RNA polymerase variants, methods for their production and their use in RNA synthesis

Abstract

The application provides an RNA polymerase variant and a preparation method thereof, and uses the RNA polymerase variant in in vitro transcription, so that low dsRNA contamination and high-integrity RNA products can be obtained, the utilization efficiency of cap analogs in a co-transcription capping reaction system is improved, and cost is saved. In addition, the application further provides an in vitro RNA generation method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to RNA polymerase variants, methods for preparing the same, and applications thereof in RNA synthesis. BACKGROUND

[0002] The mRNA vaccine development cycle is relatively short, and new candidate vaccines can be quickly developed to respond to viral mutations. Through the dual mechanisms of humoral immunity and T cell immunity, the immunogenicity is strong, the effect is significant, and the production process is simple, which is easy for efficient research and large-scale production, and can achieve rapid and rapid global supply for viral epidemics and the like.

[0003] According to the latest news, in addition to mRNA, the research on circular RNA (circRNA) related drugs has also made a breakthrough. Orna company (Orna Therapeutics) has developed an in vivo cell therapy product using circRNA, and the research report published at the 2022 American Society for Gene and Cell Therapy (ASGCT) annual meeting has proved that it also has great application potential in other fields such as tumor treatment.

[0004] RNA has made a great contribution in the field of drug research and development such as vaccines, but the removal of some impurities in the actual production process still needs further research. Double-stranded RNA (dsRNA) impurities can cause strong immunogenicity (Goubau et al., 2014; Kato et al., 2006; Mu et al., 2018), so it is urgent to develop an effective method to reduce double-stranded RNA impurities. SUMMARY

[0005] The present application provides an RNA polymerase variant, a method for preparing the same and its application in RNA synthesis. In the first aspect of the application, the present application provides an RNA polymerase variant, which has an amino acid sequence comprising at least one, at least two, at least three, at least four, at least five or at least six mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388 or F880, relative to SEQ ID NO: 1, wherein the type of mutation is selected from deletion or substitution, and the amino acid sequence of the variant 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 mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388 or F880; and the type of mutation is selected from deletion or substitution. In some embodiments, the type of mutation is deletion (denoted as DEL, for example, DEL5, indicating deletion of an amino acid at the 5th position). In some embodiments, the type of mutation is substitution (for example, K5A, indicating substitution of lysine to alanine at the 5th position). In some embodiments, the type of mutation comprises both deletion and substitution, i.e. deletion at some positions and substitution at some positions.

[0006] In some embodiments, the present application provides an RNA polymerase variant, which has one amino acid mutation relative to SEQ ID NO: 1, wherein the mutated amino acid position is selected from N171, K172, R173, Y178, R298, Y385, K387, D388 or F880, and the type of mutation is selected from substitution or deletion. In some embodiments, the amino acid sequence of the variant has one amino acid mutation relative to SEQ ID NO: 1, wherein the mutation is selected from:

[0007] (1) substitution at the N171 position, and the substituted amino acid is G; or

[0008] (2) substitution or deletion at the K172 position, and the substituted amino acid can be selected from A, G, E, D, H, Y, S, W, P, I, M, V, F, T, C, N, L; or

[0009] (3) substitution or deletion at the R173 position, and the substituted amino acid can be selected from A, C, G, E, D, H, Y, S, W, P, N, Q; or

[0010] (4) substitution at the Y178 position, and the substituted amino acid is H; or

[0011] (5) a substitution at position R298, the substituting amino acid being A; or

[0012] (6) a substitution at position Y385, the substituting amino acid being selected from the group consisting of A, E, D; or

[0013] (7) a substitution at position K387, the substituting amino acid being selected from the group consisting of A, Y, S, Q; or

[0014] (8) a substitution at position D388, the substituting amino acid being selected from the group consisting of A, G, L; or

[0015] (9) a substitution at position F880, the substituting amino acid being selected from the group consisting of A, G, W.

[0016] In some embodiments, the RNA polymerase variant provided herein has an amino acid sequence comprising two, three, four, or five mutations at positions selected from D130, K172, R173, Y178, R298, Y385, K387, D388, or F880 relative to SEQ ID NO: 1, 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: (1) a mutation at the K172 position and further comprises one, two, three, or four mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388, or F880, the mutations being substitution or deletion; or (2) a mutation at the R173 position and further comprises one, two, or three mutations at positions selected from D130, Y178, R298, K387, or D388, the mutations being substitution or deletion; or (3) two or three mutations at positions R298, Y385, K387, or D388, the mutations being substitution or deletion. In some embodiments, the amino acid sequence of the variant has a combination of mutation points selected from any one of the following 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+R298, Y385+K387+D388, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y178, D130+R173+Y178+K387, D130+K172+Y178+D388, D130+K172+R173+Y178+D388.In some embodiments, further, the mutation type of the variant at the D130 position can be D130E. In some embodiments, further, the mutation type of the variant at the K172 position can be selected from DEL172, K172A, or K172G. In some embodiments, further, the mutation type of the variant at the R173 position can be selected from DEL173, R173A, R173G, or R173C. In some embodiments, further, the mutation type of the variant at the Y178 position can be selected from Y178H or Y178P. In some embodiments, further, the mutation type of the variant at the R298 position is R298A. In some embodiments, further, the mutation type of the variant at the Y385 position is Y385A. In some embodiments, further, the mutation type of the variant at the K387 position can be selected from K387S, K387Y, or K387G. In some embodiments, further, the mutation type of the variant at the D388 position can be selected from D388Y, D388A, or D388G. In some embodiments, further, the mutation type of the variant at the F880 position can be selected from F880A or F880Y.

[0017] In some embodiments, the variant comprises a mutation at the K172 position and further comprises one, two, three, or four amino acid mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388, or F880, the mutation type is 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 a mutation set selected from any one of the following: 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+Y178+D388, D130+K172+D388, K172+K387+R298, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y178, D130+K172+Y178+D388, D130+K172+R173+Y178+D388. In some embodiments, further, the mutation at the D130 position of the variant is D130E. In some embodiments, further, the mutation at the K172 position of the variant can be selected from DEL172, K172A, or K172G. In some embodiments, further, the mutation at the R173 position of the variant can be selected from DEL173, R173A, R173G, or R173C. In some embodiments, further, the mutation at the Y178 position of the variant is Y178H. Further, the mutation at the R298 position of the variant is R298A. In some embodiments, further, the mutation at the Y385 position of the variant is Y385A. In some embodiments, further, the mutation at the K387 position of the variant can be selected from K387S, K387Y, or K387G. In some embodiments, further, the mutation at the D388 position of the variant can be selected from D388Y, D388A, or D388G. In some embodiments, further, the mutation at the F880 position of the variant can be selected from F880A or F880Y.

[0018] In some embodiments, the variant comprises a mutation at the R173 position and further comprises one, two or three mutations of amino acids selected from the group consisting of D130, Y178, R298, K387, D388 or F880, the mutation type is 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 a mutation group selected from any one of the following: D130+R173, R173+Y178, R173+D388, R173+R298, D130+R173+D388, D130+R173+Y178+K387. In some embodiments, further, the mutation type of the variant at the D130 position is D130E. In some embodiments, further, the mutation type of the variant at the R173 position can be selected from DEL173, R173A, R173G or R173C. In some embodiments, further, the mutation type of the variant at the Y178 position is Y178H or Y178P. In some embodiments, further, the mutation type of the variant at the R298 position is R298A. In some embodiments, further, the mutation type of the variant at the K387 position is K387Y. In some embodiments, further, the mutation type of the variant at the D388 position can be selected from D388Y or D388G.

[0019] In some embodiments, the variant comprises a mutation of any two or three amino acids selected from the group consisting of R298, Y385, K387 or D388, the mutation type is 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 a mutation group selected from any one of the following: K387+R298, Y385+R298, D388+R298, Y385+K387, Y385+D388, K387+D388, Y385+K387+D388. In some embodiments, further, the mutation type of the variant at the R298 position is R298A. In some embodiments, further, the substitution of the variant at the Y385 position is Y385A. In some embodiments, further, the substitution of the variant at the K387 position can be selected from K387S or K387Y. In some embodiments, further, the substitution of the variant at the D388 position can be selected from D388A or D388G.

[0020] In some embodiments, the amino acid sequence of the 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 set forth in SEQ ID NOs: 2-141. In some embodiments, the amino acid sequence of the variant described herein is selected from the group consisting of SEQ ID NOs: 2-141.

[0021] In a second aspect, the present application provides a method of producing an RNA polymerase variant, comprising producing at least one RNA polymerase variant described herein in a host cell. In some embodiments, the host cell contains an expression vector carrying a nucleotide sequence corresponding to the RNA polymerase variant described herein. In some embodiments, various modifications can be present in the polynucleotide sequence in the coding region, as long as the variant amino acid sequence of the present application is not changed by the degeneracy of the codon or by the preferred codon in the organism expressing the variant. In some embodiments, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 143-282.

[0022] In a third aspect, the present application provides a method of reducing the generation of dsRNA impurities in the process of in vitro transcription to produce RNA, comprising contacting a DNA template with one or more RNA polymerase variants described herein and incubating in an in vitro transcription system.

[0023] In a fourth aspect, the present application provides a method of improving the integrity of the in vitro transcribed RNA product, comprising contacting a DNA template with one or more RNA polymerase variants described herein and incubating in an in vitro transcription system.

[0024] In a fifth aspect, the present application provides a method of generating RNA, comprising contacting a DNA template with one or more RNA polymerase variants described herein and incubating in an in vitro transcription system under conditions that result in the generation of a RNA transcription product.

[0025] In some embodiments, the RNA produced by the methods described herein can 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.

[0026] In some embodiments, the DNA template length can be selected from 1000-13000 bp. In some embodiments, the DNA template length can be selected from 8000-13000 bp. In some embodiments, the DNA template length can be selected from 10000-13000 bp.

[0027] 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 reduction in residual dsRNA impurities after purification as compared to using wild-type RNA polymerase (SEQ ID NO: 1). In some embodiments, the RNA produced using the methods described herein has a residual dsRNA impurity level (residual dsRNA amount / total RNA amount) 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.

[0028] In some embodiments, the mRNA produced using the methods described herein has 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% improvement in mRNA product integrity after purification as compared to using wild-type T7 RNA polymerase (SEQ ID NO: 1).

[0029] In some embodiments, the saRNA produced using the methods described herein has 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% improvement in saRNA product integrity after purification as compared to using wild-type T7 RNA polymerase.

[0030] In a sixth aspect, the present application provides a method of generating capped RNA in vitro, comprising incubating a DNA template with one or more RNA polymerase variants described herein and a cap analog in an in vitro transcription reaction system.

[0031] In some embodiments, the cap analog is a di-nucleotide cap, a tri-nucleotide cap, or a tetra-nucleotide cap. In some embodiments, the cap analog is a tri-nucleotide cap. In some embodiments, the tri-nucleotide cap is selected from the group consisting of GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU, GGA, GG C, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, the tri-nucleotide cap is selected from the group consisting of m 7 GpppApA, m 7 GpppApC, m 7 GpppApG, m 7 GpppApU, m 7 GpppCpA, m 7 GpppCpC, m 7 GpppCpG, m 7 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 GpppUpU. In some embodiments, the tri-nucleotide cap is selected from the group consisting of 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′OMepppUpA, m 7 G 3′OMe pppUpC, m 7 G 3′OMe pppUpG, and m 7 G 3′OMe pppUpU. 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′OMe pppA 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 pU. In some embodiments, the trinucleotide cap is selected from m 7 GpppA 2′OMe pA, m 7 GpppA2′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 7 GpppG 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 pU. In some embodiments, the trinucleotide cap is preferably m7GpppA2ΌMepG.

[0032] In some embodiments, the in vitro transcription capping reaction is performed using the RNA polymerase variant described herein, and the capping rate of the mRNA product is increased to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the use of wild-type T7 RNA polymerase (SEQ ID NO: 1). In some embodiments, the capping rate of the mRNA product can be increased to 100%.

[0033] In a seventh aspect, the present application provides a method for in vitro transcription, comprising contacting a DNA template with one or more RNA polymerase variants described herein under conditions that result in the production of an RNA transcript, incubating in an in vitro transcription system. In some embodiments, the method comprises the following steps: 1) providing a DNA template comprising a T7 promoter functionally linked to a target nucleotide sequence to be transcribed; 2) contacting the DNA template of step 1) with one or more RNA polymerase variants described herein; 3) incubating the DNA template and the RNA polymerase variant in an in vitro transcription system.

[0034] In some embodiments, the incubation temperature of step 3) is 30-50 °C, preferably 37 °C. In some embodiments, the incubation time of step 3) is 20-240 min, preferably 60 min.

[0035] The in vitro transcription system described herein comprises nucleotide triphosphates and buffer components.

[0036] In some embodiments, the nucleotide triphosphates can be selected from modified or unmodified nucleotide triphosphates (including analogs thereof). In some embodiments, the nucleotide triphosphates can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the nucleotide triphosphates can be selected from modified nucleotide triphosphates, including but not limited to mlA (Nl-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), mlψ (Nl-methyl-pseudouridine), nucleotide triphosphates with labels (the labels can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0037] In some embodiments, the in vitro transcription system further comprises RNase inhibitors, inorganic pyrophosphatases, magnesium ions. In some embodiments, the in vitro transcription system further comprises DEPC water.

[0038] In an eighth aspect, the present application provides a composition or kit comprising one or more RNA polymerase variants described herein.

[0039] 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, wherein the in vitro transcription components can be selected from nucleotide triphosphates, RNase inhibitors, inorganic pyrophosphatases, magnesium ions, etc. In one embodiment, the in vitro transcription system components can be selected from commercially available mRNA in vitro transcription reagents.

[0040] 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 m7GpppA2’OMepG.

[0041] In a ninth aspect, 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 herein. In some embodiments, the RNA product is not chemically modified. In some embodiments, the RNA product is chemically modified.

[0042] Other embodiments:

[0043] 1. An RNA polymerase variant comprising one, two, three, four, or five mutations at positions selected from D130, N171, K172, R173, Y178, R298, Y385, K387, D388, or F880 relative to SEQ ID NO: 1, the mutations being of the type substitution or deletion, and the amino acid sequence of the variant having at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1.

[0044] 2. The variant of item 1, the amino acid sequence of the variant having one mutation relative to SEQ ID NO: 1, the position of the mutated amino acid being selected from N171, K172, R173, Y178, R298, Y385, K387, D388, or F880.

[0045] 3. The variant of item 1, the amino acid sequence of the variant comprising, relative to SEQ ID NO: 1:

[0046] (1) a mutation at the K172 position, and further comprising one, two, three, or four mutations at positions selected from D130, R173, Y178, R298, Y385, K387, D388, or F880, the mutations being of the type substitution or deletion; or

[0047] (2) a mutation at the K173 position, and further comprising one, two, or three mutations at positions selected from D130, Y178, R298, K387, or D388, the mutations being of the type substitution or deletion; or

[0048] (3) two or three mutations at positions R298, Y385, K387, or D388, the mutations being of the type substitution.

[0049] 4. The variant of item 3, comprising a mutation set selected from any of: 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+R298, Y385+K387+D388, K172+R173+Y385+F880, K172+R173+D388+F880, K172+R173+Y178+D388, D130+K172+R173+D388, D130+K172+R173+Y178, D130+R173+Y178+K387, D130+K172+Y178+D388, or D130+K172+R173+Y178+D388.

[0050] 5. The variant of item 2, wherein,

[0051] 1) the mutation type at position N171 is N171G;

[0052] 2) the mutation type 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;

[0053] 3) the mutation type 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;

[0054] 4) the type of mutation at position Y178 is Y178H;

[0055] 5) the type of mutation at position R298 is R298A;

[0056] 6) the type of mutation at position Y385 is selected from Y385A, Y385D or Y385E;

[0057] 7) the type of mutation at position K387 is selected from K387Q, K387Y, K387S or K387A;

[0058] 8) the type of mutation at position D388 is selected from D388A, D388G or D388L; and

[0059] 9) the type of mutation at position F880 is selected from F880A, F880G or F880W.

[0060] 6. The variant as described in item 3 or item 4, wherein,

[0061] 1) the type of mutation at position D130 is D130E;

[0062] 2) the type of mutation at position K172 is selected from DEL172, K172A or K172G;

[0063] 3) the type of mutation at position R173 is selected from DEL173, R173A, R173G or R173C;

[0064] 4) the type of mutation at position Y178 is selected from Y178H or Y178P;

[0065] 5) the type of mutation at position R298 is R298A;

[0066] 6) the type of mutation at position Y385 is Y385A;

[0067] 7) the type of mutation at position K387 is selected from K387S, K387Y or K387G;

[0068] 8) the type of mutation at position D388 is selected from D388Y, D388A or D388G; and

[0069] 9) the type of mutation at position F880 is selected from F880A or F880Y.

[0070] 7. The variant as described in item 1, having an amino acid sequence as set forth in any one of SEQ ID NOs: 2-141.

[0071] 8. A nucleotide sequence encoding the variant as described in any one of items 1-7.

[0072] 9. A method for preparing RNA polymerase variants, comprising producing at least one RNA polymerase variant as described in any of items 1-7 in a host cell; wherein the host cell contains an expression vector carrying a nucleotide sequence as described in item 8.

[0073] 10. A method for reducing the generation of dsRNA impurities in the preparation of RNA by in vitro transcription, comprising contacting a DNA template with a variant as described in any of items 1-7 and incubating in an in vitro transcription system.

[0074] 11. A method for improving the integrity of RNA products of in vitro transcription, comprising contacting a DNA template with a variant as described in any of items 1-7 and incubating in an in vitro transcription system.

[0075] 12. Use of at least one variant as described in items 1-7 or a method as described in items 10-11 for the generation of RNA by in vitro transcription.

[0076] 13. A method for the generation of RNA by in vitro transcription, comprising contacting a DNA template with at least one variant as described in items 1-7 and incubating in an in vitro transcription system.

[0077] 14. A method for the generation of capped RNA by in vitro transcription, comprising contacting a DNA template with at least one variant as described in items 1-7 and a cap analog and incubating in an in vitro transcription reaction system.

[0078] 15. A composition or kit comprising at least one RNA polymerase variant as described in any of items 1-7 and at least one buffer component.

[0079] 16. A composition or kit as described in item 15, further comprising a cap analog.

[0080] The present application has the following beneficial effects:

[0081] The RNA polymerase variants of the present application have high catalytic efficiency. When used in the synthesis of mRNA, the generation of dsRNA impurities is reduced. Compared with the currently reported methods for the preparation of mRNA, the present application produces the least dsRNA impurities, and is a safe, green and low-dsRNA-impurity mRNA preparation method. The use of the mRNA preparation method of the present application can greatly reduce the possibility of immunogenicity caused by dsRNA impurities. In addition, some of the RNA polymerase variants provided by the present application can improve the integrity of the in vitro transcription product RNA; the variant DEL172+K387S can also improve the utilization rate of cap analogs in the cotranscriptional capping reaction, thereby saving costs. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1A schematic diagram for construction of recombinant plasmid. DETAILED DESCRIPTION

[0083] In the embodiments of the present application, the definition of enzyme activity is: the amount of enzyme required to generate 1 μmol of product or to convert 1 μmol of substrate in 1 minute under specific reaction conditions.

[0084] Example 1 Preparation of RNA polymerase variant

[0085] The RNA polymerases and variants shown in Table 1 were subjected to DNA sequence synthesis (SEQ ID NO: 142-282) and then PCR amplified, and then introduced into the BseRI and HindIII enzyme digestion sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was introduced into E. coli BL21 (DE3) by chemical transformation technique, plated on LB plates containing ampicillin resistance, and placed in a 37°C incubator overnight. Single colonies that grew were subjected to plasmid extraction and sequencing, and finally a recombinant engineering bacterium containing the target gene was obtained. The successfully sequenced E. coli recombinant strain was inoculated into LB medium for overnight activation culture, then 1-5% V / V was inoculated into fermentation broth (LB medium), and cultured until the OD 600 value was 0.6-0.8. Then 0.5 mol / L IPTG was added to a final concentration, and the culture was continued for 4-6 h. Then the strain was collected by centrifugation at 12000 rpm and 5°C, and the collected strain was washed with 0.2 M PBS buffer at pH 7.0. The bacterial cells were obtained. After ultrasonic disruption, affinity chromatography purification was performed to obtain the RNA polymerase stock solution.

[0086] WT is a wild-type T7 RNA polymerase variant, and its amino acid sequence is:

[0087] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1)

[0088] RNA polymerase variants and their mutation sites are shown in Table 1:

[0089] Table 1: RNA polymerase variant mutation sites and corresponding amino acid sequence numbers

[0090]

[0091]

[0092]

[0093] Example 2: dsRNA impurity generation in in vitro transcription

[0094] 2.1 Unmodified NTP

[0095] (1) The enzyme stock solution obtained in Example 1 was diluted with storage buffer (Vazyme, item number: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 2 were loaded into an eight-row array and mixed, and centrifuged; the eight-row array was placed in a PCR instrument for 1 h of 37°C reaction, then 36 μL of magnetic beads were mixed and incubated at room temperature for 2-5 min; the mixture was placed on a magnetic stand to purify mRNA (Vazyme, item number: N412), and after purification, the purified mRNA was transferred to an RNase-free centrifuge tube to obtain purified mRNA;

[0096] (2) The dsRNA impurity content was tested by a dsRNA detection kit (Vazyme, item number: DD3509).

[0097] Table 2: Reaction system ratio

[0098]

[0099]

[0100] The dsRNA detection results are shown in Table 3. Compared with the WT group, most of the polymerase variants in Example 1 can effectively reduce the generation of dsRNA impurities in in vitro transcription, and the variants in the DEL172-173+Y385A, DEL172-173+D388A, and DEL172-173+D388G groups can reduce the residual amount of dsRNA by 99%.

[0101] Table 3: dsRNA residual amount

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] 2.2 Modified NTP (m1ψ)

[0108] (1) The stock solution of RNA polymerase variants DEL172-173+Y385A, DEL172-173+D388A and DEL172-173+D388G was diluted with storage buffer (Vazyme, item number: DD4101) to an enzyme activity of 300 U / μL. The reaction components (20 μL) in Table 4 were loaded into an eight-row array and mixed, and centrifuged; the eight-row array was placed in a PCR instrument for 1 h of reaction at 37℃, then 36 μL of magnetic beads were added and mixed, and incubated at room temperature for 2-5 min; the mixture was placed on a magnetic stand to purify mRNA (Vazyme, item number: N412), and after purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain purified mRNA;

[0109] (2) The dsRNA impurity content was tested by a dsRNA detection kit (Vazyme, item number: DD3509).

[0110] Table 4: Reaction system ratio

[0111]

[0112]

[0113] The dsRNA detection results showed that the in vitro transcription reaction involving enzyme variants DEL172-173+D388A and DEL172-173+D388G could reduce the residual rate of dsRNA (residual amount of dsRNA / total amount of RNA) to 0.0003%; the in vitro transcription reaction involving enzyme variant DEL172-173+Y385A could reduce the residual rate of dsRNA to 0.0001%.

[0114] Example 3 Comparison of the integrity of in vitro transcribed RNA products

[0115] 3.1 Unmodified NTP (template SEQ ID NO: 283)

[0116] (1) The purified mRNA was obtained according to the procedure (1) of 2.1 in Reference Example 2;

[0117] (2) Capillary electrophoresis was performed on a Qsep400 full-automatic nucleic acid analyzer to detect the integrity of the mRNA (intact RNA product / total RNA).

[0118] The detection results are shown in Tables 5-1 and 5-2.

[0119] 3.2 Modified NTP (m1ψ, template SEQ ID NO: 283)

[0120] (1) Obtain purified mRNA by following the procedure (1) of 2.2 in Reference Example 2;

[0121] (2) Perform capillary electrophoresis by Qsep400 automatic nucleic acid analyzer to detect the integrity of mRNA.

[0122] The detection results are shown in Table 5-1 and Table 5-2.

[0123] Table 5-1: Integrity of mRNA product

[0124]

[0125]

[0126] Table 5-2: Integrity of mRNA product

[0127]

[0128]

[0129] 3.3 saRNA template I (template SEQ ID NO: 284)

[0130] (1) Dilute the stock solution of RNA polymerase variant K387Y to an enzyme activity of 300 U / μL with storage buffer (Vazyme, item number: DD4101). Perform in vitro transcription according to the reaction system ratio in Table 2 to obtain purified mRNA;

[0131] (2) Perform capillary electrophoresis by Qsep400 automatic nucleic acid analyzer to detect the integrity of saRNA product.

[0132] The detection results show that the integrity of the transcription product is only 56.4% when using wild-type T7 RNA polymerase (WT) for in vitro transcription reaction, while the integrity of saRNA product can be improved to 73.1% by using K387Y variant.

[0133] 3.4 saRNA template II (>10000 nt)

[0134] (1) Dilute the stock solution of RNA polymerase variants K387A and DEL172+K387Y to an enzyme activity of 300 U / μL with storage buffer (Vazyme, item number: DD4101). Perform in vitro transcription according to the reaction system ratio in Table 6 to obtain purified mRNA;

[0135] Table 6: Reaction system

[0136]

[0137] (2) Cap rate detection: The purified mRNA obtained in step (1) was combined with the probe, and the reaction system and reaction conditions are shown in Tables 8 and 9, respectively.

[0138] The detection results showed that, compared with the saRNA product obtained by using the WT group polymerase, the variants of the K387A group and the DEL172+K387Y group could increase the integrity of the saRNA product by 3.1% and 4.9%, respectively.

[0139] Example 4: Preparation of capped RNA by in vitro transcription

[0140] (1) The stock solution of the K387Y and DEL172+K387S variant enzymes was diluted with storage buffer (Vazyme, item number: DD4101) to have an enzyme activity of 300 U / μL. The MIX solution was prepared in the reaction system (20 μL) in Table 7 and transferred to an EP tube, and then the MIX solution was aliquoted into an eight-row array, mixed, and centrifuged. The eight-row array was placed in a PCR instrument at 37°C for 1 h, and then 36 μL of magnetic beads were added and mixed, followed by incubation at room temperature for 2-5 min. The mixture was placed on a magnetic stand to purify the mRNA (Vazyme, item number: N412), and after purification, the mRNA was transferred to an RNase-free centrifuge tube to obtain the purified mRNA.

[0141] Table 7: Reaction system

[0142]

[0143] (2) Cap rate detection:

[0144] ① The purified mRNA obtained in step (1) was combined with the probe, and the reaction system and reaction conditions are shown in Tables 8 and 9, respectively.

[0145] Table 8: Reaction system

[0146]

[0147]

[0148] Table 9: Reaction conditions

[0149] Temperature Time 95℃ 2 mins 70～16℃ -0.1 °C / s, ~180 cycles 16℃ ∞

[0150] ② RNase H enzyme digestion: The reaction enzyme digestion reaction system was prepared according to Table 10 (Thermo Scientific, item number: EN0201), and after being mixed uniformly by vortexing, the mixture was placed in a PCR instrument and reacted at 25°C for 20 min.

[0151] Table 10: Reaction system

[0152] Components 25 pmol volume Product from previous step 21 μL RNase H Reaction Buffer (10x) 3 μL RNase H (5 U / μl) 3 μL RNase-free H2O 3 μL Total system 30 μL

[0153] ③SA magnetic beads binding:

[0154] A. Magnetic beads washing: take 9 μL SA magnetic beads into a centrifuge tube, place it on a magnetic stand, and after the solution becomes clear, use a pipette to discard the supernatant; take the centrifuge tube off the magnetic stand, add 200 μL RNase-free H2O, place it on the magnetic stand, and after the solution becomes clear, use a pipette to discard the supernatant; add 200 μL RNase-free H2O again and repeat the washing once.

[0155] B. Reaction conditions: take the centrifuge tube off the magnetic stand, add the enzyme digestion product to the SA magnetic beads, use a pipette to blow and beat 20-30 times, mix thoroughly, place it in a rolling instrument for incubation at room temperature for 30 min, so that the magnetic beads and enzyme digestion product are fully combined.

[0156] ④Rinsing and elution:

[0157] A. Place the product of step ③ on a magnetic stand for 2-3 min, and after the solution becomes clear, use a pipette to discard the supernatant;

[0158] B. Add 200 μL rinsing solution, be careful not to blow the magnetic beads apart, stand for 0.5-1 min, and use a pipette to discard the supernatant;

[0159] C. Repeat step B;

[0160] D. Take the centrifuge tube off the magnetic stand, add 30 uL elution solution, mix evenly with a pipette for 10-20 times, and elute the magnetic beads evenly;

[0161] E. Place it in a PCR instrument, react at 85℃ for 3 min, then place it on a magnetic stand immediately, and after the solution becomes clear (0.5-1 min), take the supernatant to a new centrifuge tube, and the supernatant is the desired product;

[0162] F. Cap1 capping rate was detected by capillary electrophoresis, and the capping rate calculation formula was:

[0163] Cap1 capping rate % = [Cap1 peak area / (Uncap peak area + Cap1 peak area)] x 100%.

[0164] The detection results show that in the cotranscriptional capping reaction, the mRNA product capping rate of the WT (wild type T7 RNA polymerase) group is only 87.2%, while the K387Y group can increase the mRNA product capping rate to 91.3%, and the DEL172+K387S group can increase it to 100%.

Claims

1. A variant of an RNA polymerase, wherein, The amino acid sequence of the variant comprises a mutation at the K172 position and further comprises a mutation at the K387 position relative to SEQ ID NO: 1, wherein the mutation at the K172 position is of the type DEL172, and the amino acid sequence of the variant is as set forth in any one of SEQ ID NOs: 45, 46, 82, 84-87, 96, 139.

2. A nucleotide molecule encoding the variant as claimed in claim 1.

3. A method of producing an RNA polymerase variant comprising producing in a host cell at least one RNA polymerase variant as described in claim 1 ; wherein, 4. A method of reducing the generation of dsRNA impurities in an in vitro transcription process for preparing RNA, comprising contacting a DNA template with the variant as claimed in claim 1, and incubating in an in vitro transcription system.

5. Use of at least one variant as claimed in claim 1 or the method as claimed in claim 4 for the in vitro transcription to generate RNA.

6. A method of in vitro transcription to generate RNA, comprising contacting a DNA template with at least one variant as claimed in claim 1, and incubating in an in vitro transcription system.

7. A method of in vitro transcription to generate capped RNA, comprising contacting a DNA template with at least one variant as claimed in claim 1 and a cap analog, and incubating in an in vitro transcription reaction system.

8. A composition or kit comprising at least one RNA polymerase variant as claimed in claim 1 and at least one buffer component. ​

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