RNA polymerase variants and uses thereof

Abstract

The invention provides the RNA polymerase variant and the application thereof, the provided RNA polymerase variant is used for carrying out in-vitro transcription reaction, the generation of truncated or over-extended impurities is reduced, the completeness of a transcribed RNA product is improved, and the RNA polymerase variant can be suitable for preparing 1000-13000nt RNA products. In addition, the invention also provides a method for synthesizing RNA.

Description

RNA polymerase variants and their applications Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to RNA polymerase variants and applications thereof. Background Art

[0002] mRNA therapy refers to the use of mRNA-based drugs to treat or prevent diseases. By introducing mRNA as a vaccine or therapeutic agent, in vitro transcribed (IVT) mRNA acts as an information carrier to guide the production of functional proteins or peptides in the human body. Since the 21st century, mRNA technology has matured, achieving remarkable progress in the research of cancer, rare diseases, genetic disorders, and infectious diseases. In particular, the outbreak of COVID-19 in late 2019 accelerated the launch of mRNA vaccines, rapidly transforming them into a revolutionary new medicine.

[0003] Currently, mRNA vaccines are mainly divided into two types: one is non-replicating mRNA, which refers to the delivery of a complete gene sequence synthesized in vitro into the body through a vector and induces 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). In addition to encoding the required target protein, saRNA also carries a sequence that can express RNA polymerase (RNA-dependent RNA polymerase, RdRP). Once the RNA polymerase is produced, more saRNA can be produced using saRNA as a template. Compared to ordinary mRNA, 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 of the antigen protein in the body. At the same time, because saRNA forms double-stranded RNA during the replication process, it may stimulate the cell's innate immune response, which can further enhance the effect of the vaccine and thus prolong the therapeutic effect. Therefore, the use of saRNA vaccines can reduce the dose and number of injections used, and reduce the possible toxic side effects of mRNA and drug delivery vehicles. Currently, there are many saRNA vaccines entering clinical trials, but none of them have been approved for marketing. However, in the saRNA production process, the integrity of the self-replicating template has always been a difficult problem to solve due to its long fragments (>8000bp).

[0004] Compared to traditional vaccines, mRNA vaccines have advantages such as a shorter R&D cycle, stronger immunogenicity, and simpler production processes. However, the production of mRNA vaccines involves multiple biological processes and raw material processing, requiring multiple quality assessments of the mRNA API's characteristics, purity, quantity, physical state (integrity), and safety. The assessment of mRNA integrity is a crucial step. Because IVT mRNA can produce truncated or over-extended impurities during synthesis, which not only reduce mRNA integrity but also lead to an adverse increase in immunogenicity, it is crucial to ensure the integrity of mRNA during in vitro RNA synthesis.

[0005] Summary of the Invention

[0006] The present application provides multiple RNA polymerase variants that can significantly reduce the truncation and overextension rates of mRNA products during in vitro transcription. The present application also provides methods for preparing these variants and their use in in vitro RNA synthesis.

[0007] In a first aspect, the present application provides an RNA polymerase variant, the variant amino acid sequence having 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 positions 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. In some embodiments, the amino acid sequence of the variant comprises a mutation selected from the group consisting of R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391, or K392, wherein the mutation is a substitution or a deletion.

[0008] In some embodiments, 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.

[0009] In some embodiments, the mutation at position R34 is R34A.

[0010] In some embodiments, the mutation at position D130 can be selected from D130E or D130C.

[0011] In some embodiments, the mutation at position V174 can be selected from V174A, V174D, V174E, V174G, or deletion (DEL174).

[0012] In some embodiments, the mutation at position Y178 can be selected from Y178H, Y178G, Y178V, Y178F, Y178D, Y178N, Y178E, Y178S, Y178T or Y178P.

[0013] In some embodiments, the mutation at position G285 is G285A.

[0014] In some embodiments, the mutation at position K303 is K303L.

[0015] In some embodiments, the mutation at position N325 can be selected from N325A or N325L.

[0016] In some embodiments, the mutation at position Y385 can be selected from Y385A, Y385E, or Y385N.

[0017] In some embodiments, the mutation at position R386 can be selected from R386A, R386L, R386I, R386M, R386V, R386S, R386T, R386H, R386Q, R386N, R386K, R386D, R386C, R386W or R386G.

[0018] In some embodiments, the mutation at the K387 position can be selected from K387G, K387Y, K387Q, K387A, K387N, K387S, or K387W.

[0019] In some embodiments, the mutation at position D388 can be selected from D388Y or D388L.

[0020] In some embodiments, the mutation at the K389 position can be selected from K389A or K389R.

[0021] In some embodiments, the mutation at point R391 can be selected from R391A, R391L, or R391K.

[0022] In some embodiments, the mutation at the K392 position can be selected from K392A, K392F, K392I, K392T, K392H, K392Q, K392E, or K392C.

[0023] 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 compared to the sequence shown in any one of SEQ ID NOs: 2-63. In some embodiments, the amino acid sequence of the variant described herein is as shown in any one of SEQ ID NOs: 2-63.

[0024] As used herein, "sequence identity" refers to the relationship between the sequences of two or more polypeptides, as determined by comparing (aligning) the sequences.

[0025] In a second aspect, the present application provides polynucleotides encoding RNA polymerase variants. Due to codon degeneracy or codon preferences of the host cell expressing the polypeptide, the polynucleotides can be any polynucleotide encoding the variants described herein without changing the amino acid sequence. In some embodiments, the polynucleotides encoding the RNA polymerase variants of the present application can be selected from SEQ ID NOs: 65-126.

[0026] In a third aspect, 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. In some embodiments, the host cell contains an expression vector carrying an RNA polymerase variant polynucleotide molecule described herein.

[0027] In a fourth aspect, 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 transcript. In some embodiments, the RNA product is mRNA. In some embodiments, the RNA product is saRNA. In some embodiments, the RNA product can also be siRNA, gRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, etc.

[0028] In a fifth aspect, the present application provides a method for performing 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. 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 in step 1) with one or more RNA polymerase variants described herein; and 3) incubating the DNA template and RNA polymerase variant in an in vitro transcription system.

[0029] In some embodiments, the DNA template is 1000-13000 bp in length. In some embodiments, the DNA template can be selected from 1000-10000 bp in length. In some embodiments, the DNA template can be selected from 8000-10000 bp in length. In some embodiments, the DNA template can be selected from 10000-13000 bp in length.

[0030] In some embodiments, the in vitro transcription system comprises a nucleoside triphosphate and a buffer component. In some embodiments, the nucleoside triphosphate can be selected from modified or unmodified nucleoside triphosphates (including analogs thereof). In some embodiments, the nucleoside triphosphate can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, 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), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), markers (markers can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0031] In some embodiments, the in vitro transcription system described in step 3) further comprises an RNase inhibitor, an inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription system described in step 3) further comprises DEPC water.

[0032] In some embodiments, RNA produced using the methods for generating RNA or in vitro transcription described herein, after purification, has improved RNA product integrity; wherein integrity refers to the proportion of intact RNA product obtained. In some embodiments, the RNA product may be coding RNA or non-coding RNA. In some embodiments, the RNA product includes, but is not limited to, mRNA, siRNA, gRNA, saRNA, dsRNA, ssRNA, miRNA, piRNA, shRNA, and the like.

[0033] In some embodiments, the mRNA produced using the methods of 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 using wild-type T7 RNA polymerase (SEQ ID NO: 1).

[0034] In some embodiments, the integrity of the saRNA product produced using the methods of generating RNA or in vitro transcription methods described herein, after purification, is 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.

[0035] In a sixth aspect, 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 herein under conditions that result in the generation of an RNA transcript.

[0036] In some embodiments, the cap analog has the structure:

[0037] Among them, B1-B n+1 Each is independently a nucleobase; R1 is H or alkyl, R2-R n+3 Each is independently H, OH, alkyl, O-alkyl, S-alkyl or NH-alkyl, X1-X n+3 Each is independently O or S, Y1-Y n+4 Each is independently O, S, NH or CH2, Z1-Z n+3 Each is independently OH, SH, BH3, alkyl or O-alkyl, M1-M n Each is independently O, S, NH or CH2; l is 0, 1 or 2, n1 is 1, n1-n n Each is independently 0 or 1.

[0038] The conditions leading to the production 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.

[0039] In a seventh aspect, the present application provides a composition or kit comprising one or more RNA polymerase variants described herein. In some embodiments, the composition or kit further comprises one or more 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. In some embodiments, the in vitro transcription system components can be selected from commercially available RNA in vitro transcription reagents.

[0040] In an eighth aspect, the RNA polymerase variant described in the present application is used in the synthesis of RNA drugs.

[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 implementation plans:

[0043] 1. An RNA polymerase variant having an amino acid sequence that is at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher identity to SEQ ID NO: 1, wherein the amino acid sequence of the variant comprises at least one mutation selected from the group consisting of R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391 or K392 relative to SEQ ID NO: 1.

[0044] 2. The variant as described in item 1, whose amino acid sequence 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.

[0045] 3. A variant according to any one of item 1 or 2, wherein:

[0046] 1) The mutation at the R34 site is R34A;

[0047] 2) The mutation at the D130 site can be selected from D130E or D130C;

[0048] 3) The mutation at the V174 site can be selected from V174A, V174D, V174E, V174G or DEL174;

[0049] 4) The mutation at the Y178 site can be selected from Y178H, Y178G, Y178V, Y178F, Y178D, Y178N, Y178E, Y178S, Y178T or Y178P;

[0050] 5) The mutation at the G285 site is G285A;

[0051] 6) The mutation at the K303 site is K303L;

[0052] 7) The mutation at the N325 site can be selected from N325A or N325L;

[0053] 8) The mutation at the Y385 site can be selected from Y385A, Y385E or Y385N;

[0054] 9) The mutation at the R386 site may be selected from R386A, R386L, R386I, R386M, R386V, R386S, R386T, R386H, R386Q, R386N, R386K, R386D, R386C, R386W or R386G;

[0055] 10) The mutation at the K387 site can be selected from K387G, K387Y, K387Q, K387A, K387N, K387S or K387W;

[0056] 11) The mutation at the D388 site can be selected from D388Y or D388L;

[0057] 12) The mutation at the K389 site can be selected from K389A or K389R;

[0058] 13) The mutation at point R391 can be selected from R391A, R391L or R391K;

[0059] 14) The mutation at the K392 site can be selected from K392A, K392F, K392I, K392T, K392H, K392Q, K392E or K392C.

[0060] 4. The variant according to any one of item 1 or 2, wherein the amino acid sequence of the variant is shown in any one of SEQ ID NOs: 2-63.

[0061] 5. A polynucleotide molecule comprising a polynucleotide molecule encoding the RNA polymerase variant according to any one of items 1 to 4.

[0062] 6. A method for preparing an RNA polymerase variant, comprising introducing the polynucleotide molecule described in item 5 into a host cell.

[0063] 7. A method for producing an RNA product, comprising contacting a DNA template with at least one RNA polymerase variant according to any one of items 1 to 4 under conditions that result in the production of an RNA transcript.

[0064] 8. As described in item 7, the RNA product can be selected from mRNA, saRNA, siRNA, gRNA, dsRNA, ssRNA, miRNA, piRNA or shRNA; preferably mRNA or saRNA.

[0065] 9. A method of producing saRNA, comprising contacting a DNA template and at least one RNA polymerase variant according to any one of items 1-4 under conditions that result in the production of an RNA transcript.

[0066] 10. A method for performing in vitro transcription, comprising reacting a DNA template with at least one RNA polymerase variant according to any one of items 1 to 4 under conditions that result in the production of an RNA transcript.

[0067] 11. A composition or kit comprising at least one RNA polymerase variant according to any one of items 1 to 4.

[0068] 12. The composition or kit as described in item 11, further comprising one or more components of an in vitro transcription system.

[0069] 13. The composition or kit according to any one of items 11 or 12, further comprising a cap analog.

[0070] Compared with the prior art, this application has the following beneficial effects:

[0071] 1. The RNA polymerase variants provided in this application have improved performance. While improving the integrity of RNA products during in vitro transcription, they can also significantly reduce the probability of RNA truncation and over-extension, reduce the production of by-products, and have adaptability to a wide range of templates and application scenarios.

[0072] 2. The 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 is a schematic diagram of the construction of the recombinant plasmid. DETAILED DESCRIPTION

[0074] The technical solutions of the present application are further described below with reference to specific examples. However, the following examples are merely examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims. In the following examples, unless otherwise specified, all reagents and consumables used were purchased from common suppliers in the field, and the experimental methods and technical means used were conventional methods and means in the field.

[0075] In the examples of the present application, enzyme activity is defined as the amount of enzyme required to generate 1 μmol of product or convert 1 μmol of substrate within 1 minute under reaction conditions of 37°C.

[0076] Example 1 Preparation of RNA polymerase variants

[0077] A DNA fragment was synthesized according to the DNA sequence shown in SEQ ID NOs: 64-126 (the encoded amino acid sequence corresponds to SEQ ID NOs: 1-63). After PCR amplification, the DNA fragment was introduced into the BseRI and HindIII restriction sites of the expression vector pQE-80L to obtain a recombinant expression vector. The constructed vector was introduced into E. coli BL21 (DE3) by transformation technology, spread on LB plates containing ampicillin resistance, and incubated in a 37°C incubator overnight. The single colonies that grew were subjected to plasmid extraction and sequencing to finally obtain recombinant engineered bacteria containing the target gene. The successfully sequenced recombinant E. coli strain was inoculated into LB medium for overnight activation culture, then inoculated into the fermentation broth (LB medium) at 1-5% v / v and cultured to an OD600 value of 0.6-0.8. IPTG was added at a final concentration of 0.5 mol / L and cultured for another 4-6 hours. The strain was then collected by centrifugation at 12,000 rpm and 5°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0) to obtain the bacterial cells. After ultrasonic disruption, the cells were purified by affinity chromatography to obtain a stock solution of RNA polymerase.

[0078] RNA polymerase variants and their mutation sites are shown in Table 1:

[0079] Table 1 RNA polymerase variants

[0080] Among them, WT is the wild-type T7 RNA polymerase, and its amino acid sequence is:

[0081] Example 2 Detection of the integrity of in vitro transcription products

[0082] 2.1 Unmodified NTP (UTP)

[0083] (1) Dilute the enzyme stock solution with storage buffer (50mM Tris-HCl (25℃, pH 7.9), 100mM NaCl, 0.1mM EDTA, 2mM DTT, 0.1% Triton X-100, 50% Glycerol) to an enzyme activity of 300U / μL. Prepare the MIX solution according to the reaction system (20μL) in Table 2, transfer the MIX solution to the eight-well strip, mix well, and centrifuge; place the eight-well strip on a PCR instrument and react at 37℃ for 1 hour, then add 36μL of magnetic beads (Vazyme, Product No.: N412), mix well, and incubate at room temperature for 2-5 minutes; place the mixed solution on a magnetic stand to purify mRNA, and after purification, transfer it to an RNase-free centrifuge tube to obtain purified mRNA;

[0084] (2) Capillary electrophoresis was performed using a Qsep400 fully automatic nucleic acid analyzer to detect the integrity of mRNA (intact RNA product / total RNA product).

[0085] Table 2 Reaction system ratio

[0086] The test results are shown in Table 3-1 and Table 3-2. Compared with the wild-type T7 RNA polymerase (WT), the T7 RNA polymerase variants in Table 3-1 and Table 3-2 can effectively increase the integrity of the mRNA product.

[0087] Table 3-1 Improvement of RNA polymerase variant integrity

[0088] Table 3-2 Improvement of RNA polymerase variant integrity

[0089] 2.2 saRNA template (template SEQ ID NO: 128)

[0090] (1) Dilute the enzyme stock solutions of the K387Y variant and the K389A variant with storage buffer to an enzyme activity of 300 U / μL; refer to the reaction system ratio and experimental method in 2.1 of Example 2 to obtain purified saRNA;

[0091] (2) Capillary electrophoresis was performed using a Qsep400 fully automatic nucleic acid analyzer to detect the integrity of the saRNA product.

[0092] The 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.

Claims

1. An RNA polymerase variant having an amino acid sequence having at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more sequence identity to SEQ ID NO: 1, wherein: The amino acid sequence of the variant comprises at least one mutation selected from the following positions relative to SEQ ID NO: 1: R34, D130, V174, Y178, G285, K303, N325, Y385, R386, K387, D388, K389, R391 or K392.

2. The RNA polymerase variant according to claim 1, wherein The amino acid sequence of the RNA polymerase 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.

3. The RNA polymerase variant according to claim 1 or 2, wherein 1) The mutation at the R34 site is R34A; 2) The mutation at the D130 site may be selected from D130E or D130C; 3) The mutation at the V174 site may be selected from V174A, V174D, V174E, V174G or DEL174; 4) The mutation at the Y178 position may be selected from Y178H, Y178G, Y178V, Y178F, Y178D, Y178N, Y178E, Y178S, Y178T or Y178P; 5) the mutation at the G285 site is G285A; 6) the mutation at the K303 site is K303L; 7) The mutation at the N325 site can be selected from N325A or N325L; 8) The mutation at the Y385 site can be selected from Y385A, Y385E or Y385N; 9) The mutation at the R386 site may be selected from R386A, R386L, R386I, R386M, R386V, R386S, R386T, R386H, R386Q, R386N, R386K, R386D, R386C, R386W or R386G; 10) The mutation at the K387 site may be selected from K387G, K387Y, K387Q, K387A, K387N, K387S or K387W; 11) The mutation at the D388 site can be selected from D388Y or D388L; 12) The mutation at the K389 site can be selected from K389A or K389R; 13) The mutation at point R391 can be selected from R391A, R391L or R391K; or 14) The mutation at the K392 site can be selected from K392A, K392F, K392I, K392T, K392H, K392Q, K392E or K392C.

4. The RNA polymerase variant according to claim 1 or 2, wherein The amino acid sequence of the RNA polymerase variant is shown in any one of SEQ ID NOs: 2-63.

5. A polynucleotide molecule comprising a polynucleotide molecule encoding the RNA polymerase variant according to any one of claims 1 to 4.

6. A method for preparing an RNA polymerase variant, comprising introducing the polynucleotide molecule of claim 5 into a host cell.

7. A method for producing an RNA product, comprising contacting a DNA template with at least one RNA polymerase variant according to any one of claims 1 to 4 under conditions that result in the production of an RNA transcript.

8. A method of producing saRNA, comprising contacting a DNA template and at least one RNA polymerase variant of any one of claims 1-4 under conditions that result in the production of an RNA transcript.

9. A method for performing in vitro transcription, comprising contacting a DNA template with at least one RNA polymerase variant according to any one of claims 1 to 4 under conditions that result in the production of an RNA transcript.

10. A composition or kit comprising at least one RNA polymerase variant according to any one of claims 1 to 4.

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