Self-replicating RNA vector, preparation method therefor, and use thereof

Abstract

The present invention relates to a self-replicating RNA vector, a preparation method therefor, and a use thereof. The sequence of the self-replicating RNA vector sequentially comprises in the direction from a 5' end to a 3 'end: a T7 promoter sequence, an ATG sequence, an SINV RNA replicase coding sequence, a target gene sequence, and a signal sequence containing poly (A). The preparation method comprises: inserting an ATG sequence at an insertion sequence position of a pKGCT7-SINV-GFP backbone to obtain pKGCT7-SINV-GFP, carrying out AscI-NotI double enzyme digestion to remove a GFP sequence, and then using a recombinase to insert a target gene sequence between AscI and NotI, so as to obtain the self-replicating RNA vector. The use is to prepare self-replicating RNA. The self-replicating RNA vector can use pseudouridine or N1-methyl-pseudouridine to achieve low immunogenicity without reducing expression efficiency.

Description

Self-replicating RNA vector, preparation method and application thereof Technical Field

[0001] The present invention relates to a self-replicating RNA vector, a preparation method and application thereof, and belongs to the field of biotechnology. Background Art

[0002] The most efficient method for mRNA production currently utilizes T7 RNA polymerase for co-transcriptional capping using cap analogs GAG (m7G(5')ppp(5')(2'OMeA)pG) or GAU (m7G(5')ppp(5')(2'OMeA)pU). Simultaneously, pseudouridine triphosphate (or its derivatives) replaces uridine triphosphate (UTP). This single-step approach yields mRNA with a very high capping efficiency and complete replacement of uracil with pseudouridine. This mRNA can be directly purified without further reactions to form an mRNA stock solution for encapsulation. The two key elements of this method are the co-transcriptional capping with cap analogs and the use of pseudouridine to reduce immunogenicity. The use of pseudouridine and its derivatives has been crucial to the current development of the mRNA field. Currently, T7 RNA polymerase is used, rather than SP6 or T3 RNA polymerase, which are less well-studied, and it is unclear whether the current T7 RNA polymerase system can be easily transplanted.

[0003] Self-replicating RNA vectors are considered to be the next generation of RNA vectors. Compared with the most widely used mRNA vectors, self-replicating RNA vectors can replicate and amplify themselves, so a small amount can produce a lot of target proteins, and have a longer duration, which can overcome the current problems of large mRNA vector usage and short duration.

[0004] The core component of self-replicating RNA is its RNA replicase system. The sequences commonly used are from the nsp1-4 proteins of the Togaviridae family. These proteins replicate by recognizing specific conserved sequence elements (CSEs, typically four in number, two at the 5' and 3' ends) and simultaneously produce the target protein's mRNA. The most common sequences are from Venezuelan equine encephalitis virus (VEEV), Semliki forest virus (SFV), and Sindbis virus (SINV); other sequences are less commonly used.

[0005] Since SINV and SFV must use SP6 RNA polymerase for transcription and then use a capping system to complete the capping, which is different from the currently commonly used T7 RNA polymerase system, the most commonly used self-replicating RNA vector currently uses VEEV's RNA replicase. However, the use of pseudouracil nucleotides (or N1-methyl pseudouracil nucleotides) for modification of this system will lead to a severe decrease in efficiency (see: McGee, Joshua E et al. "Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA." bioRxiv: the preprint server for biology 2023.09.15.557994.17 Sep.2023, doi: 10.1101 / 2023.09.15.557994.Preprint.); in addition, other modified nucleotides are also used (see: Aboshi, M. et al. "Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine, in healthy individuals." medRxiv (2023).), but the expression efficiency of modification and non-modification was not compared in this document. However, unmodified RNA can induce strong innate immunity and produce strong side effects. Therefore, there is an urgent need to develop a self-replicating RNA vector that can be modified with pseudouridine nucleotides (or N1-methyl pseudouridine nucleotides) without reducing expression efficiency.

[0006] Summary of the Invention

[0007] The primary objective of the present invention is to overcome the problems of the prior art by providing a self-replicating RNA vector capable of utilizing pseudouridine or N1-methyl-pseudouridine without compromising expression efficiency. Also provided are methods for preparing the self-replicating RNA vector and its application, as well as methods for preparing self-replicating RNA.

[0008] The technical solution of the present invention to solve the technical problem is as follows:

[0009] A self-replicating RNA vector comprises the following sequence from 5' to 3': a T7 promoter sequence, an ATG sequence, a SINV RNA replicase coding sequence, a target gene sequence, and a poly(A) sequence.

[0010] Wherein, the T7 promoter sequence is 1309bp-1325bp of SEQ ID No.5.

[0011] The SINV RNA replicase coding sequence is 1329bp-8986bp of SEQ ID No. 5, or a sequence obtained by replacing degenerate codons on the basis of 1329bp-8986bp of SEQ ID No. 5.

[0012] Preferably, the poly(A) sequence is at least 25 bp in length.

[0013] Preferably, the number of non-A bases contained in the poly(A) sequence is greater than or equal to 0.

[0014] Preferably, in the sequence of the self-replicating RNA vector, an AscI restriction site is provided at the 5' end of the target gene sequence, and a NotI restriction site is provided at the 3' end, and an XbaI restriction site is used as a vector linearization site.

[0015] Preferably, in the sequence of the self-replicating RNA vector, before the T7 promoter sequence, there are also provided in order from the 5' end to the 3' end: a CMV enhancer sequence, a CMV promoter sequence, and a β-globin intron sequence; after the poly (A) sequence, there are also provided in order from the 5' end to the 3' end: a bGH poly (A) signal sequence, an ori sequence, and a resistance gene sequence.

[0016] Preferably, the resistance gene sequence is a KanR sequence.

[0017] Preferably, the sequence of the self-replicating RNA vector is shown as SEQ ID No. 5.

[0018] The present invention also provides:

[0019] A method for preparing a self-replicating RNA vector comprises the following steps:

[0020] Step 1: Use the pKGCT7-SINV-GFP backbone (SEQ ID No. 2); the insert sequence is located between 1325bp and 1326bp of the sequence; the sequence between 1309bp and 1325bp is the T7 promoter, the sequence between 8984bp and 8991bp is the AscI restriction site, the sequence between 9713bp and 9720bp is the NotI restriction site, and the sequence between 10101bp and 10106bp is the XbaI restriction site.

[0021] Step 2: insert the ATG sequence into the insertion sequence position of the pKGCT7-SINV-GFP backbone to obtain pKGCT7-SINV-GFP, the sequence of which is SEQ ID No. 5;

[0022] The third step is to perform double digestion of pKGCT7-SINV-GFP with AscI-NotI to cut off the GFP sequence, and then use recombinase to insert the target gene sequence between AscI and NotI to obtain a self-replicating RNA vector.

[0023] The present invention also provides:

[0024] The self-replicating RNA vector described above is used for preparing self-replicating RNA.

[0025] Preferably, pseudouridine or N1-methyl-pseudouridine is used in the preparation of self-replicating RNA.

[0026] The present invention also provides:

[0027] A method for preparing self-replicating RNA comprises the following steps:

[0028] The first step is to use a self-replicating RNA vector as described above; linearize the self-replicating RNA vector by enzyme digestion and use it as a linearization template;

[0029] Step 2: Perform in vitro transcription using an in vitro transcription system; the in vitro transcription system includes: the linearized template obtained in the first step, transcription buffer, ATP, GTP, CTP, pseudouridine or N1-methyl-pseudouridine, GAU cap analog, T7 RNA polymerase, and nuclease-free water;

[0030] In the third step, the transcription product obtained in the second step is purified to obtain a self-replicating RNA stock solution.

[0031] Preferably, the GAU cap analog is: m7G(5')ppp(5')(2'OMeA)pU or m7G(5')ppp(5')(2'OMeA)pG.

[0032] Compared with the prior art, the present invention changes SINV by one base (specifically: its initial three bases are replaced by natural AUU to AUG, and the original ATT is deleted and ATG is inserted in the corresponding plasmid sequence), so that it can directly use the T7 RNA polymerase system and the GAU cap analog to prepare self-replicating RNA; and through further testing, it was found that the expression efficiency of pseudouridine or N1-methyl-pseudouridine using the improved vector is the same as that of uridine nucleotides. Thus, the present invention obtains a self-replicating RNA vector that can use pseudouridine or N1-methyl-pseudouridine without reducing expression efficiency, solving the problem that self-replicating RNA expression efficiency and immunogenicity cannot be achieved at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a plasmid map of pKGCT7-SINV-GFP of Example 1 of the present invention.

[0034] FIG2 is a plasmid map of pKGCT7-VEEV-GFP of Example 1 of the present invention.

[0035] FIG3 is a plasmid map of pKGCT7-SINV-GFP-Fluc of Example 2 of the present invention.

[0036] FIG4 is a plasmid map of pKGCT7-VEEV-GFP-Fluc of Example 2 of the present invention. DETAILED DESCRIPTION

[0037] In specific implementation, the sequence of the self-replicating RNA vector of the present invention is provided with the following sequence from 5' to 3': T7 promoter sequence, ATG sequence, SINV RNA replicase coding sequence, target gene sequence, and poly(A) sequence.

[0038] In the sequence of the self-replicating RNA vector, an AscI restriction site is provided at the 5' end of the target gene sequence, and a NotI restriction site is provided at the 3' end, and an XbaI restriction site is used as a vector linearization site.

[0039] In the sequence of the self-replicating RNA vector, before the T7 promoter sequence, there are also arranged in order from the 5' end to the 3' end: a CMV enhancer sequence, a CMV promoter sequence, and a β-globin intron sequence.

[0040] In the sequence of the self-replicating RNA vector, after the poly(A) sequence, there are also arranged in order from the 5' end to the 3' end: bGH poly(A) signal sequence, ori sequence, and resistance gene sequence; wherein the resistance gene sequence is KanR sequence.

[0041] As a specific example, the sequence of the self-replicating RNA vector is shown as SEQ ID No. 5, and the plasmid map is shown in FIG1 .

[0042] In the sequence of the self-replicating RNA vector, the T7 promoter sequence is 1309 bp to 1325 bp of SEQ ID No. 5.

[0043] The SINV RNA replicase coding sequence is 1329 bp-8986 bp of SEQ ID No. 5, or a sequence obtained by replacing degenerate codons on the basis of 1329 bp-8986 bp of SEQ ID No. 5.

[0044] The length of the poly(A) sequence is at least 25 bp, and the number of non-A bases contained in the poly(A) sequence is greater than or equal to 0.

[0045] The preparation method of the self-replicating RNA vector of the present invention comprises:

[0046] Step 1: Use the pKGCT7-SINV-GFP backbone (SEQ ID No. 2); the insert sequence is located between 1325bp and 1326bp of the sequence; the sequence between 1309bp and 1325bp is the T7 promoter, the sequence between 8984bp and 8991bp is the AscI restriction site, the sequence between 9713bp and 9720bp is the NotI restriction site, and the sequence between 10101bp and 10106bp is the XbaI restriction site.

[0047] Step 2: insert the ATG sequence into the insertion sequence position of the pKGCT7-SINV-GFP backbone to obtain pKGCT7-SINV-GFP, the sequence of which is SEQ ID No. 5;

[0048] The third step is to perform double digestion of pKGCT7-SINV-GFP with AscI-NotI to cut off the GFP sequence, and then use recombinase to insert the target gene sequence between AscI and NotI to obtain a self-replicating RNA vector.

[0049] The self-replicating RNA vector of the present invention is used for preparing self-replicating RNA, wherein pseudouridine or N1-methyl-pseudouridine is used in preparing the self-replicating RNA.

[0050] The method for preparing the self-replicating RNA of the present invention comprises:

[0051] The first step is to linearize the self-replicating RNA vector by enzyme digestion and use it as a linearization template;

[0052] Step 2: Perform in vitro transcription using an in vitro transcription system; the in vitro transcription system includes: the linearized template obtained in the first step, transcription buffer, ATP, GTP, CTP, pseudouridine or N1-methyl-pseudouridine, GAU cap analog, T7 RNA polymerase, and nuclease-free water;

[0053] In the third step, the transcription product obtained in the second step is purified to obtain a self-replicating RNA stock solution.

[0054] Among them, the GAU cap analogs are: m7G(5')ppp(5')(2'OMeA)pU or m7G(5')ppp(5')(2'OMeA)pG.

[0055] The present invention will be described in further detail below with reference to the accompanying drawings and in conjunction with embodiments, but the present invention is not limited to the examples given.

[0056] Example 1

[0057] This example is about modifying SINV so that it can be used for in vitro transcription and achieve effective expression.

[0058] The specific contents of this embodiment are as follows:

[0059] The applicant synthesized the following plasmids:

[0060] pKGCT7-VEEV-GFP: The plasmid map is shown in Figure 2, and the sequence is SEQ ID No. 1.

[0061] pKGCT7-SINV-GFP backbone: Sequence No. 2; insert sequence located between 1325 and 1326 bp. The sequence taatacgactcactata (1309-1325 bp) represents the T7 promoter (all published vectors use the sp6 promoter), the sequence ggcgcgcc (8984-8991 bp) represents the AscI restriction site, the sequence gcggccgc (9713-9720 bp) represents the NotI restriction site, and the sequence tctaga (10101-10106 bp) represents the XbaI restriction site.

[0062] Insert the following into the insertion sequence of the pKGCT7-SINV-GFP backbone:

[0063] ATT, to obtain plasmid SINV-ATT;

[0064] AGGATT, to obtain plasmid SINV-AGGATT;

[0065] AGG, to obtain plasmid SINV-AGG;

[0066] ATG was used to obtain the plasmid pKGCT7-SINV-GFP. The plasmid map is shown in Figure 1, and the sequence is SEQ ID No. 5. In pKGCT7-SINV-GFP, the critical region is 1309 bp to 10109 bp, from the T7 promoter AT to the XbaI restriction site (Note: the T7 promoter AT is taatacgactcactataAT). When replacing the target gene, the GFP sequence is cut off by double digestion with AscI and NotI, and then the target sequence is inserted between the AscI and NotI residues using a recombinase.

[0067] The five plasmids obtained above, pKGCT7-VEEV-GFP, pKGCT7-SINV-GFP, SINV-ATT, SINV-AGGATT, and SINV-AGG, were linearized by XbaI digestion.

[0068] The in vitro transcription system for pKGCT7-VEEV-GFP, pKGCT7-SINV-GFP, and SINV-ATT is as follows:

[0069] In this in vitro transcription system, the Jiangsu Shenji Biotechnology Co., Ltd. T7 in vitro transcription kit (AGCU), catalog number 10110U, was used; the GAU Clean CAP was m7G(5')ppp(5')(2'OMeA)pU 100mM Ammonium Solution, catalog number CAP30112.

[0070] The in vitro transcription system for SINV-AGGATT and SINV-AGG is as follows:

[0071] In this in vitro transcription system, the GAG ​​CAP is m7G(5')ppp(5')(2'OMeA)pG 100mM Ammonium Solution, catalog number CAP3011.

[0072] Each in vitro transcription system was incubated at 37°C for 4 hours, followed by DNase I digestion to remove the template and purification using Norvegren RNA Clean Beads (N412).

[0073] The RNA yield was detected and calculated, and the results are shown in the following table:

[0074] The results show that SINV-ATT (i.e., the natural sequence of SINV and the sequence of the published vector) has an extremely low yield and is extremely unsuitable for the T7 RNA polymerase system. This also proves that the vectors published in the prior art should all use the SP6 promoter-SP6 RNA polymerase system.

[0075] 293T cells were transfected using Lipo2000 (Thermo, Cat. No. 11668019). 10 ng of RNA (except SINV-ATT, RNA transcribed from the above vectors) was added to each well. Green fluorescence was observed 48 hours after transfection. The results are as follows:

[0076] The results showed that replacing the first three bases of SINV RNA from AUU to AUG could achieve effective expression, but if AUU was replaced by AGGAUU or AGG, expression could not be achieved.

[0077] Example 2

[0078] This example is a verification comparison of expression efficiency.

[0079] The specific contents of this embodiment are as follows:

[0080] The use of N1-methylpseudouridine in VEEV significantly reduces the expression efficiency, but the expression efficiency of SINV using N1-methylpseudouridine is not significantly different from that using uridine.

[0081] For better quantification, this example uses GFP-Fluc to replace the GFP sequence and uses a luciferase reporter system for quantification.

[0082] pKGCT7-SINV-GFP and pKGCT-VEEV-GFP were double-digested with AscI and NotI, respectively, to replace GFP with GFP-Fluc. The final sequences were:

[0083] pKGCT7-SINV-GFP-Fluc: The plasmid map is shown in Figure 3, and the sequence is SEQ ID No. 3.

[0084] pKGCT7-VEEV-GFP-Fluc: The plasmid map is shown in FIG4 , and the sequence is SEQ ID No. 4.

[0085] The two plasmids were linearized with XbaI and then transcribed in vitro using the following two systems to obtain unmodified RNA (i.e., using uracil UTP) and N1-methylpseudouracil (i.e., N1-Me-pUTP) modified RNA:

[0086] To obtain unmodified RNA, use the T7 in vitro transcription kit (AGCU) from Jiangsu Shenji Biotechnology Co., Ltd., catalog number 10110U.

[0087] Obtain N1-methylpseudouracil-modified RNA: Use Jiangsu Shenji Biotechnology Co., Ltd. T7 in vitro transcription kit (AGCN), catalog number 10110N:

[0088] Each in vitro transcription system was incubated at 37°C for 4 hours, followed by DNase I digestion to remove the template and purification using Norvegren RNA Clean Beads (N412).

[0089] The RNA yield was detected and calculated, and the results are shown in the following table:

[0090] This result indicates that the use of N1-methylpseudouracil slightly reduces the yield, but this modification is crucial for its in vivo effect (reduced immunogenicity).

[0091] 293T cells were transfected using Lipo2000 (Thermo, Cat. No. 11668019). 10 ng of RNA (transcribed using the above vectors) was added to each well. The green fluorescence intensity was observed 48 hours after transfection. The results are as follows:

[0092] The results showed that, consistent with previous reports, the expression efficiency of VEEV vectors was significantly reduced after modification with pseudouridine nucleoside.

[0093] For accurate quantification, this example uses a luciferase reporter system for detection, and the results are as follows:

[0094] The results showed that, consistent with the fluorescence results above, pseudouridine modification of the VEEV vector significantly reduced expression efficiency by approximately 90%. In contrast, there was no significant difference between the SINV vectors with and without pseudouridine modification.

[0095] From the above embodiments, we can see that:

[0096] (1) The present invention replaces the third base of SINV from U to G, thereby obtaining a self-replicating RNA vector that is efficiently prepared using the T7 RNA polymerase system. The self-replicating RNA vector can utilize pseudouridine or N1-methyl-pseudouridine without reducing the expression efficiency.

[0097] Specifically, the present invention changes one base of SINV (specifically, its starting three bases are replaced by natural AUU to AUG, and the original ATT is deleted and ATG is inserted in the corresponding plasmid sequence), so that it can directly use T7 RNA polymerase (without having to use the SP6 RNA polymerase used in the existing SINV vector, reference: English, Justin G et al. "VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells." Cell vol.178, 3(2019):748-761.e17.doi:10.1016 / j.cell.2019.05.051) system and GAU cap analogs to prepare self-replicating RNA; and further testing found that the expression efficiency of pseudouridine or N1-methyl-pseudouridine using the above-mentioned improved vector is the same as that of uracil nucleotides. Thus, the present invention obtains a self-replicating RNA vector that can use pseudouridine or N1-methyl-pseudouridine without reducing the expression efficiency.

[0098] (2) The process of preparing self-replicating RNA in the present invention mainly includes: (i) using XbaI enzyme digestion to linearize the plasmid; (ii) in vitro transcription, the key components of which include the linearized template in the first step, T7 RNA polymerase, reaction buffer, ATP, GTP, CTP, and pseudouridine triphosphate (or its derivatives), and GAU cap analogs. The SINV vector obtained by the present invention through single base substitution modification can perfectly utilize the current T7 RNA polymerase co-transcription capping system. Its preparation process is consistent with the currently improved VEEV vector. After in vitro transcription, direct purification can obtain the self-replicating RNA stock solution.

[0099] (3) The present invention solves the problem of the inability to achieve both high expression efficiency and high immunogenicity of self-replicating RNA. The prior art uses common VEEV sequences. If uridine is used, the immunogenicity is strong, but if pseudouridine or N1-methyl-pseudouridine is used, the expression efficiency is low. The SINV vector modified by the present invention has the same expression efficiency when using pseudouridine or N1-methyl-pseudouridine as when using uridine, thus achieving both high expression efficiency and low immunogenicity.

[0100] In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. A self-replicating RNA vector, characterized in that: The sequence of the self-replicating RNA vector is sequentially provided with: T7 promoter sequence, ATG sequence, SINV RNA replicase coding sequence, target gene sequence, and poly (A) sequence in the direction from 5' end to 3' end. Wherein, the T7 promoter sequence is 1309bp-1325bp of SEQ ID No.

5. The SINV RNA replicase coding sequence is 1329bp-8986bp of SEQ ID No. 5, or a sequence obtained by replacing degenerate codons on the basis of 1329bp-8986bp of SEQ ID No.

5.

2. A self-replicating RNA vector according to claim 1, characterized in that: The poly(A) sequence is at least 25 bp in length.

3. A self-replicating RNA vector according to claim 2, characterized in that: The number of non-A bases contained in the poly(A) sequence is greater than or equal to 0.

4. A self-replicating RNA vector according to claim 1, characterized in that: In the sequence of the self-replicating RNA vector, an AscI restriction site is provided at the 5' end of the target gene sequence, and a NotI restriction site is provided at the 3' end, and an XbaI restriction site is used as a vector linearization site.

5. A self-replicating RNA vector according to claim 4, characterized in that: In the sequence of the self-replicating RNA vector, before the T7 promoter sequence, there are also the following sequences in the 5' to 3' direction: a CMV enhancer sequence, a CMV promoter sequence, and a β-globin intron sequence; after the poly(A) sequence, there are also the following sequences in the 5' to 3' direction: a bGH poly(A) signal sequence, an ori sequence, and a resistance gene sequence.

6. A self-replicating RNA vector according to claim 5, characterized in that: The resistance gene sequence is a KanR sequence.

7. A self-replicating RNA vector according to claim 1, characterized in that: The sequence of the self-replicating RNA vector is shown as SEQ ID No.

5.

8. A method for preparing a self-replicating RNA vector, characterized in that: The following steps are involved: Step 1: Use the pKGCT7-SINV-GFP backbone (SEQ ID No. 2); the insert sequence is located between 1325bp and 1326bp of the sequence; the sequence between 1309bp and 1325bp is the T7 promoter, the sequence between 8984bp and 8991bp is the AscI restriction site, the sequence between 9713bp and 9720bp is the NotI restriction site, and the sequence between 10101bp and 10106bp is the XbaI restriction site. Step 2: insert the ATG sequence into the insertion sequence position of the pKGCT7-SINV-GFP backbone to obtain pKGCT7-SINV-GFP, the sequence of which is SEQ ID No. 5; The third step is to perform double digestion of pKGCT7-SINV-GFP with AscI-NotI to cut off the GFP sequence, and then use recombinase to insert the target gene sequence between AscI and NotI to obtain a self-replicating RNA vector.

9. Use of the self-replicating RNA vector according to any one of claims 1 to 7 for preparing self-replicating RNA.

10. The use according to claim 9, characterized in that: Pseudouridine or N1-methyl-pseudouridine is used in the preparation of self-replicating RNA.

11. A method for preparing self-replicating RNA, characterized in that: The following steps are involved: The first step is to use a self-replicating RNA vector according to any one of claims 1 to 7; The self-replicating RNA vector is linearized by enzyme digestion and used as a linearization template; Step 2: Perform in vitro transcription using an in vitro transcription system; the in vitro transcription system includes: the linearized template obtained in the first step, transcription buffer, ATP, GTP, CTP, pseudouridine or N1-methyl-pseudouridine, GAU cap analog, T7 RNA polymerase, and nuclease-free water; Step 3: Purify the transcription product obtained in step 2 to obtain a self-replicating RNA stock solution.

12. The method for preparing a self-replicating RNA according to claim 11, wherein: The GAU cap analogs are: m7G(5')ppp(5')(2'OMeA)pU or m7G(5')ppp(5')(2'OMeA)pG.

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