RNA polymerase variants
Patent Information
- Application Number
- CN202511292587.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2024-09-24
- Filing Date
- 2025-09-11
- Publication Date
- 2026-10-09
- Estimated Expiration
- 2045-09-11
AI Technical Summary
[0004]RNA在疫苗等药物研发领域大放异彩，但在实际生产过程中的一些杂质的去除还有待进一步研究，其中双链RNA(dsRNA)杂质会引起强烈的免疫原性(Goubau et al.,2014；Kato et al.,2006；Mu et al.,2018)，因此亟急需开发有效降低双链RNA杂质的方法
[0060] The RNA polymerase variant described in this application exhibits high catalytic efficiency. Its use in mRNA synthesis reduces the generation of double-stranded RNA contaminants. Compared to existing reported mRNA preparation methods, it produces the lowest levels of double-stranded RNA contaminants, making it a safe, green, and low-double-stranded RNA contaminant mRNA preparation method. Utilizing this mRNA preparation method can significantly reduce the possibility of immunogenicity caused by double-stranded RNA impurities.
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Figure CN121406603B_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of biotechnology, specifically relating to RNA polymerase variants, their preparation methods, and their applications in RNA synthesis. Background of the Invention
[0002] mRNA vaccines have a relatively short development cycle, enabling the rapid development of novel candidate vaccines to address viral mutations. Through a dual mechanism of humoral and T-cell immunity, they exhibit strong immunogenicity and significant efficacy. Furthermore, their production process is simple, facilitating efficient research and development and large-scale production.
[0003] According to the latest news, in addition to mRNA, research on circular RNA (circRNA)-related drugs has also made breakthrough progress. Orna Therapeutics has developed in vivo cell therapy products using circRNA, and a research report presented at the 2022 American Society for Gene and Cell Therapy (ASGCT) Annual Meeting has demonstrated its great application potential in other fields such as cancer treatment.
[0004] RNA has played a significant role in the development of vaccines and other drugs, but the removal of some impurities in the actual production process still requires further research. Among them, double-stranded RNA (dsRNA) impurities can cause strong immunogenicity (Goubau et al., 2014; Kato et al., 2006; Mu et al., 2018). Therefore, there is an urgent need to develop effective methods to reduce double-stranded RNA impurities. Summary of the Invention
[0005] In a first aspect, this disclosure 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: G753 or K389.
[0006] Secondly, this disclosure provides a class of biological materials selected from one or more of the following:
[0007] 1) Encoding the polynucleotide molecules of the above variants;
[0008] 2) Expression vectors containing polynucleotide molecules as described in 1);
[0009] 3) Host cells containing the polynucleotide molecules described in 1), or host cells containing the expression vectors described in 2).
[0010] Thirdly, this disclosure provides a method for preparing the aforementioned RNA polymerase variant.
[0011] Fourthly, this disclosure provides a composition comprising at least one RNA polymerase variant as described in this disclosure.
[0012] Fifthly, this disclosure provides a kit comprising at least one RNA polymerase variant as described in this disclosure.
[0013] Sixthly, this disclosure provides the use of the above-described RNA polymerase variants, compositions, or kits in the in vitro transcription preparation of RNA.
[0014] In a seventh aspect, this disclosure also provides a method for preparing RNA. Invention Details
[0016] RNA polymerase variants
[0017] The RNA polymerase variant disclosed herein 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: G753 or K389; the mutation type may be either substitution or deletion.
[0018] In some embodiments, the amino acid sequence of the variant contains a mutation selected from any of the following sites compared to SEQ ID NO: 1: G753C, G753A+K389A.
[0019] In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-3.
[0020] biomaterials
[0021] This disclosure provides a polynucleotide encoding an RNA polymerase variant. Due to codon degeneracy or codon bias in 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 disclosure may be selected from SEQ ID NO: 5-6.
[0022] The expression vectors provided in this disclosure comprise a multinucleotide molecule encoding a variant of the RNA polymerase disclosed herein. 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.
[0023] 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 origin site. In some embodiments, the expression vector described in this disclosure is preferably pQE-80L.
[0024] The host cell provided in this disclosure refers to any cell that is favorable for expression of the variants of this disclosure, i.e., any cell that is susceptible after being transformed, transfected or transduced with the expression vectors described in this disclosure, and includes any daughter 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, 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(DE3).
[0026] Preparation methods of RNA polymerase variants
[0027] The method for preparing RNA polymerase variants provided in this disclosure includes 1) culturing the host cells described in this disclosure under suitable variant expression conditions; and 2) recovering the variant.
[0028] 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.
[0029] 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), ammonium sulfate precipitation, etc.
[0030] Composition
[0031] The compositions provided in this disclosure comprise at least one RNA polymerase variant described in this disclosure.
[0032] The compositions described in this disclosure can be compositions for storing RNA polymerase variants. In some embodiments, in addition to the RNA polymerase variants described above, the compositions described in this disclosure 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 compositions for storing RNA polymerase variants described in this disclosure contain: RNA polymerase variants, Tris-HCl, NaCl, EDTA, DTT, Triton X-100, and glycerol.
[0033] 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.
[0034] Reagent test kit
[0035] The kits provided in this disclosure contain at least one RNA polymerase variant as described in this disclosure.
[0036] In some embodiments, the kit may further comprise one or more in vitro transcription reaction reagents, such as buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the kit further comprises a cap analog, which may be selected from unmethylated cap analogs, dimethylated cap analogs, trimethylated cap analogs, dimethylated symmetrical cap analogs, or anti-reverse cap analogs. 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 pyrophosphatase, magnesium ions, etc. In one embodiment, the in vitro transcription system components may be selected from commercially available RNA in vitro transcription reagents.
[0037] application
[0038] This disclosure provides the use of the above-described RNA polymerase variants, compositions, or kits in the in vitro transcription preparation of RNA. This disclosure also provides the use of at least one variant as described herein in reducing the generation of dsRNA impurities in the in vitro transcription preparation of RNA.
[0039] 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 disclosure, incubating in an in vitro transcription reaction system to obtain the target product.
[0040] Preparation method
[0041] This disclosure 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 herein, incubating in an in vitro transcription reaction system to obtain a target product.
[0042] Suitable in vitro transcription reaction systems and incubation conditions for generating RNA products are well known in the art. Those skilled in the art can determine appropriate reaction system pH, reaction temperature, reaction time, salt concentration, or whether to add exogenous cofactors, taking into account the optimal activity of RNA polymerase. In some embodiments, the in vitro transcription reaction system described in this disclosure includes in vitro transcription reaction reagents: one or more buffer components, modified or unmodified nucleoside triphosphates, RNase inhibitors, pyrophosphatase, magnesium ions, water, etc. In some embodiments, the incubation temperature in the incubation step described in this disclosure is 30–50°C, preferably 37°C. In some embodiments, the incubation time in the incubation step described in this disclosure is 20–240 min, preferably 60 min.
[0043] In some embodiments, RNA products prepared using the methods described in this disclosure have higher yields, and / or higher integrity, and / or lower dsRNA impurity content, and / or more capped mRNA products compared to those prepared using wild-type RNA polymerase (SEQ ID NO: 1).
[0044] In some embodiments, the relative residual amount of dsRNA impurities (the ratio of residual dsRNA in the variant group to residual dsRNA in the WT group) of the RNA product prepared using the methods described in this disclosure 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 lower, compared to the RNA product prepared using the methods described in this disclosure.
[0045] 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, an 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.
[0046] Other implementation plans
[0047] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant, relative to SEQ ID NO: 1, contains a mutation selected from the following sites: G753C, G753A, or K389A.
[0048] 2. The variants described in item 1, whose amino acid sequences are shown in any of SEQ ID NO: 2-3.
[0049] 3. Biological materials, selected from one or more of the following:
[0050] 1) Encoding a polynucleotide molecule of an RNA polymerase variant as described in either item 1 or item 2;
[0051] 2) Expression vectors containing polynucleotide molecules as described in 1);
[0052] 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).
[0053] 4. A composition comprising a variant as described in either item 1 or item 2.
[0054] 5. A kit containing a variant as described in either item 1 or item 2.
[0055] 6. Use of any variant described in item 1 or 2, the composition described in item 4, or the kit described in item 5 in in vitro transcription.
[0056] 7. Applications of reducing the generation of dsRNA impurities during in vitro transcription to prepare RNA, as described in any variant of item 1 or 2, the composition described in item 4, or the kit described in item 5.
[0057] 8. As in the application of item 7, the RNA preparation process includes contacting a DNA template with a variant as described in either item 1 or 2, and incubating it in an in vitro transcription system.
[0058] 9. A method for preparing RNA, the method comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, and an RNA polymerase variant, incubating in an in vitro transcription reaction system to obtain a target RNA product; wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NO: 2-3.
[0059] Beneficial effects
[0060] The RNA polymerase variant described in this application exhibits high catalytic efficiency. Its use in mRNA synthesis reduces the generation of double-stranded RNA contaminants. Compared to existing reported mRNA preparation methods, it produces the lowest levels of double-stranded RNA contaminants, making it a safe, green, and low-double-stranded RNA contaminant mRNA preparation method. Utilizing this mRNA preparation method can significantly reduce the possibility of immunogenicity caused by double-stranded RNA impurities. Attached Figure Description
[0061] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids;
[0062] Figure 2 The effect of RNA polymerase variants on dsRNA impurity residues. Detailed Implementation
[0063] The technical solutions of this disclosure are further illustrated below with reference to specific embodiments. However, the following embodiments are merely examples of this disclosure and do not represent or limit the scope of protection of this disclosure. The scope of protection of this disclosure 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.
[0064] In this embodiment of the application, enzyme activity is defined as: at 37°C and pH 8.0, the enzyme activity is 1 nmol / mL within 1 hour. 3 The amount of enzyme required to incorporate H]ATP into an acid-insoluble precipitate is defined as one active unit.
[0065] Example 1: Preparation of RNA polymerase variants
[0066] The RNA polymerases and their variants shown in Table 1 were synthesized using DNA sequences (SEQ ID NO: 4-6) and then amplified by PCR. The DNA was then introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. This constructed vector was then introduced into *E. coli* BL21(DE3) via chemical transformation. The vector was plated on LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies were then subjected to plasmid extraction and sequencing to obtain recombinant engineered bacteria containing the target gene. The successfully sequenced *E. coli* recombinant strains were inoculated into LB medium for overnight activation culture, followed by inoculation into the fermentation broth (LB medium) at 1–5% v / v and cultured until OD500. With a 600 value of 0.6–0.8, after adding IPTG to a final concentration of 0.5 mol / L and culturing for 4–6 h, the strain was collected by centrifugation at 12000 rpm and 5 °C. The collected strain was washed with 0.2 M PBS buffer at pH 7.0 to obtain bacterial cells. After sonication, affinity chromatography was performed to purify the RNA polymerase and its variants.
[0067] The correspondence between RNA polymerases and their variants and amino acid sequences is shown in Table 1:
[0068] Table 1:
[0069]
[0070]
[0071] Example 2: Generation of dsRNA impurities during in vitro transcription
[0072] (1) The enzyme stock solution obtained in Example 1 was diluted with storage buffer (Vazyme, catalog number: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 2 were loaded into an octet and mixed well, then centrifuged. The octet was placed on a PCR instrument and reacted at 37°C for 1 h. Then 36 μL of magnetic beads (Vazyme, catalog number: N412) were added and mixed well. The mixture was then incubated at room temperature for 2–5 min. The mixture was placed on a magnetic rack to purify the mRNA. After purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.
[0073] (2) The content of dsRNA impurities was tested using a dsRNA detection kit (Vazyme, catalog number: DD3509).
[0074] Table 2: Reaction System Proportions
[0075]
[0076]
[0077] dsRNA detection results are shown in Figure 2 Compared with the WT group, the polymerase variants in Example 1 were able to effectively reduce the generation of dsRNA impurities during in vitro transcription.
Claims
1. An RNA polymerase variant, characterized in that, The amino acid sequences of the variants are shown in any of SEQ ID NO: 2-3.
2. A biomaterial, characterized in that, The biomaterial is selected from one or more of the following: 1) Encoding a polynucleotide molecule that is a variant of the RNA polymerase as described in claim 1; 2) Expression vectors containing polynucleotide molecules as described in 1); 3) A host cell containing a polynucleotide molecule as described in 1), or a host cell containing an expression vector as described in 2).
3. The composition, characterized in that, Includes the variant as described in claim 1.
4. A reagent kit, characterized in that, Includes the variant as described in claim 1.
5. The application of the variant of claim 1, the composition of claim 3, or the kit of claim 4 in reducing the generation of dsRNA impurities during in vitro transcription to prepare RNA.
6. The application of claim 5, wherein the RNA preparation process comprises contacting a DNA template with the variant of claim 1 and incubating it in an in vitro transcription system.
7. A method for preparing RNA, characterized in that, The method includes 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 polymerase variant is as shown in any one of SEQ ID NO: 2-3.