RNA polymerase variants and uses thereof

Abstract

The application provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. The RNA polymerase variant provided by the application has the performance of significantly improving the integrity of an ultralong fragment mRNA transcription product, and can be used for preparing mRNA by in vitro transcription, especially for preparing saRNA of more than 10000 nt, which can improve the integrity of the product, and is of great significance for the industrialized production of saRNA.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to RNA polymerase variants and their applications. Background Technology

[0002] mRNA vaccines have recently become a hot research area due to their high potency, safety and efficacy, rapid clinical development capabilities, and potential for rapid and low-cost production. Two forms of mRNA vaccines have been developed: one is traditional mRNA, consisting 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 mRNAs are unstable and rapidly degrade within cells, resulting in inconsistent protein expression levels. For long-term treatment, this may require multiple, high-dose injections, increasing the risk of toxic side effects. Another option is self-amplifying RNA (saRNA) derived from the genome of a positive-sense RNA virus. The key characteristic of saRNA is that it not only retains the self-replicating activity of the RNA virus vector but also carries a sequence capable of expressing RNA-dependent RNA polymerase (RdRP). Once this enzyme is generated, it can use the saRNA as a template to produce more saRNA, meaning that only a low dose of saRNA is needed to achieve high levels of protein expression. Furthermore, the self-replication of saRNA in vivo leads to longer-lasting antigen protein expression, resulting in a more sustained immune response. In addition, saRNA replication produces double-stranded RNA structures that can be recognized by pattern recognition receptors (PRRs) within cells, activating the cellular innate immune response and further enhancing the immune response of saRNA vaccines.

[0003] Existing studies have shown that saRNA can still produce the same immune response compared to traditional mRNA vaccines, even with injection doses hundreds or even thousands of times smaller. For therapeutic RNA, fewer doses and administrations can reduce the potential toxic side effects of mRNA delivery vectors; for RNA vaccine production, lower doses can reduce saRNA production costs. However, due to the complexity of the encoding gene, saRNA has a much larger molecular weight than traditional mRNA. Maintaining product integrity during saRNA synthesis has always been a challenging issue, especially for ultra-long fragments >10,000 nt, and is a primary concern in optimizing saRNA synthesis processes. Summary of the Invention

[0004] In a first aspect, this application provides a class of RNA polymerase variants whose amino acid sequences contain at least one mutation selected from the following amino acid sites compared to SEQ ID NO: 1: L680, I605, K71, E350, M369, N370, A373, A382, K642, F644.

[0005] Secondly, this application provides a class of biological materials selected from one or more of the following:

[0006] 1) The polynucleotide molecule encoding the above variants;

[0007] 2) Expression vectors containing polynucleotide molecules as described in 1);

[0008] 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).

[0009] Thirdly, this application provides a method for preparing the aforementioned RNA polymerase variant.

[0010] Fourthly, this application provides a composition comprising at least one RNA polymerase variant as described in this application.

[0011] Fifthly, this application provides a kit comprising at least one RNA polymerase variant as described in this application.

[0012] Sixthly, this application provides the application of the above-mentioned RNA polymerase variant in the in vitro transcription preparation of RNA.

[0013] Seventhly, this application also provides a method for preparing RNA. Invention Details

[0015] RNA polymerase variants

[0016] The RNA polymerase variant provided in this application contains at least one mutation selected from the following amino acid sites compared to the amino acid sequence SEQ ID NO: 1 of the wild-type T7 RNA polymerase: L680, I605, K71, E350, M369, N370, A373, A382, K642, F644; the mutation type may be substitution or deletion.

[0017] In some embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:1.

[0018] In some embodiments, the variant comprises a mutation at amino acid site L680, and also comprises one, two, or three mutations selected from I605, K71, E350, M369, N370, A373, A382, K642, or F644. In some embodiments, the mutation at L680 is L680I or L680V. In some embodiments, the one, two, or three mutations selected from I605, K71, E350, M369, N370, A373, A382, K642, or F644 are one, two, or three mutations selected from I605P, K71M, E350A, M369K, N370P, A373K, A382K, K642R, or F644Y.

[0019] In some embodiments, the variant contains a mutation at amino acid site L680 and has an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1.

[0020] In some embodiments, the amino acid sequence of the variant, relative to SEQ ID NO: 1, includes any mutation selected from the following: I605P, L680V, ​​L680I, K71M+I605P+L680V, ​​E350A+I605P+L680V, ​​M369K+A382K+L680V, ​​M369K+I605P+L680V, ​​M369K+A382K+I605P+L680V, ​​N370P+I605P+L680V, ​​A373K+L680I, A382K+L680I, I605P+L680V, ​​K642R+F644Y+L680V.

[0021] In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-14.

[0022] In some embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:2-14.

[0023] biomaterials

[0024] This application provides a polynucleotide encoding the RNA polymerase variant described in this application. Due to codon degeneracy or codon bias of the host cell expressing the polypeptide, the polynucleotide sequence can be any polynucleotide sequence encoding the variant without altering the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of this application may be selected from SEQ ID NO: 16-28.

[0025] The expression vector provided in this application comprises a multinucleotide molecule encoding the RNA polymerase variant described in this application. In some embodiments, the expression vector further comprises one or more regulatory sequences, including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequences are operatively linked to the multinucleotide molecule encoding the variant.

[0026] In some embodiments, the expression vector may be a linear or circular DNA molecule, typically containing elements such as a multiple cloning site, an antibiotic resistance gene, and a replication initiation site. In some embodiments, the expression vector described in this application is preferably pQE-80L.

[0027] The host cell provided in this application refers to any cell that is favorable for the expression of the RNA polymerase variants described in this application, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vectors described in this application, and includes any daughter cells that are different from the parent cells due to mutations that occur during replication.

[0028] In some embodiments, the host cell is a prokaryotic cell, selectable from Gram-positive or Gram-negative bacteria. In some embodiments, the host cell is a Gram-positive bacterium, including but not limited to: *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *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*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*. In some embodiments, the host cell is *Escherichia coli* BL21.

[0029] Preparation methods of RNA polymerase variants

[0030] This application provides a method for preparing the RNA polymerase variant described in this application, comprising: 1) culturing the host cell described in this application under suitable variant expression conditions; and 2) recovering the variant.

[0031] In some embodiments, the method for recovering variants can be a method known in the art, such as centrifugation, filtration, treatment with a crystalline 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.

[0032] In some embodiments, the preparation method further includes a purification step of the variant, which can be a method known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), isoelectric point focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0033] Composition

[0034] The composition provided in this application comprises at least one RNA polymerase variant described in this application.

[0035] The composition described in this application can be a composition for storing RNA polymerase variants. In some embodiments, in addition to the aforementioned RNA polymerase variants, the composition described in this application 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 Triton X-100), stabilizers (such as glycerol), and other components. In some embodiments, the composition for storing RNA polymerase variants described in this application comprises: RNA polymerase variant, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.

[0036] In some embodiments, the composition further comprises 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 pyrophosphatases, magnesium ions, etc.

[0037] Reagent test kit

[0038] The kit provided in this application contains at least one RNA polymerase variant as described in this application.

[0039] In some embodiments, the kit further comprises a cap analogue, which may be selected from unmethylated cap analogues, dimethylated cap analogues, trimethylated cap analogues, dimethylated symmetrical cap analogues, or anti-reverse cap analogues. In some embodiments, the trinucleotide cap is selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap is preferably m7GpppA2′OMepG.

[0040] In some embodiments, the kit 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 pyrophosphatases, magnesium ions, etc. In some embodiments, the in vitro transcription system components may be selected from commercially available RNA in vitro transcription reagents.

[0041] application

[0042] This application provides the use of at least one variant as described herein in the in vitro transcription preparation of RNA. In some embodiments, the length of the RNA is between 1000-13000 nt. In some embodiments, the length of the RNA is between 8000-13000 nt. In some embodiments, the length of the RNA is between 10000-13000 bp.

[0043] Compared with RNA produced by in vitro transcription reaction involving the RNA polymerase variant described in this application (SEQ ID NO: 1), RNA has at least 5% higher integrity, preferably at least 10%, and more preferably at least 15% higher integrity.

[0044] In some embodiments, the integrity of the saRNA product produced by an in vitro transcription reaction involving the RNA polymerase variant described in this application is improved by at least about 5%, about 10%, or about 15% compared to that produced using wild-type RNA polymerase (SEQ ID NO: 1).

[0045] In some embodiments, the in vitro transcription preparation of RNA includes contacting a DNA template, a modified or unmodified nucleoside triphosphate, with at least one RNA polymerase variant described in this application, and incubating in an in vitro transcription reaction system to obtain the target product.

[0046] In some embodiments, the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the trinucleotide cap is selected from GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the trinucleotide cap is selected from m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GppppUpC, m7GppppUpG, and m7GppppUpU. In some embodiments, the trinucleotide cap is selected from m7G3′OMepppApA, m7G3′OMepppApC, m7G3′OMepppApG, m7G3′OMepppApU, m7G3′OMepppCpA, m7G3′OMepppCpC, m7G3′OMepppCpG, m7G3′OMepppCpU, m7G3′OMepppGpA, m7G3′OMepppGpC, m7G3′OMepppGpG, m7G3′OMepppGpU, m7G3′OMepppUpA, m7G3′OMepppUpC, m7G3′OMepppUpG, and m7G3′OMepppUpU. In some embodiments, the trinucleotide cap is selected from m7G3′OMepppA2′OMepA, m7G3′OMepppA2′OMepC, m7G3′OMepppA2′OMepG, m7G3′OMepppA2′OMepU, m7G3′OMepppC2′OMepA, m7G3′OMepppC2′OMepC, m7G3′OMepppC2′OMepG, m7G3′OMeppp C2′OMepU、m7G3′OMepppG2′OMepA、m7G3′OMepppG2′OMepC、m7G3′OMepppG2′OMepG、m7G3′OMepppG2′OMepU、m7G3′OMepppU2′OMepA、m7G3′OMepppU2′OMepC、m7G3′OMepppU2′OMepG、and m7G3′OMepppU2′OMepU。In some embodiments, the trinucleotide cap is selected from m7GpppA2′OMepA, m7GpppA2′OMepC, m7GpppA2′OMepG, m7GpppA2′OMepU, m7GpppC2′OMepA, m7GpppC2′OMepC, m7GpppC2′OMepG, m7GpppC2′OMepU, m7GpppG2′OMepA, m7GpppG2′OMepC, m7GpppG2′OMepG, m7GpppG2′OMepU, m7GpppU2′OMepA, m7GpppU2′OMepC, m7GpppU2′OMepG, and m7GpppU2′OMepU. In some embodiments, the trinucleotide cap is preferably m7GpppA2′OMepG.

[0047] This application also provides the use of at least one RNA polymerase variant as described in this application in the synthesis of RNA drugs.

[0048] Preparation method

[0049] This application provides a method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate with at least one RNA polymerase variant described in this application, incubating in an in vitro transcription reaction system to obtain a target product.

[0050] In some embodiments, the target product is saRNA. In some embodiments, the DNA template length is 1000-13000 bp. In some embodiments, the DNA template length is 8000-13000 bp. In some embodiments, the DNA template length is 10000-13000 bp. In some embodiments, the preparation of saRNA using the RNA polymerase variant described in this application results in an improvement in product integrity of 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% compared to using wild-type RNA polymerase (SEQ ID NO: 1).

[0051] In some embodiments, the in vitro transcription reaction system includes one or more buffer components. In some embodiments, the buffer component may be selected from Tris-HCl, Hepes, citric acid, or commercially available buffer components. In some embodiments, the in vitro transcription buffer system also includes an RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription buffer system also includes water (e.g., 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 analogue, which may be selected from unmethylated cap analogues, dimethylated cap analogues, trimethylated cap analogues, dimethylated symmetrical cap analogues, or anti-reverse cap analogues.

[0052] In some embodiments, the cap analogue is selected from m7GpppA2′OMepA, m7GpppA2′OMepC, m7GpppA2′OMepG, m7GpppA2′OMepU, m7GpppC2′OMepA, m7GpppC2′OMepC, m7GpppC2′OMepG, m7GpppC2′OMepU, m7GpppG2′OMepA, m7GpppG2′OMepC, m7GpppG2′OMepG, m7GpppG2′OMepU, m7GpppU2′OMepA, m7GpppU2′OMepC, m7GpppU2′OMepG, and m7GpppU2′OMepU.

[0053] Other implementation plans

[0054] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant includes any mutation selected from the following sites relative to SEQ ID NO: 1: L680, I605, K71, E350, M369, N370, A373, A382, K642, F644, wherein the mutation type may be substitution or deletion.

[0055] 2. A variant as described in item 1, wherein:

[0056] (1) The substitution at position L680 is I or V;

[0057] (2) The substitution at position I605 is P;

[0058] (3) The substitution at position K71 is M;

[0059] (4) Replace A at position E350;

[0060] (5) The substitution at position M369 is K;

[0061] (6) The substitution at position N370 is P;

[0062] (7) The substitution at position A373 is K;

[0063] (8) The substitution at position A382 is K;

[0064] (9) The substitution at position K642 is R;

[0065] (10) Replace Y at position F644.

[0066] 3. The variant described in item 1 contains a mutation at the L680 site, and also contains one, two or three mutations selected from I605, K71, E350, M369, N370, A373, A382, K642 or F644.

[0067] 4. The variant as described in item 3, wherein the mutation at the L680 site of the variant is L680I or L680V.

[0068] 5. The variant as described in item 3, wherein one, two, or three mutations selected from I605, K71, E350, M369, N370, A373, A382, K642, or F644 are selected from one, two, or three mutations selected from I605P, K71M, E350A, M369K, N370P, A373K, A382K, K642R, or F644Y.

[0069] 6. The variant as described in claim 1, characterized in that the amino acid sequence of the variant contains, relative to SEQ ID NO: 1, any mutation selected from the following sites: I605P, L680V, ​​L680I, K71M+I605P+L680V, ​​E350A+I605P+L680V, ​​M369K+A382K+L680V, ​​M369K+I605P+L680V, ​​M369K+A382K+I605P+L680V, ​​N370P+I605P+L680V, ​​A373K+L680I, A382K+L680I, I605P+L680V, ​​K642R+F644Y+L680V.

[0070] 7. The variants described in item 1, having an amino acid sequence as shown in any of SEQ ID NO: 2-14.

[0071] 8. Biological materials, selected from one or more of the following:

[0072] 1) A polynucleotide molecule encoding an RNA polymerase variant as described in any of items 1-7;

[0073] 2) Expression vectors containing polynucleotide molecules as described in 1);

[0074] 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).

[0075] 9. A composition comprising any of the variants described in items 1-7.

[0076] 10. A kit containing any of the variants described in items 1-7.

[0077] 11. Any variant described in any of items 1-7, the composition described in item 9, or the kit described in item 10.

[0078] 12. A method for preparing RNA, characterized in that the method comprises contacting a DNA template, a modified or unmodified nucleoside triphosphate, and an RNA polymerase variant, incubating them in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the variant is shown in SEQ ID NO: 2-14.

[0079] 13. As described in item 12, the in vitro transcription system also includes a cap analogue.

[0080] 14. As described in item 12, the target RNA is saRNA.

[0081] 15. Application of RNA polymerase variants as described in items 1-7 in RNA drug synthesis.

[0082] Beneficial effects

[0083] The RNA polymerase variant provided in this application significantly improves the integrity of ultra-long mRNA transcripts and can be used for in vitro transcription to prepare mRNA, especially saRNA exceeding 10,000 nt. Furthermore, this application also provides a method for saRNA preparation that efficiently produces complete ultra-long saRNA while ensuring saRNA expression efficacy, which is of great significance for the industrial production of saRNA. Attached Figure Description

[0084] Figure 1 This is a schematic diagram of the construction of recombinant plasmids.

[0085] Figure 2 The effect of RNA polymerase variants on the integrity of saRNA products. Detailed Implementation

[0086] The technical solution of this application will be further described below with reference to specific embodiments. However, the following embodiments are merely examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application is determined by 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 techniques used are conventional methods and techniques in the art.

[0087] Example 1: Preparation of RNA polymerase variants

[0088] DNA fragments were synthesized according to the DNA sequences shown in SEQ ID NO:15~28. After PCR amplification, the fragments were introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector, which was transformed into E. coli BL21 (Novagen Inc.). After screening by antibiotic resistance plate plating, cloned strains were obtained. After confirming successful recombination, the obtained strains were activated overnight at 37°C in LB medium and then added to fermentation broth (LB medium). The strains were cultured until the OD 600 value reached 0.6~0.8. IPTG was added to a final concentration of 0.5 mol / L and the strains were cultured for another 4-6 h. The strains were collected by centrifugation at 12000 rpm and 5°C. The collected strains were washed with 0.2 M PBS buffer at pH 7.0 to obtain bacterial cells. After sonication, the cells were purified by affinity chromatography to obtain the RNA polymerase stock solution.

[0089] Table 1-1: Correspondence between RNA polymerase and amino acid sequence

[0090]

[0091] Table 1-2: Correspondence between RNA polymerase and amino acid sequence

[0092]

[0093] Table 1-3: Correspondence between RNA polymerase and amino acid sequence

[0094]

[0095] Table 1-4: Correspondence between RNA polymerase and amino acid sequence

[0096]

[0097] Example 2: Validation of in vitro transcription reaction

[0098] The enzyme stock solution was diluted with storage buffer (50 mM Tris-HCl (25℃, 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 the MIX solution (excluding T7 RNA polymerase) in EP tubes according to the reaction system (20 μL) in Table 2; aliquot the MIX solution into octuplets, and then add 1 μL of diluted T7 RNA polymerase to each octuplet containing the MIX solution, mix well, and centrifuge; place the octuplets on a PCR instrument and react at 37℃ for 1.5 h, then add 36 μL of magnetic beads and mix well, incubate at room temperature for 2-5 min; place the mixture on a magnetic rack to purify mRNA (Vazyme, catalog number: N412), and transfer the purified mRNA to an RNase-free centrifuge tube. The purified mRNA is then detected by capillary electrophoresis using a Qsep400 fully automated nucleic acid analyzer with N3 clips (injection 4KV and separation 2KV, 20 nt Marker) to check the integrity of the mRNA.

[0099] Table 2: Reaction System Proportions

[0100]

[0101] Test results as follows Figure 2 As shown in Table 3, with wild-type T7 RNA polymerase (WT) as the control, all mutants significantly improved product integrity. Among them, M369K+A382K+L680V significantly improved product integrity from 66.9% in the wild type to 85.3%.

[0102] Table 3 RNA Product Integrity

[0103]

Claims

1. A variant of RNA polymerase characterized in that, The amino acid sequence of the variant is mutated at the following positions relative to SEQ ID NO: 1, I605P, or L680V, or L680I, or K71M+I605P+L680V, or E350A+I605P+L680V, or M369K+A382K+L680V, or M369K+I605P+L680V, or M369K+A382K+I605P+L680V, or N370P+I605P+L680V, or A373K+L680I, or A382K+L680I, or I605P+L680V or K642R+F644Y+L680V, and the amino acid sequence of the variant is represented by SEQ ID NO: 2-14, respectively.

2. 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 of claim 1; 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).

3. Composition, characterized in that, comprising the variant of claim 1.

4. A kit, characterized in that, comprising the variant of claim 1.

5. Use of the variant of claim 1, the composition of claim 3, or the kit of claim 4 in in vitro transcription for non-diagnostic or therapeutic purposes.

6. A method of preparing RNA, characterized by, The method comprises contacting a DNA template, modified or unmodified triphosphate nucleotides with the RNA polymerase variant of claim 1, incubating in an in vitro transcription reaction system, and obtaining a target RNA product.

7. The method of claim 6, wherein the target RNA is a saRNA.

8. Use of the RNA polymerase variant of claim 1 in the synthesis of RNA drugs.

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