultrasonication, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., HPLC, and combinations of the above methods.

[0029] In some embodiments, the preparation method also 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), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0030] Composition

[0031] The present disclosure provides a composition comprising at least one RNA polymerase variant described in the present disclosure.

[0032] The composition described in the present disclosure may be a composition for storing RNA polymerase variants. In some embodiments, the composition described in the present disclosure, in addition to the above-mentioned RNA polymerase variants, may optionally include: 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 described in the present disclosure includes: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, TritonX-100 and glycerol.

[0033] 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 may be selected from one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and the like.

[0034] Reagent test kit

[0035] The present disclosure provides a kit comprising at least one RNA polymerase variant as described in the present disclosure.

[0036] In some embodiments, the kit further comprises a cap analog, which can be selected from unmethylated cap analogs, dimethylated cap analogs, trimethylated cap analogs, dimethylated symmetric cap analogs, or anti-reverse cap analogs. 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 pyrophosphatase, magnesium ions, etc. In one embodiment, the in vitro transcription system components can be selected from commercially available RNA in vitro transcription reagents.

[0037] application

[0038] The present disclosure provides the use of at least one variant as described in the present disclosure in preparing RNA by in vitro transcription. In some embodiments, the length of the RNA is between 1000-13000nt. In some embodiments, the length of the RNA is between 8000-13000nt.

[0039] In some embodiments, the in vitro transcription to prepare RNA comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant described in the present disclosure, and incubating them in an in vitro transcription reaction system to obtain a target product.

[0040] The present disclosure also provides use of at least one RNA polymerase variant as described in the present disclosure in the synthesis of RNA drugs.

[0041] Preparation method

[0042] The present disclosure provides a method for preparing RNA, comprising contacting a DNA template, modified or unmodified nucleoside triphosphates with at least one RNA polymerase variant described in the present disclosure, and incubating them in an in vitro transcription reaction system to obtain a target product.

[0043] In some embodiments, the target product is saRNA. In some embodiments, the DNA template is 1000-13000bp in length. In some embodiments, the DNA template is 8000-13000bp in length. In some embodiments, the DNA template is 10000-13000bp in length. In some embodiments, saRNA is prepared using the RNA polymerase variants described in the present disclosure, and the integrity of its product is increased by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% relative to the use of wild-type RNA polymerase (SEQ ID NO: 1).

[0044] In some embodiments, the RNA products prepared by the methods described in the present disclosure have higher yields, and / or higher integrity, and / or less dsRNA impurity content, and / or more capped mRNA products, etc., compared to using wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, the relative residual amount of dsRNA impurities (ratio of dsRNA residual amount of the variant group / dsRNA residual amount of the WT group) of the RNA products prepared by the scheme described in the present disclosure compared to using wild-type RNA polymerase is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, less than 1% or less.

[0045] In some embodiments, the in vitro transcription reaction system comprises one or more buffer components. 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 also comprises RNase inhibitors, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system also comprises water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.). In some embodiments, the in vitro transcription buffer system also includes 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.

[0046] Other Implementations

[0047] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant comprises a mutation at any one of the following sites relative to SEQ ID NO: 1: H176, H300.

[0048] 2. The variant as described in item 1, whose amino acid sequence is shown in any one of SEQ ID NOs: 2-3.

[0049] 3. Biomaterials selected from one or more of the following:

[0050] 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any one of item 1 or 2;

[0051] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0052] 3) A host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

[0053] 4. A composition comprising a variant as described in any one of item 1 or 2.

[0054] 5. A kit comprising the variant as described in any one of item 1 or 2.

[0055] 6. Use of the variant described in any one of item 1 or 2, the composition described in item 4 or the kit described in item 5 in in vitro transcription.

[0056] 7. Use of an RNA polymerase variant in preparing RNA by in vitro transcription, wherein the length of the RNA is between 8000-13000 nt; and the amino acid sequence of the variant is shown in SEQ ID NO: 2-3.

[0057] 8. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates with an RNA polymerase variant, incubating them in an in vitro transcription reaction system, and obtaining a target RNA product; wherein the amino acid sequence of the variant is as shown in SEQ ID NO: 2-3.

[0058] 9. The method as described in item 8, wherein the length of the DNA template is 8000-13000 bp, preferably 10000-13000 bp.

[0059] 10. The method as described in item 8 or 9, wherein the in vitro transcription system further comprises a cap analog.

[0060] 11. The method of item 8, wherein the target RNA is saRNA.

[0061] 12. Use of the RNA polymerase variant as described in item 1-2 in the synthesis of RNA drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Schematic diagram of the construction of recombinant plasmid.

[0064] Figure 2 The effect of RNA polymerase variants on the integrity of saRNA products.

[0065] Figure 3 The effect of RNA polymerase variants on residual dsRNA impurities.

[0066] Beneficial Effects

[0067] The RNA polymerase variant provided by the present disclosure has the performance of significantly reducing the generation of dsRNA impurities during transcription and improving the integrity of ultra-long fragment mRNA transcription products, and can be used for in vitro transcription to prepare mRNA, especially in the preparation of saRNAs exceeding 10000nt, which can improve product integrity. In addition, the present disclosure also provides a saRNA preparation method, which can efficiently produce complete ultra-long saRNAs while ensuring the expression efficiency of saRNAs, which is of great significance for the industrial production of saRNAs. DETAILED DESCRIPTION

[0068] The technical solution of the present disclosure is further described below in conjunction with specific embodiments. However, the following embodiments are merely examples of the present disclosure and do not represent or limit the scope of protection of the present disclosure. The scope of protection of the present disclosure shall be subject to the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0069] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of product or convert 1 μmol of substrate within 1 minute under specific reaction conditions.

[0070] Example 1 Preparation of RNA polymerase variants

[0071] A DNA fragment was synthesized according to the DNA sequence shown in SEQ ID NO: 4-6, and after PCR amplification, the BseRI and HindIII restriction sites of the expression vector pQE-80L were introduced to obtain a recombinant expression vector, which was transformed into E. coli BL21 (Novagen Inc.), and cloned strains were obtained after antibiotic resistance plate coating screening; after successful recombination was verified, the obtained strains were activated overnight in LB medium at 37°C and added to fermentation liquid (LB medium), cultured to an OD600 value of 0.6-0.8, IPTG with a final concentration of 0.5 mol / L was added and continued to be cultured for 4-6 hours, and the strains were collected by centrifugation at 12000 rpm and 5°C, and the collected strains were washed with a 0.2 M PBS buffer with a pH value of 7.0 to obtain bacterial cells; after ultrasonic disruption, affinity chromatography purification was performed to obtain RNA polymerase and its variants.

[0072] The correspondence between RNA polymerase and its variants and amino acid sequences is shown in Table 1:

[0073] Table 1:

[0074]

[0075]

[0076] Example 2 In vitro transcription reaction verification I (DNA template I)

[0077] The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25°C, pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300 U / μL. Prepare MIX solution (except T7 RNA polymerase) according to the reaction system (20 μL) in Table 2 and put it into EP tube; dispense MIX solution into eight strips, and then use a gun to take 1 μL of the diluted T7 RNA polymerase into the eight strips containing MIX solution, mix well, and centrifuge; place the eight strips on a PCR instrument, react at 37°C for 1.5h, add 36 μL of magnetic beads, mix well, and incubate at room temperature for 2-5min; place the mixed solution on a magnetic rack to purify mRNA (Vazyme, catalog number: N412), and transfer it to an RNase-free centrifuge tube after purification to obtain purified mRNA, and perform capillary electrophoresis detection on a Qsep400 fully automatic nucleic acid analyzer, N3 cassette (injection 4KV and separation 2KV, 20ntMarker), to detect the integrity of mRNA.

[0078] Table 2: Reaction system ratio

[0079]

[0080]

[0081] Test results such as Figure 2 As shown, with the wild-type T7 RNA polymerase (WT) as the control, both H300D and H176D mutants can significantly improve the integrity of saRNA products.

[0082] Example 3 Verification II of impurity generation by in vitro transcription of dsRNA

[0083] (1) The enzyme stock solution obtained in Example 1 was diluted with storage buffer (50mM Tris-HCl (25°C, pH 7.9), 100mM NaCl, 0.1mM EDTA, 2mM DTT, 0.1% TritonX-100, 50% Glycerol) to an enzyme activity of 300U / μL. The reaction components (20μL) in Table 3 were added to the eight-row strip, mixed, and centrifuged; the eight-row strip was placed on a PCR instrument and reacted at 37°C for 1h, and then 36μL of magnetic beads were added, mixed, and incubated at room temperature for 2-5min; the mixed solution was placed on a magnetic stand to purify mRNA (Vazyme, catalog number: N412), and after purification, it was transferred to an RNase-free centrifuge tube to obtain purified mRNA;

[0084] (2) The dsRNA impurity content was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).

[0085] Table 3: Reaction system ratio

[0086]

[0087]

[0088] After testing, it was found that the H176D site significantly reduced the production of dsRNA impurities.

Claims

1. An RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises a mutation at any one of the following sites relative to SEQ ID NO: 1: H176, H300.

2. The variant according to claim 1, wherein the amino acid sequence of the variant is shown in any one of SEQ ID NOs: 2-3.

3. Biomaterial, characterized in that The biological material is selected from one or more of the following: 1) A polynucleotide molecule encoding the RNA polymerase variant according to any one of claims 1 or 2; 2) an expression vector comprising the polynucleotide molecule as described in 1); 3) A host cell comprising the polynucleotide molecule as described in 1), or a host cell comprising the expression vector as described in 2).

4. A composition, characterized in that The invention comprises a variant as claimed in any one of claims 1 or 2.

5. A kit, characterized in that: The invention comprises a variant as claimed in any one of claims 1 or 2.

6. Use of the variant according to any one of claims 1 or 2, the composition according to claim 4 or the kit according to claim 5 in in vitro transcription.

7. Use of an RNA polymerase variant in preparing RNA by in vitro transcription, characterized in that: The length of the RNA is between 8000-13000 nt; and the amino acid sequence of the polymerase variant is shown in SEQ ID NO: 2-3.

8. A method for preparing RNA, characterized in that: The method comprises contacting a DNA template, modified or unmodified nucleoside triphosphates and an RNA polymerase variant, and incubating them in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the polymerase variant is shown in SEQ ID NO: 2-3.

9. The method according to claim 8, wherein the length of the DNA template is 8000-13000 bp, preferably 10000-13000 bp.

10. The method according to claim 8 or 9, wherein the in vitro transcription reaction system further comprises a cap analog.

11. The method of claim 8, wherein the target RNA is saRNA.

12. Use of the RNA polymerase variant according to claims 1-2 in the synthesis of RNA drugs.

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