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WO2024230665A1 - Variant d'arn polymérase et son utilisation - Google Patents

Variant d'arn polymérase et son utilisation Download PDF

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Publication number
WO2024230665A1
WO2024230665A1 PCT/CN2024/091329 CN2024091329W WO2024230665A1 WO 2024230665 A1 WO2024230665 A1 WO 2024230665A1 CN 2024091329 W CN2024091329 W CN 2024091329W WO 2024230665 A1 WO2024230665 A1 WO 2024230665A1
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Prior art keywords
mutation
rna polymerase
rna
site
polymerase variant
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Chinese (zh)
Inventor
徐晓昱
金秋恒
何伟
王冲
胡荟雪
张新亚
任渊渊
耿琪
周红莉
邹杨逍宇
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Nanjing Vazyme Biotech Co Ltd
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Nanjing Vazyme Biotech Co Ltd
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Priority to CN202480009399.1A priority Critical patent/CN120641560A/zh
Publication of WO2024230665A1 publication Critical patent/WO2024230665A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)

Definitions

  • the present application relates to the field of biotechnology, and in particular to RNA polymerase variants and applications thereof.
  • mRNA therapy refers to the use of mRNA-based drugs to treat or prevent diseases.
  • IVTT in vitro transcribed
  • mRNA technology has become increasingly mature and has made remarkable progress in the research of cancer, rare diseases, genetic diseases and infectious diseases.
  • COVID-19 novel coronavirus pneumonia
  • mRNA vaccines there are two main types of mRNA vaccines: one is non-replicating mRNA, which refers to the delivery of a complete gene sequence synthesized in vitro into the body through a vector to induce a certain immune response.
  • the mRNA vaccines Comirnaty (developed by Pfizer/BioNTech) and Spikevax (developed by Moderna) that were conditionally approved by the FDA for marketing after 2020 are non-replicating vaccines; the other is virus-derived self-amplifying mRNA (saRNA).
  • saRNA virus-derived self-amplifying mRNA
  • saRNA virus-derived self-amplifying mRNA
  • saRNA In addition to encoding the required target protein, saRNA also carries a sequence that can express RNA polymerase (RNA-dependent RNA polymerase, RdRP).
  • saRNA can be produced using saRNA as a template.
  • saRNA can replicate autonomously in the body, and only a very small amount is needed to induce a stronger immune response and prolong the existence time of the antigen protein in the body.
  • saRNA forms double-stranded RNA during the replication process, it may stimulate the inherent immune response of cells, which can further enhance the effect of the vaccine and thus prolong the therapeutic effect; therefore, the use of saRNA vaccines can reduce the injection dose and number of injections used, and reduce the possible toxic side effects of mRNA and drug delivery vehicles.
  • saRNA vaccines entering clinical trials, but none of them have been approved for marketing.
  • the integrity of the self-replicating template has always been a difficult problem to solve due to its long fragments (>8000bp).
  • mRNA vaccines Compared with traditional vaccines, mRNA vaccines have the advantages of short R&D cycle, strong immunogenicity, simple production process, etc.
  • the production of mRNA vaccines involves multiple biological processes and raw material processing, which requires quality assessment of mRNA API characteristics, purity, quantity, physical state (integrity) and safety, among which the assessment of mRNA integrity is a crucial step.
  • In vitro RNA synthesis will produce truncated or over-extended impurities, which will not only reduce the integrity of mRNA, but also lead to an increase in adverse immunogenicity. Therefore, it is crucial to ensure the integrity of mRNA during in vitro RNA synthesis.
  • the present application provides a plurality of RNA polymerase variants, which can significantly reduce the truncation and over-extension probability of mRNA products in in vitro transcription.
  • the present application also provides methods for preparing these variants and their use in in vitro RNA synthesis.
  • the present application provides an RNA polymerase variant, the amino acid sequence of which has at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity compared to SEQ ID NO: 1, wherein the amino acid sequence of the RNA polymerase variant comprises at least one mutation selected from the following amino acid sites relative to SEQ ID NO: 1: R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391 or K392, wherein the mutation type is substitution or deletion.
  • the amino acid sequence of the variant comprises a mutation selected from the following sites: R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391 or K392, wherein the mutation type is substitution or deletion.
  • the amino acid sequence of the variant has an amino acid mutation relative to SEQ ID NO: 1, and the mutation site can be selected from: R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391 or K392, wherein the mutation type is substitution or deletion.
  • the mutation at position R34 is R34A.
  • the mutation at position D130 can be selected from D130E or D130C.
  • the mutation at position V174 can be selected from V174A, V174D, V174E, V174G or deletion (DEL174).
  • the mutation at position Y178 can be selected from Y178H, Y178G, Y178V, Y178F, Y178D, Y178N, Y178E, Y178S, Y178T or Y178P.
  • the mutation at position G285 is G285A.
  • the mutation at position K303 is K303L.
  • the mutation at position N325 can be selected from N325A or N325L.
  • the mutation at position Y385 can be selected from Y385A, Y385E or Y385N.
  • the mutation at the R386 position can be selected from R386A, R386L, R386I, R386M, R386V, R386S, R386T, R386H, R386Q, R386N, R386K, R386D, R386C, R386W or R386G.
  • the mutation at the K387 position can be selected from K387G, K387Y, K387Q, K387A, K387N, K387S or K387W.
  • the mutation at position D388 can be selected from D388Y or D388L.
  • the mutation at position K389 can be selected from K389A or K389R.
  • the mutation at point R391 can be selected from R391A, R391L or R391K.
  • the mutation at the K392 position can be selected from K392A, K392F, K392I, K392T, K392H, K392Q, K392E or K392C.
  • the amino acid sequence of the variant described in the present application has at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or higher sequence identity compared to the sequence shown in any one of SEQ ID NO: 2-63. In some embodiments, the amino acid sequence of the variant described in the present application is as shown in any one of SEQ ID NO: 2-63.
  • sequence identity refers to the relationship between the sequences of two or more polypeptides, as determined by comparing (aligning) the sequences.
  • the present application provides a polynucleotide encoding an RNA polymerase variant. Due to codon degeneracy or codon preference of a host cell expressing a polypeptide, the polynucleotide is any polynucleotide encoding the variant described in the present application without changing the amino acid sequence. In some embodiments, the polynucleotide encoding the RNA polymerase variant of the present application can be selected from SEQ ID NO: 65-126.
  • the present application provides a method for preparing an RNA polymerase variant, comprising generating at least one RNA polymerase variant described herein in a host cell.
  • the host cell contains an expression vector carrying an RNA polymerase variant polynucleotide molecule described herein.
  • the present application provides a method for generating an RNA product, comprising contacting a template DNA molecule with one or more RNA polymerase variants described herein under conditions that result in the generation of an RNA transcription product.
  • the RNA product is mRNA.
  • the RNA product is saRNA.
  • the RNA product can also be siRNA, gRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc.
  • the present application provides a method for in vitro transcription, which includes contacting a DNA template with one or more RNA polymerase variants described in the present application under conditions that result in the generation of RNA transcription products.
  • the method includes the following steps: 1) providing a DNA template comprising a T7 promoter, wherein the T7 promoter is functionally linked to a target nucleotide sequence to be transcribed; 2) contacting the DNA template in step 1) with one or more RNA polymerase variants described in the present application; and 3) incubating the DNA template and RNA polymerase variant in an in vitro transcription system.
  • the length of the DNA template is 1000-13000bp. In some embodiments, the length of the DNA template can be selected from 1000-10000bp. In some embodiments, the length of the DNA template can be selected from 8000-10000bp. In some embodiments, the length of the DNA template can be selected from 10000-13000bp.
  • the in vitro transcription system comprises a nucleoside triphosphate and a buffer component.
  • the nucleoside triphosphate can be selected from modified or unmodified nucleoside triphosphates (including analogs thereof).
  • the nucleoside triphosphate can be selected from unmodified ATP, GTP, CTP, UTP.
  • the nucleoside triphosphate can be selected from modified nucleoside triphosphates, and the modification types on the nucleoside include but are not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ⁇ (pseudouridine), m1 ⁇ (N1-methyl-pseudouridine), markers (markers can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).
  • the in vitro transcription system described in step 3) further comprises RNase inhibitor, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription system described in step 3) further comprises DEPC water.
  • the RNA produced by the method for generating RNA or the method for in vitro transcription described in the present application is purified, and the integrity of the RNA product is improved; wherein the integrity is the proportion of the complete RNA product obtained.
  • the RNA product may be a coding RNA or a non-coding RNA.
  • the RNA product includes but is not limited to mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc.
  • the mRNA produced by the methods for generating RNA or in vitro transcription described herein, after purification, has an mRNA product integrity that is improved 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% compared to that produced by wild-type T7 RNA polymerase (SEQ ID NO: 1).
  • the saRNA produced by the method for generating RNA or the in vitro transcription method described in the present application after purification, has an integrity of the saRNA product improved by at least about 3%, about 5%, about 10%, about 12%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24% or about 25% compared to using wild-type T7 RNA polymerase.
  • the present application provides a method for co-transcriptional capping, comprising reacting a DNA template with one or more RNA polymerase variants and cap analogs described in the present application under conditions that result in the generation of an RNA transcription product.
  • the cap analog has the structure:
  • B 1 -B n+1 are each independently a nucleobase;
  • R 1 is H or alkyl,
  • R 2 -R n+3 are each independently H, OH, alkyl, O-alkyl, S-alkyl or NH-alkyl,
  • X 1 -X n+3 are each independently O or S,
  • Y 1 -Y n+4 are each independently O, S, NH or CH 2 ,
  • Z 1 -Z n+3 are each independently OH, SH, BH 3 , alkyl or O-alkyl
  • M 1 -M n are each independently O, S, NH or CH 2 ;
  • l is 0, 1 or 2
  • n 1 is 1
  • n 1 -nn are each independently 0 or 1.
  • RNA transcription products The conditions leading to the generation of RNA transcription products described in this application are well known in the art. A person of ordinary skill in the art can determine the appropriate pH value, reaction temperature, reaction time, salt concentration of the reaction system, or whether to add exogenous auxiliary factors, etc., taking into account the optimal activity of RNA polymerase.
  • the present application provides a composition or kit comprising one or more RNA polymerase variants described in the present application.
  • the composition or kit further comprises one or more A variety of in vitro transcription system components; wherein the in vitro transcription system components can be selected from nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, buffer components, magnesium ions, etc.
  • the in vitro transcription system components can be selected from commercially available RNA in vitro transcription reagents.
  • RNA polymerase variant described in the present application is used in the synthesis of RNA drugs.
  • the present application provides a composition comprising RNA and a pharmaceutically acceptable excipient, wherein the RNA is produced by the in vitro transcription method described in the present application.
  • the RNA product is not chemically modified.
  • the RNA product is chemically modified.
  • RNA polymerase variant whose amino acid sequence has at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher sequence identity compared to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises at least one mutation selected from the following sites relative to SEQ ID NO: 1: R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391 or K392.
  • the mutation at the D130 site may be selected from D130E or D130C;
  • the mutation at the V174 site may be selected from V174A, V174D, V174E, V174G or DEL174;
  • the mutation at the Y178 position may be selected from Y178H, Y178G, Y178V, Y178F, Y178D, Y178N, Y178E, Y178S, Y178T or Y178P;
  • the mutation at the N325 site can be selected from N325A or N325L;
  • the mutation at the Y385 site can be selected from Y385A, Y385E or Y385N;
  • the mutation at the R386 site can be selected from R386A, R386L, R386I, R386M, R386V, R386S, R386T, R386H, R386Q, R386N, R386K, R386D, R386C, R386W or R386G;
  • the mutation at the K387 site may be selected from K387G, K387Y, K387Q, K387A, K387N, K387S or K387W;
  • the mutation at the D388 site can be selected from D388Y or D388L;
  • the mutation at the K389 site can be selected from K389A or K389R;
  • the mutation at point R391 can be selected from R391A, R391L or R391K;
  • the mutation at the K392 site can be selected from K392A, K392F, K392I, K392T, K392H, K392Q, K392E or K392C.
  • a polynucleotide molecule comprising a polynucleotide molecule encoding the RNA polymerase variant described in any one of items 1 to 4.
  • a method for preparing an RNA polymerase variant comprising introducing the polynucleotide molecule described in item 5 into a host cell.
  • a method for producing an RNA product comprising contacting a DNA template with at least one RNA polymerase variant as described in any one of items 1 to 4 under conditions that result in the production of an RNA transcript.
  • the RNA product can be selected from mRNA, saRNA, siRNA, gRNA, dsRNA, ssRNA, miRNA, piRNA or shRNA; preferably mRNA or saRNA.
  • a method for producing saRNA comprising contacting a DNA template with at least one RNA polymerase variant as described in any one of items 1-4 under conditions that result in the production of an RNA transcript.
  • a method for performing in vitro transcription comprising reacting a DNA template with at least one RNA polymerase variant as described in any one of items 1 to 4 under conditions resulting in the production of an RNA transcript.
  • composition or kit comprising at least one RNA polymerase variant as described in any one of items 1 to 4.
  • composition or kit as described in item 11 further comprises one or more components of an in vitro transcription system.
  • RNA polymerase variants provided in this application have improved performance and can be used in in vitro transcription. While improving the integrity of RNA products, it can also significantly reduce the chances of RNA truncation and over-extension, reduce the production of by-products, and has adaptability to a wide range of templates and application scenarios.
  • RNA polymerase mutants provided in this application can reduce the purification cost in the production process of RNA drugs, quickly prepare RNA drugs that meet the requirements for use, and provide an efficient tool enzyme for the large-scale production of RNA drugs.
  • Figure 1 is a schematic diagram of the construction of the recombinant plasmid.
  • the enzyme activity is defined as the amount of enzyme required to generate 1 ⁇ mol of product or convert 1 ⁇ mol of substrate within 1 minute under the reaction condition of 37°C.
  • SEQ ID NO: 64-126 the encoded amino acid sequence corresponds to SEQ ID NO: 1-63
  • a DNA fragment was synthesized and introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L after PCR amplification to obtain a recombinant expression vector.
  • the constructed vector was introduced into E. coli BL21 (DE3) by transformation technology, spread on an LB plate containing ampicillin resistance, and incubated in a 37°C incubator overnight. The grown single colonies were subjected to plasmid extraction and sequencing to finally obtain recombinant engineered bacteria containing the target gene.
  • coli strain was inoculated into LB medium for overnight activation culture, and then inoculated into the fermentation liquid (LB medium) at 1% V/V and cultured to an OD 600 value of 0.6-0.8. After adding IPTG with a final concentration of 0.5 mol/L and continuing to culture for 4-6 hours, the strain was collected by centrifugation at 12000 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 bacteria; after ultrasonic disruption, affinity chromatography purification was performed to obtain RNA polymerase stock solution.
  • RNA polymerase variants and their mutation sites are shown in Table 1:
  • WT is the wild-type T7 RNA polymerase, and its amino acid sequence is:
  • MIX solution Prepare MIX solution according to the reaction system (20 ⁇ L) in Table 2, transfer the MIX solution to the eight-row strip, mix well, and centrifuge; place the eight-row strip on a PCR instrument and react at 37°C for 1h, then add 36 ⁇ L of magnetic beads (Vazyme, catalog number: N412), mix well, and incubate at room temperature for 2-5min; place the mixed solution on a magnetic rack to purify mRNA, and after purification, transfer it to an RNase-free centrifuge tube to obtain purified mRNA;
  • test results are shown in Tables 3-1 and 3-2.
  • T7 RNA polymerase variants in Tables 3-1 and 3-2 can effectively increase the integrity of the mRNA product.
  • test results showed that the integrity of saRNA in the transcription products of the WT group was only 56.4%, while the variants of the K387Y group and K389A group could increase the integrity of saRNA products to 73.1% and 75.5%, respectively, achieving a significant improvement in the integrity of mRNA products.

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Abstract

L'invention concerne un variant d'ARN polymérase et son utilisation. Une réaction de transcription in vitro est effectuée à l'aide du variant d'ARN polymérase de la présente invention, de telle sorte que la génération d'impuretés tronquées ou trop étendues est réduite, et l'intégrité du produit d'ARN de transcription est améliorée. Le variant d'ARN polymérase est approprié pour la préparation d'un produit d'ARN de 1000 à 13000 nt. En outre, La présente invention porte sur un procédé de synthèse d'un ARN.
PCT/CN2024/091329 2023-05-09 2024-05-07 Variant d'arn polymérase et son utilisation Pending WO2024230665A1 (fr)

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CN202480009399.1A CN120641560A (zh) 2023-05-09 2024-05-07 Rna聚合酶变体及其应用

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CN202310515121.9A CN120366260A (zh) 2023-05-09 2023-05-09 Rna聚合酶变体及其应用
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WO2025152833A1 (fr) * 2024-01-19 2025-07-24 南京诺唯赞生物科技股份有限公司 Variant d'arn polymérase et son utilisation

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