RNA polymerase mutants, methods for their preparation and their use in RNA synthesis

Abstract

The present invention provides an RNA polymerase mutant and a method for producing the same. The use of the RNA polymerase mutant in in vitro transcription can produce RNA products with low dsRNA impurity content and high integrity, while also improving the utilization efficiency of cap analogs in co-transcriptional capping reaction systems and reducing costs. A method for producing RNA by in vitro transcription is also provided.

Description

[Technical Field]

[0001] This application is in the field of biotechnology, and specifically relates to RNA polymerase mutants, methods for their production, and their use in RNA synthesis. [Background technology]

[0002] At the end of 2019, a global pandemic of coronavirus disease 2019 (COVID-19) occurred, infecting billions of people worldwide. Prior to the launch of Paxlovid (a combination of nilmatrervir and ritonavir) (which received emergency marketing authorization from the FDA on November 22, 2021), vaccines were the most effective defense against infection. In this massive global battle against infectious diseases, billions of people have been administered COVID-19 vaccines, including BNT162b2 (mRNA vaccine) developed by BioNTech and Pfizer, mRNA-1273 (mRNA vaccine) developed by Moderna, and AZD1222 (adenovirus vector vaccine) developed by AstraZeneca. Among these, RNA vaccines boast the highest preventive efficacy. mRNA vaccines have made significant contributions to infectious disease prevention by preventing infection, reducing the severity of illness, and suppressing the spread of the virus.

[0003] The relatively short development cycle of mRNA vaccines makes it possible to rapidly develop new vaccine candidates that respond to viral mutations. Their dual mechanisms of humoral immunity and T cell immunity result in strong immunogenicity and pronounced efficacy. Their simple production process allows for efficient development and large-scale production, enabling rapid global supply in response to infectious diseases such as the new coronavirus.

[0004] According to the latest news, in addition to mRNA, there has also been significant progress in the research of circular RNA (circRNA)-related drugs. Orna Therapeutics has developed an in vivo cell therapy product using circRNA. A research report presented at the 2022 American Society for Gene and Cell Therapy (ASGCT) Annual Meeting demonstrated the significant potential for application in other fields, such as oncology treatment.

[0005] RNA has made great progress in the development of pharmaceuticals such as vaccines. However, further research is needed to remove impurities in actual manufacturing processes. 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 methods to effectively reduce dsRNA impurities. Summary of the Invention [Problem to be solved by the invention]

[0006] The present application provides RNA polymerase mutants, methods for their production, and their use in RNA synthesis. [Means for solving the problem]

[0007] According to a first aspect of the present application, there is provided an RNA polymerase mutant, whose amino acid sequence comprises at least one, at least two, at least three, at least four, at least five, or at least six amino acid mutations relative to SEQ ID NO:1 at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, wherein the type of mutation is selected from deletion or substitution, and the amino acid sequence of the mutant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some embodiments, the variant comprises one, two, three, four, five, or six amino acid mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, where the type of mutation is selected from deletions or substitutions. In some embodiments, the type of mutation is a deletion (indicated by DEL, such as DEL5, indicating deletion of an amino acid at position 5). In some embodiments, the type of mutation is a substitution (e.g., K5A, indicating a lysine at position 5 has been mutated to alanine). In some embodiments, the type of mutation includes deletions and substitutions, i.e., deletions occur at some positions and substitutions occur at some positions.

[0008] In some embodiments, the RNA polymerase mutants provided herein have an amino acid sequence comprising a single amino acid mutation relative to SEQ ID NO:1, wherein the mutation occurs at an amino acid position selected from N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, and the type of mutation is a substitution or deletion. In some embodiments, the amino acid sequence of the mutant comprises a single amino acid mutation relative to SEQ ID NO:1, wherein the mutation is selected from: (1) a substitution at position N171: the substituted amino acid is G; or (2) a substitution or deletion at position K172: the substituted amino acid may be selected from A, G, E, D, H, Y, S, W, P, I, M, V, F, T, C, N, L; or (3) a substitution or deletion at position R173: the substituted amino acid may be selected from A, C, G, E, D, H, Y, S, W, P, N, Q; or (4) a substitution at position Y178: the substituted amino acid is H; or (5) a substitution at position R298: the substituted amino acid is A; or (6) a substitution at position Y385: the substituted amino acid may be selected from A, E, D, or (7) a substitution at position K387: the substituted amino acid may be selected from A, Y, S, Q; or (8) a substitution at position D388: the substituted amino acid may be selected from A, G, L; or (9) Substitution at position F880: The substituted amino acid may be selected from A, G, and W.

[0009] In some embodiments, the RNA polymerase mutants provided herein comprise two, three, four, or five amino acid mutations relative to SEQ ID NO:1 at positions selected from D130, K172, R173, Y178, R298, Y385, K387, D388, or F880, and the amino acid sequence of the mutant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity compared to SEQ ID NO:1. In some embodiments, the mutant comprises: (1) a mutation at position K172 and one, two, three, or four amino acid mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388, or F880, wherein the type of mutation is a substitution or deletion; or (2) a mutation at position R173 and one, two, or three amino acid mutations at positions selected from D130, Y178, R298, K387, or D388, wherein the type of mutation is a substitution or deletion; or (3) two or three amino acid mutations at positions R298, Y385, K387, or D388, wherein the type of mutation is a substitution or deletion.In some embodiments, the amino acid sequence of the variants is, relative to SEQ ID NO: 1, K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, D130+R173, R173+Y178, R173+D388, R173+R298, K387+R298, Y385+R298, D388+R298, Y385+K387, Y385+D388, K387+D388, K172+R173+Y385, K172+R173+D388, K172+R173 +K387, K172+R173+F880, K172+R173+Y178, D130+K172+R173, D130+K172+Y178, D130 +K172+K387, D130+R173+D388, K172+Y178+D388, D130+K172+D388, K172+K387+R29 8, Y385+K387+D388, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y17 The mutant may further comprise any one of the following combinations of mutations: D130+K172+R173+D388, D130+K172+R173+Y178, D130+R173+Y178+K387, D130+K172+Y178+D388, and D130+K172+R173+Y178+D388. In some embodiments, the mutant further comprises a D130E mutation at position D130. In some embodiments, the mutant further comprises a D130E mutation at position K172 selected from DEL172, K172A, or K172G. In some embodiments, the mutant further comprises a DEL173 mutation at position R173 selected from DEL173, R173A, R173G, or R173C. In some embodiments, the mutant may further comprise a mutation at position Y178 selected from Y178H or Y178P. In some embodiments, the mutant may further comprise a mutation at position R298 that is R298A. In some embodiments, the mutant may further comprise a mutation at position Y385 that is Y385A.In some embodiments, the mutant may further comprise a mutation at position K387 selected from K387S, K387Y, or K387G. In some embodiments, the mutant may further comprise a mutation at position D388 selected from D388Y, D388A, or D388G. In some embodiments, the mutant may further comprise a mutation at position F880 selected from F880A or F880Y.

[0010] In some embodiments, the variant comprises a mutation at position K172 and further comprises one, two, three or four amino acid mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388 or F880, wherein the type of mutation is a substitution or deletion, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity compared to SEQ ID NO:1. In some embodiments, the variants are K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, K172+R173+Y385, K172+R173+D388, K172+R173+K387, K172+R173+F880, K172+R173+Y178, D130+K172+R173, D130+K172+Y178, D130+K172+K387, K172+Y In some embodiments, the mutant further comprises any one of the following mutation groups: K172+R173+Y385+F880, K172+R173+D388, D130+K172+D388, K172+K387+R298, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y178+D388, D130+K172+R173+Y178, D130+K172+Y178+D388, and D130+K172+R173+Y178+D388. In some embodiments, the mutant further comprises any one of the following mutation groups: K172+R173+Y385+F880, K172+R173+Y178+D388, D130+K172+R173+Y178, D130+K172+Y178+D388, and D130+K172+R173+Y178+D388. In some embodiments, the mutation at position D130 is D130E. In some embodiments, the mutant may further comprise a mutation at position K172 selected from DEL172, K172A, or K172G. In some embodiments, the mutant may further comprise a mutation at position R173 selected from DEL173, R173A, R173G, or R173C. In some embodiments, the mutant may further comprise a mutation at position Y178 of Y178H. In some embodiments, the mutant may further comprise a mutation at position R298 of R298A. In some embodiments, the mutant may further comprise a mutation at position Y385 of Y385A.In some embodiments, the mutant may further comprise a mutation at position K387 selected from K387S, K387Y, or K387G. In some embodiments, the mutant may further comprise a mutation at position D388 selected from D388Y, D388A, or D388G. In some embodiments, the mutant may further comprise a mutation at position F880 selected from F880A or F880Y.

[0011] In some embodiments, the variant comprises a mutation at position R173 and further comprises one, two, or three amino acid mutations at positions selected from D130, Y178, R298, K387, D388, or F880, wherein the type of mutation is a substitution or deletion, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity compared to SEQ ID NO: 1. In some embodiments, the variant comprises any one of the following mutations selected from the group consisting of D130+R173, R173+Y178, R173+D388, R173+R298, D130+R173+D388, and D130+R173+Y178+K387. In some embodiments, the variant further comprises a mutation at position D130 that is D130E. In some embodiments, the mutant may further comprise a mutation at position R173 selected from DEL173, R173A, R173G, or R173C. In some embodiments, the mutant may further comprise a mutation at position Y178 selected from Y178H or Y178P. In some embodiments, the mutant may further comprise a mutation at position R298 selected from R298A. In some embodiments, the mutant may further comprise a mutation at position K387 selected from K387Y. In some embodiments, the mutant may further comprise a mutation at position D388 selected from D388Y or D388G.

[0012] In some embodiments, the variant comprises any two or three amino acid mutations at positions selected from R298, Y385, K387, or D388, wherein the type of mutation is a substitution or deletion, and the amino acid sequence of the variant has at least 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the variant comprises any one of the following mutation groups: K387+R298, Y385+R298, D388+R298, Y385+K387, Y385+D388, K387+D388, and Y385+K387+D388. In some embodiments, the variant further comprises R298A as the mutation at position R298. In some embodiments, the variant further comprises Y385A as the substitution at position Y385. In some embodiments, the mutant may further comprise a substitution at position K387 selected from K387S or K387Y, and in some embodiments, the mutant may further comprise a substitution at position D388 selected from D388A or D388G.

[0013] In some embodiments, the amino acid sequence of a variant described herein 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 more sequence identity to any one of the amino acid sequences selected from SEQ ID NOs: 2-141. In some embodiments, the amino acid sequence of a variant described herein is selected from SEQ ID NOs: 2-141.

[0014] In a second aspect, the present application provides methods for producing an RNA polymerase mutant, comprising producing at least one of the RNA polymerase mutants described herein in a host cell. In some embodiments, the host cell comprises an expression vector having a nucleotide sequence corresponding to an RNA polymerase mutant described herein. In some embodiments, various modifications may be present in the coding region of the polynucleotide sequence, as long as the amino acid sequence of the mutant does not change due to codon degeneracy or preferred codons in the organism in which the mutant is expressed. In some embodiments, the nucleotide sequence is selected from SEQ ID NOs: 143-282.

[0015] In a third aspect, the present application provides a method for reducing the production of dsRNA impurities in RNA production by in vitro transcription, comprising contacting a DNA template with one or more RNA polymerase mutants described herein and incubating them in an in vitro transcription system.

[0016] In a fourth aspect, the present application provides a method for improving the integrity of an RNA product obtained by in vitro transcription, comprising contacting a DNA template with one or more RNA polymerase mutants described herein and incubating them in an in vitro transcription system.

[0017] In a fifth aspect, the present application provides a method of producing RNA, comprising contacting a DNA template with one or more RNA polymerase mutants described herein and incubating in an in vitro transcription system under conditions that result in the production of an RNA transcript.

[0018] In some embodiments, the RNA produced using the methods described herein may be coding or non-coding RNA, including, but not limited to, mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc. In some embodiments, the RNA product is mRNA. In some embodiments, the RNA product is saRNA.

[0019] In some embodiments, the length of the DNA template may be selected from 1,000 to 13,000 bp. In some embodiments, the length of the DNA template may be selected from 8,000 to 13,000 bp. In some embodiments, the length of the DNA template may be selected from 10,000 to 13,000 bp.

[0020] In some embodiments, the RNA produced using the methods described herein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more reduced residual dsRNA impurities after purification compared to when a wild-type RNA polymerase (SEQ ID NO: 1) is used. In some embodiments, the RNA produced using the methods described herein has a residual dsRNA impurity rate (residual dsRNA / total RNA) of less than 0.04%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, less than 0.0005%, less than 0.0003%, or less than 0.0001% after purification.

[0021] In some embodiments, the mRNA produced using the methods described herein exhibits 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% improved mRNA product integrity after purification compared to using wild-type T7 RNA polymerase (SEQ ID NO:1).

[0022] In some embodiments, the saRNA produced using the methods described herein exhibits 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%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% improved saRNA product integrity after purification compared to that produced using wild-type T7 RNA polymerase.

[0023] In a sixth aspect, the present application provides a method for producing capped RNA by in vitro transcription, comprising incubating a DNA template with one or more RNA polymerase mutants and cap analogs described herein in an in vitro transcription reaction system.

[0024] 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 m 7 GpppApA, m 7 GpppApC, m 7 GpppApG, m 7 GpppApU, m 7 GpppCpA, m 7 GpppCpC, m 7 GpppCpG, m7 GpppCpU, m 7 GpppGpA, m 7 GpppGpC, m 7 GpppGpG, m 7 GpppGpU, m 7 GpppUpA,m 7 GpppUpC, m 7 GpppUpG, and m 7 In some embodiments, the trinucleotide cap is selected from m 7 G 3′OMe pppApA, m 7 G 3′OMe pppApC, m 7 G 3′OMe pppApG, m 7 G 3′OMe pppApU, m 7 G 3′OMe pppCpA, m 7 G 3′OMe pppCpC, m 7 G 3′OMe pppCpG, m 7 G 3′OMe pppCpU, m 7 G 3′OMe pppGpA, m 7 G 3′OMe pppGpC, m 7 G 3′OMe pppGpG, m 7 G 3′OMe pppGpU,m 7 G 3′OMe pppUpA,m 7 G 3′OMe pppUpC,m 7 G 3′OMe pppUpG, and m 7 G 3′OMe In some embodiments, the trinucleotide cap is selected from m 7 G 3′OMe pppA 2′OMe pA, m 7 G 3′OMe pppA 2′OMe pC, m 7 G 3′OMe pppA 2′OMe pG, m 7 G 3′OMepppA 2′OMe pU, m 7 G 3′OMe pppC 2′OMe pA, m 7 G 3′OMe pppC 2′OMe pC, m 7 G 3′OMe pppC 2′OMe pG, m 7 G 3′OMe pppC 2′OMe pU, m 7 G 3′OMe pppG 2′OMe pA, m 7 G 3′OMe pppG 2′OMe pC, m 7 G 3′OMe pppG 2′OMe pG, m 7 G 3′OMe pppG 2′OMe pU, m 7 G 3′OMe pppU 2′OMe pA, m 7 G 3′OMe pppU 2′OMe pC, m 7 G 3′OMe pppU 2′OMe pG, and m 7 G 3′OMe pppU 2′OMe In some embodiments, the trinucleotide cap is selected from m 7 GpppA 2′OMe pA, m 7 GpppA 2′OMe pC, m 7 GpppA 2′OMe pG, m 7 GpppA 2′OMe pU, m 7 GpppC 2′OMe pA, m 7 GpppC 2′OMe pC, m 7 GpppC 2′OMe pG, m 7 GpppC 2′OMe pU, m 7 GpppG 2′OMe pA, m 7 GpppG 2′OMe pC, m 7GpppG 2′OMe pG, m 7 GpppG 2′OMe pU, m 7 GpppU 2′OMe pA, m 7 GpppU 2′OMe pC, m 7 GpppU 2′OMe pG, and m 7 GpppU 2′OMe In some embodiments, the trinucleotide cap is preferably selected from m 7 GpppA 2’OMe pG.

[0025] In some embodiments, in vitro transcription capping reactions are performed using the polymerase mutants described herein, resulting in an increase in capping rate of the mRNA product of up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to using wild-type T7 RNA polymerase (SEQ ID NO: 1). In some embodiments, the increase in capping rate of the mRNA product can be up to 100%.

[0026] In a seventh aspect, the present application provides in vitro transcription methods comprising contacting a DNA template with one or more RNA polymerase mutants described herein and incubating in an in vitro transcription system under conditions that result in the production of an RNA transcript. In some embodiments, the method comprises the steps of: 1) providing a DNA template comprising a T7 promoter operably linked to a target nucleotide sequence to be transcribed; 2) contacting the DNA template from step 1 with one or more RNA polymerase mutants described herein; and 3) incubating the DNA template and RNA polymerase mutants in the in vitro transcription system.

[0027] In some embodiments, the incubation temperature in step 3) is 30° C. to 50° C., preferably 37° C. In some embodiments, the incubation time in step 3) is 20 to 240 minutes, preferably 60 minutes.

[0028] The in vitro transcription system described herein comprises nucleoside triphosphates and buffering components.

[0029] In some embodiments, the nucleoside triphosphate may be selected from modified or unmodified nucleoside triphosphates (including analogs thereof). In some embodiments, the nucleoside triphosphate may be selected from unmodified ATP, GTP, CTP, and UTP. In some embodiments, the nucleoside triphosphate may be selected from modified nucleoside triphosphates, including, but not limited to, m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methylpseudouridine), and labeled nucleoside triphosphates (the label may be biotin, a fluorescent substance, digoxin, a radioactive element, etc.).

[0030] In some embodiments, the in vitro transcription system further comprises an RNase inhibitor, inorganic pyrophosphatase, and magnesium ions, hi some embodiments, the in vitro transcription system further comprises DEPC water.

[0031] In an eighth aspect, the present application provides a composition or kit comprising one or more RNA polymerase mutants described herein.

[0032] In one embodiment, the composition or kit further comprises one or more buffer components. In one embodiment, the composition or kit further comprises one or more in vitro transcription system components, which may be selected from nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, and the like. In one embodiment, the in vitro transcription system components may be selected from commercially available mRNA in vitro transcription reagents.

[0033] In some embodiments, the composition or kit further comprises a cap analog. 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 preferably selected from the group consisting of m 7 GpppA 2’OMe pG.

[0034] In a ninth aspect, the present application provides a composition comprising RNA and a pharmaceutically acceptable excipient, wherein the RNA is produced by an in vitro transcription method described herein. In some embodiments, the RNA product is chemically unmodified. In some embodiments, the RNA product is chemically modified.

[0035] Other embodiments: 1. An RNA polymerase mutant, the amino acid sequence of which comprises one, two, three, four, or five amino acid mutations relative to SEQ ID NO:1 at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880, wherein the type of mutation is a substitution or deletion, and the amino acid sequence of the mutant has at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1. 2. The variant of paragraph 1, wherein the amino acid sequence of the variant comprises one amino acid mutation relative to SEQ ID NO:1, and the mutated amino acid position is selected from N171, K172, R173, Y178, R298, Y385, K387, D388, or F880. 3. The variant of paragraph 1, wherein the amino acid sequence of the variant, relative to SEQ ID NO:1, comprises: (1) a mutation at position K172 and any one, two, three, or four amino acid mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388, or F880, wherein the type of mutation is a substitution or deletion; or (2) a mutation at K173 and any one, two, or three amino acid mutations at positions selected from D130, Y178, R298, K387, or D388, wherein the type of mutation is a substitution or deletion; or (3) Any two or three amino acid mutations at positions R298, Y385, K387, or D388: the type of mutation is a substitution. 4. The mutants according to paragraph 3, comprising K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, D130+R173, R173+Y178, R173+D388, R173+R298, K387+R298, Y385+R298, D38 8+R298, Y385+K387, Y385+D388, K387+D388, K172+R173+Y385, K172+R173+D388, K17 2+R173+K387, K172+R173+F880, K172+R173+Y178, D130+K172+R173, D130+K172+Y17 8, D130+K172+K387, D130+R173+D388, K172+Y178+D388, D130+K172+D388, K172+K38 7+R298, Y385+K387+D388, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R17 3+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y178, D130+R173+Y178+K387, D130+K172+Y178+D388, or D130+K172+R173+Y178+D388. 5. The mutant according to paragraph 2, 1) The type of mutation at position N171 is N171G, 2) the type of mutation at position K172 is selected from DEL172, K172A, K172H, K172R, K172Y, K172S, K172E, K172D, K172W, K172F, K172I, K172M, K172V, K172P, K172T, K172C, K172N, K172Q, K172G, or K172L; 3) the type of mutation at position R173 is selected from DEL173, R173A, R173H, R173R, R173Y, R173S, R173E, R173D, R173W, R173F, R173I, R173M, R173V, R173P, R173T, R173C, R173N, R173Q, R173G, or R173L; 4) the type of mutation at position Y178 is Y178H; 5) The type of mutation at position R298 is R298A, 6) the type of mutation at position Y385 is selected from Y385A, Y385D, or Y385E; 7) The type of mutation at position K387 is selected from K387Q, K387Y, K387S, or K387A; 8) The type of mutation at position D388 is selected from D388A, D388G, or D388L; 9) The type of mutation at position F880 is selected from F880A, F880G, and F880W. 6. The mutant according to paragraph 3 or 4, 1) The type of mutation at position D130 is D130E, 2) The type of mutation at position K172 may be selected from DEL172, K172A, or K172G; 3) the type of mutation at position R173 may be selected from DEL173, R173A, R173G, or R173C; 4) The type of mutation at position Y178 may be selected from Y178H or Y178P; 5) The type of mutation at position R298 is R298A, 6) the type of mutation at position Y385 is Y385A; 7) The type of mutation at position K387 may be selected from K387S, K387Y, or K387G; 8) The type of mutation at position D388 may be selected from D388Y, D388A, or D388G; 9) The type of mutation at position F880 may be selected from F880A or F880Y. 7. The mutant according to item 1, wherein the amino acid sequence of the mutant is set forth in any one of SEQ ID NOs: 2 to 141. 8. A nucleotide sequence encoding a variant according to any one of clauses 1 to 7. 9. A method for producing an RNA polymerase mutant, comprising producing at least one RNA polymerase mutant described in any one of items 1 to 7 in a host cell, wherein the host cell contains an expression vector having a nucleotide sequence described in item 8. 10. A method for reducing the production of dsRNA impurities in RNA production by in vitro transcription, comprising contacting a DNA template with a mutant according to any one of items 1 to 7 and incubating in an in vitro transcription system. 11. A method for improving the integrity of RNA products produced by in vitro transcription, comprising contacting a DNA template with a mutant described in any one of paragraphs 1 to 7 and incubating in an in vitro transcription system. 12. Use of at least one variant according to paragraphs 1 to 7 or the method according to paragraphs 10 to 11 in the production of RNA by in vitro transcription. 13. A method for producing RNA by in vitro transcription, comprising contacting a DNA template with at least one mutant according to paragraphs 1-7 and incubating in an in vitro transcription system. 14. A method for producing capped RNA by in vitro transcription, comprising contacting a DNA template with at least one mutant and cap analog described in paragraphs 1-7 and incubating in an in vitro transcription reaction system. 15. A composition or kit comprising at least one RNA polymerase mutant according to any one of items 1 to 7 and at least one buffer component. 16. The composition or kit according to paragraph 15, further comprising a cap analogue. [Effects of the Invention]

[0036] The present application has the following beneficial effects:

[0037] The RNA polymerase mutants of the present application have high catalytic efficiency and can reduce the production of double-stranded RNA impurities when used in mRNA synthesis. Compared to previously reported mRNA production methods, the present application produces the lowest amount of double-stranded RNA impurities, making it a safe, environmentally friendly mRNA production method with low double-stranded RNA impurities. The mRNA production method of the present application can significantly reduce the potential for immunogenicity due to double-stranded RNA impurities. Furthermore, some of the RNA polymerase mutants provided by the present application can improve the integrity of in vitro transcribed RNA. Furthermore, the DEL172+K387S mutant can increase the utilization rate of cap analogs in co-transcriptional capping reactions, reducing costs. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 is a schematic diagram of the construction of recombinant plasmids. DETAILED DESCRIPTION OF THE INVENTION

[0039] In the examples of this application, enzyme activity is defined as the amount of enzyme required to produce 1 μmol of product or convert 1 μmol of substrate within 1 minute under specified reaction conditions.

[0040] Example 1: Preparation of RNA polymerase mutants The RNA polymerases and their mutants shown in Table 1 were synthesized according to their DNA sequences (SEQ ID NOs: 142-282), PCR-amplified, and then inserted into the BseRI and HindIII restriction enzyme sites of the expression vector pQE-80L to obtain recombinant expression vectors. The constructed vectors were then transformed into E. coli BL21(DE3) by chemical transformation, plated on ampicillin-containing LB plates, and incubated overnight in a 37°C incubator. Plasmids were extracted from the grown single colonies and sequenced to obtain recombinant engineered bacteria containing the target gene. The sequenced E. coli recombinant strains were inoculated into LB medium and cultured overnight for activation, then inoculated into fermentation broth (LB medium) at 1-5% V / V and cultured until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 0.5 mol / L, and the mixture was cultured for 4 to 6 hours. After centrifugation at 12,000 rpm and 5°C, the culture was harvested and washed with 0.2 M PBS buffer (pH 7.0) to obtain the cells. After ultrasonic disruption, the cells were purified by affinity chromatography to obtain the RNA polymerase stock solution.

[0041] WT is a wild-type T7 RNA polymerase mutant, and its amino acid sequence is as follows: (SEQ ID NO: 1)

[0042] The RNA polymerase mutants and their mutation sites are shown in Table 1.

[0043] [Table 1] JPEG2026507701000019.jpg231170

[0044] Example 2: dsRNA impurity formation during in vitro transcription 2.1 Unqualified NTP (1) The enzyme stock solution obtained in Example 1 was diluted with a storage buffer (Vazyme, product number: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) shown in Table 2 were added to an 8-strip tube and centrifuged. The 8-strip tube was placed in a PCR device and reacted at 37°C for 1 hour. 36 μL of magnetic beads were then added, mixed uniformly, and incubated at room temperature for 2 to 5 minutes. The mixture was then placed in a magnetic rack and the mRNA (Vazyme, product number N412) was purified. After purification, the mixture was transferred to an RNase-free centrifuge tube to obtain purified mRNA. (2) The content of dsRNA impurities was detected using a dsRNA detection kit (Vazyme, product number: DD3509).

[0045] [Table 2]

[0046] The dsRNA detection results are shown in Table 3. Compared with the WT group, most of the polymerase mutants in Example 1 were able to effectively reduce the production of dsRNA impurities during in vitro transcription. Among them, the mutants in the DEL172-173+Y385A group, DEL172-173+D388A group, and DEL172-173+D388G group were able to reduce the amount of residual dsRNA by 99%.

[0047] [Table 3] JPEG2026507701000022.jpg249165 JPEG2026507701000023.jpg249165 JPEG2026507701000024.jpg71169

[0048] 2.2 Modified NTP(m1ψ) (1) Enzyme stock solutions of the RNA polymerase mutants DEL172-173+Y385A, DEL172-173+D388A, and DEL172-173+D388G were diluted with storage buffer (Vazyme, product number DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) listed in Table 4 were added to an 8-strip tube, mixed uniformly, and centrifuged. The 8-strip tube was placed in a PCR instrument and incubated at 37°C for 1 hour. After that, 36 μL of magnetic beads were added, mixed uniformly, and incubated at room temperature for 2–5 minutes. The mixture was then placed in a magnetic rack and purified with mRNA (Vazyme, product number N412). After purification, the mixture was transferred to an RNase-free centrifuge tube to obtain purified mRNA. (2) The content of dsRNA impurities was detected using a dsRNA detection kit (Vazyme, product number: DD3509).

[0049] [Table 4]

[0050] The dsRNA detection results showed that in vitro transcription reactions using the enzyme mutants DEL172-173+D388A and DEL172-173+D388G reduced the residual dsRNA rate (residual dsRNA / total RNA) to 0.0003%, and in vitro transcription reactions using the enzyme mutant DEL172-173+Y385A reduced the residual dsRNA rate to 0.0001%.

[0051] Example 3 Comparison of the integrity of in vitro transcribed RNA products 3.1 Unmodified NTP (Template SEQ ID NO: 283) (1) Purified mRNA is obtained by referring to step (1) of 2.1 of Example 2; (2) Capillary electrophoresis was performed using a Qsep400 fully automated nucleic acid analyzer to detect the integrity of mRNA (intact RNA product / total RNA). The detection results are shown in Tables 5-1 and 5-2.

[0052] 3.2 Modified NTP (mψ, template SEQ ID NO:283) (1) Purified mRNA was obtained by referring to step (1) of 2.2 in Example 2; (2) Capillary electrophoresis was performed using a Qsep400 fully automated nucleic acid analyzer to detect the integrity of mRNA. The detection results are shown in Tables 5-1 and 5-2.

[0053] [Table 5-1]

[0054] [Table 5-2] JPEG2026507701000028.jpg96169

[0055] 3.3 saRNA template I (template SEQ ID NO: 284) (1) The enzyme stock solution of RNA polymerase mutant K387Y was diluted with a storage buffer (Vazyme, product number: DD4101) to an enzyme activity of 300 U / μL. In vitro transcription was performed according to the reaction system composition in Table 2, and purified mRNA was obtained. (2) Capillary electrophoresis was performed using a Qsep400 fully automated nucleic acid analyzer to detect the integrity of the saRNA product. The detection results showed that in vitro transcription reactions using wild-type T7 RNA polymerase (WT) resulted in only 56.4% transcript integrity, whereas the K387Y mutant improved the saRNA product integrity to 73.1%.

[0056] 3.4 saRNA template II (>10,000nt) (1) The enzyme stock solutions of the RNA polymerase mutants K387A and DEL172+K387Y were diluted with a storage buffer (Vazyme, product number: DD4101) to an enzyme activity of 300 U / μL. In vitro transcription was performed according to the reaction system composition in Table 6, and purified mRNA was obtained.

[0057] [Table 6]

[0058] (2) Capillary electrophoresis was performed using a Qsep400 fully automated nucleic acid analyzer to detect the integrity of the saRNA product. The detection results showed that compared with the saRNA products obtained using the WT polymerase, the K387A and DEL172+K387Y mutants could improve the integrity of the saRNA products by 3.1% and 4.9%, respectively.

[0059] Example 4 Production of capped RNA by in vitro transcription (1) Stock solutions of the K387Y and DEL172+K387S mutant enzymes were diluted with storage buffer (Vazyme, product number DD4101) to an enzyme activity of 300 U / μL. A mix solution was prepared according to the reaction system (20 μL) in Table 7 and transferred to an EP tube. The mix solution was added to an 8-strip tube, mixed uniformly, and centrifuged. The 8-strip tube was placed in a PCR machine and incubated at 37°C for 1 hour. 36 μL of magnetic beads were added, mixed uniformly, and incubated at room temperature for 2–5 minutes. The mixture was then placed in a magnetic rack and purified with mRNA (Vazyme, product number N412). After purification, the mixture was transferred to an RNase-free centrifuge tube to obtain purified mRNA.

[0060] [Table 7]

[0061] (2) Capping rate detection: <1> The purified mRNA obtained in (1) was bound to a probe. The reaction system is shown in Table 8, and the reaction conditions are shown in Table 9.

[0062] [Table 8]

[0063] [Table 9]

[0064] <2> RNase H enzyme cleavage: The enzyme cleavage reaction system (Thermo Scientific, product number: EN0201) was prepared according to Table 10, thoroughly mixed by vortexing, and placed in a PCR device for reaction at 25°C for 20 minutes.

[0065] [Table 10]

[0066] <3> SA Magnetic Bead Binding: A. Washing magnetic beads: Place 9 μL of SA magnetic beads in a centrifuge tube and place it on a magnetic rack. Once the solution is clear, aspirate and discard the supernatant. Remove the centrifuge tube from the magnetic rack and add 200 μL of RNase-free H2O to wash. Place the tube on the magnetic rack. Once the solution is clear, aspirate and discard the supernatant with a pipette. Add 200 μL of RNase-free H2O to wash again. B. Reaction conditions: Remove the centrifuge tube from the magnetic rack, add the enzyme cleavage product to the SA magnetic beads, and mix evenly by pipetting 20-30 times. Place on a roller machine and incubate for 30 minutes at room temperature to completely bind the magnetic beads and the enzyme cleavage product.

[0067] <4> Rinse and Elute: A. Step <3> Place the product on a magnetic rack, and after 2-3 minutes, when the solution becomes clear, aspirate the supernatant with a pipette and discard. B. Rinse the beads by adding 200 μL of rinse solution, being careful not to disperse them. After leaving the beads for 0.5 to 1 minute, aspirate the supernatant with a pipette and discard. C. Repeat step B. D. Remove the centrifuge tube from the magnetic rack, add 30 μL of elution solution, and pipette up and down 10 to 20 times to mix evenly and thoroughly disperse the beads for complete elution. E. Place the PCR instrument in the reaction chamber at 85°C for 3 minutes, then immediately place it on the magnetic rack. Once the solution becomes clear (0.5-1 minute), aspirate the supernatant and transfer it to a new centrifuge tube. This supernatant is the desired product. F. Capillary electrophoresis was used to measure the capping rate of the product obtained in step E, and the calculation formula for the capping rate was as follows: Cap1 capping rate % = [Cap1 peak area / (Uncapped peak area + Cap1 peak area)] × 100% The detection results showed that in the co-transcriptional capping reaction, the capping rate of the mRNA product in the WT (wild-type T7 RNA polymerase) group was only 87.2%, whereas the K387Y group mutants could improve the capping rate of the mRNA product to 91.3%, and the DEL172+K387S group mutants could improve it to 100%.

Claims

1. 1. An RNA polymerase mutant, the amino acid sequence of which comprises one, two, three, four, or five amino acid mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388, and F880 relative to SEQ ID NO: 1, wherein the types of mutations are substitutions or deletions, and the amino acid sequence of the mutant has at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

2. 2. The mutant of claim 1, wherein the amino acid sequence of the mutant comprises one amino acid mutation relative to SEQ ID NO: 1, and the mutated amino acid position is selected from N171, K172, R173, Y178, R298, Y385, K387, D388, or F880.

3. The variant of claim 1, wherein the amino acid sequence of the variant, relative to SEQ ID NO: 1, comprises: (1) a mutation at position K172 and any one, two, three, or four amino acid mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388, or F880; the type of mutation is a substitution or deletion; or (2) a mutation at position K173 and any one, two, or three amino acid mutations at positions selected from D130, Y178, R298, K387, or D388; the type of mutation is a substitution or deletion; or (3) Any two or three amino acid mutations at positions selected from R298, Y385, K387, or D388: the type of mutation is a substitution.

4. The mutants are K172+R173, D130+K172, K172+K387, K172+F880, K172+D388, K172+R298, D130+R173, R173+Y178, R173+D388, R173+R298, K387+R298, Y385+R298, D388+R298, and Y38 5+K387, Y385+D388, K387+D388, K172+R173+Y385, K172+R173+D388, K172+R173+K38 7, K172+R173+F880, K172+R173+Y178, D130+K172+R173, D130+K172+Y178, D130+K172 +K387, D130+R173+D388, K172+Y178+D388, D130+K172+D388, K172+K387+R298, Y385 +K387+D388, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y178+D388 4. The mutant of claim 3, comprising any one of a group of mutations selected from: D130 + K172 + R173 + D388, D130 + K172 + R173 + Y178, D130 + R173 + Y178 + K387, D130 + K172 + Y178 + D388, or D130 + K172 + R173 + Y178 + D388.

5. 1) The type of mutation at position N171 is N171G; 2) the type of mutation at position K172 is selected from DEL172, K172A, K172G, K172E, K172D, K172H, K172Y, K172S, K172W, K172P, K172I, K172M, K172V, K172F, K172T, K172C, K172N, or K172L; 3) The type of mutation at position R173 is selected from DEL173, R173A, R173C, R173G, R173E, R173D, R173H, R173Y, R173S, R173W, R173P, R173N, or R173Q; 4) the type of mutation at position Y178 is Y178H; 5) The type of mutation at position R298 is R298A; 6) The type of mutation at position Y385 is selected from Y385A, Y385D, or Y385E; 7) The type of mutation at position K387 is selected from K387Q, K387Y, K387S, or K387A; 8) The type of mutation at position D388 is selected from D388A, D388G, or D388L; 9) The mutant according to claim 2, wherein the type of mutation at position F880 is selected from F880A, F880G, and F880W.

6. 1) The type of mutation at position D130 is D130E; 2) The type of mutation at position K172 may be selected from DEL172, K172A, or K172G; 3) The type of mutation at position R173 may be selected from DEL173, R173A, R173G, or R173C; 4) The type of mutation at position Y178 may be selected from Y178H or Y178P; 5) The type of mutation at position R298 is R298A; 6) The type of mutation at position Y385 is Y385A; 7) The type of mutation at position K387 may be selected from K387S, K387Y, or K387G; 8) The type of mutation at position D388 may be selected from D388Y, D388A, or D388G; 9) The mutant according to claim 3 or 4, wherein the type of mutation at position F880 may be selected from F880A or F880Y.

7. The mutant of claim 1, wherein the amino acid sequence of the mutant is set forth in any one of SEQ ID NOs: 2 to 141.

8. A nucleotide sequence encoding a variant according to any one of claims 1 to 7.

9. A method for producing an RNA polymerase mutant, comprising producing at least one RNA polymerase mutant described in claims 1 to 7 in a host cell, wherein the host cell comprises an expression vector having a nucleotide sequence described in claim 8.

10. A method for reducing the production of dsRNA impurities in RNA production by in vitro transcription, comprising contacting a DNA template with a mutant of any one of claims 1 to 7 and incubating in an in vitro transcription system.

11. Use of at least one variant according to claims 1 to 7 or the method according to claim 10 in the production of RNA by in vitro transcription.

12. A method for producing RNA by in vitro transcription, comprising contacting a DNA template with at least one mutant according to claims 1 to 7 and incubating in an in vitro transcription system.

13. A method for producing capped RNA by in vitro transcription, comprising contacting a DNA template with at least one mutant and cap analog described in claims 1 to 7 and incubating in an in vitro transcription reaction system.

14. A composition or kit comprising at least one RNA polymerase mutant according to any one of claims 1 to 7 and at least one buffer component.

Images