Method for isolating and purifying circular RNA (Ribonucleic Acid)
Patent Information
- Application Number
- CN202380087552.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-12-20
- Filing Date
- 2023-10-17
- Publication Date
- 2025-07-29
AI Technical Summary
The in vitro synthesis process of circRNA in the prior art has shortcomings such as low reaction concentration, low connection efficiency, and easy generation of intermolecular connections, and existing purification methods such as RNase R digestion and size exclusion chromatography are difficult to meet GMP requirements, resulting in Circular RNA has low purity and low recovery.
By adding a polyA sequence to the in vitro transcription template, circular RNA is circularized by intron self-splicing, and polyA tags are used for affinity chromatography to remove uncirculated linear RNA precursors and intron parts to achieve efficient purification. .
It improves the purity and recovery rate of circular RNA, meets GMP production requirements, reduces costs, and simplifies the production process.
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Abstract
Description
Circular RNA Isolation and Purification Methods Technical Field
[0001] This application relates to the fields of biotechnology and biopharmaceutical or vaccine production. Specifically, it relates to the production and purification of circular RNA (e.g., mRNA vaccines), by efficiently isolating circular RNA by adding polyA sequences to in vitro transcription templates, introns after in vitro transcription, and / or circular RNA precursors during the circular RNA production process. Background Art
[0002] In recent years, mRNA vaccines have emerged as the most promising and effective platform for combating a variety of diseases, including the COVID-19 pandemic. With the advancement of RNA research, mRNA vaccines currently fall into three main categories: traditional mRNA, self-amplifying RNA, and circular RNA (circRNA). CircRNA, among these, is gaining increasing attention among researchers and holds great potential.
[0003] Circular RNA is a special type of non-coding RNA molecule that also exists in nature and is the latest research hotspot in the RNA field. Unlike traditional linear RNA (linear RNA, containing two ends, 5' and 3'), circular molecules have a closed ring structure, are not easily affected by RNA exonucleases, are more stable in expression, and are not easily degraded. In addition, circular RNA also has the advantages of low immunogenicity and low required dosage. In addition, circular RNA vaccines do not require additional steps such as capping and base modification during the synthesis process, which also makes the production cost of circular RNA lower.
[0004] Circular RNA technology offers significant advantages as an alternative to mRNA, and its industrialization prospects are promising. However, the key to its effectiveness lies in the artificial preparation and purification of circular RNA. Currently, there are two main approaches for the in vitro synthesis of circular RNA: RNA ligase and ribozyme self-splicing. The RNA ligase-based cyclization method suffers from drawbacks such as low reaction concentration, low ligation efficiency, and the susceptibility to intermolecular ligation during the cyclization reaction. Ribozyme self-splicing, on the other hand, primarily achieves RNA circularization through intronic splicing, currently encompassing Type I and Type II intronic splicing.
[0005] Group I introns are enzymatically active introns that can self-splice after transcription into RNA. This self-splicing is catalyzed by the activity of sequence-specific RNA endonucleases. While this self-splicing does not require any protein enzymes in vitro, it does require proteins to fold into secondary structures in vivo.
[0006] Although RNA circularization via type I / II intron self-splicing can achieve a very high circularization rate, a small amount of linear RNA cannot be completely circularized during this process, and self-splicing introns are also produced. Therefore, purification is required to remove the residual linear RNA precursors and the introns spliced during the circularization process.
[0007] The most successful purification method reported in the literature is to first specifically digest linear RNA with RNase R, and then further separate and purify circular RNA through size exclusion chromatography (SEC). Due to the small difference in molecular weight between the circular RNA product and its precursor RNA, the separation effect of size exclusion chromatography is limited. Secondly, it is understood that there is currently no GMP-grade RNase R, which cannot meet the GMP requirements for circular RNA production; and RNase R will also degrade the target circular RNA to a certain extent, resulting in a low circular RNA yield. In addition, this part of the operation requires a large amount of RNase R, which will have a huge cost burden, and the removal of RNase R also requires additional consideration.
[0008] In summary, there is an urgent need in this field to develop an efficient separation and purification method that can be used for the in vitro synthesis of circular RNA molecules.
[0009] Summary of the Invention
[0010] This article provides a highly efficient isolation and purification method for in vitro synthesis of circular RNA molecules. This method addresses the problems of difficult purification, low purity, and low recovery of in vitro synthesized circular RNA.
[0011] In a first aspect of the present invention, a method for preparing a circular RNA of interest in vitro by intron self-splicing is provided, the method comprising:
[0012] A method for preparing a target circular RNA in vitro by intron self-splicing, the method comprising:
[0013] (a) providing a circular RNA in vitro transcription template, wherein the template comprises: a 3′-intron and a 5′-intron, and a target gene (GOI) located therebetween;
[0014] (b) performing in vitro transcription using the template to form an in vitro transcription product;
[0015] (c) allowing the in vitro transcription product to self-splice and circularize to form an in vitro circularized product;
[0016] In any one or more steps of steps (a) to (c), the introns cut out from the in vitro cyclization product and / or the uncyclized linear RNA precursor are poly(X)-dylated, thereby carrying a poly(X)-nucleotide (poly) tag, wherein the poly(X)-nucleotide is selected from the group consisting of poly(adenylic acid) (polyA), poly(uridine) (polyU), poly(cytidylic acid) (polyC), and poly(guanylic acid) (polyG);
[0017] (d) contacting the in vitro cyclized product with a poly(X)-specific binding substance to remove the excised intron with a poly(X) tag and the uncyclized linear RNA precursor with a poly(X) tag in the product to obtain the target circular RNA.
[0018] In some aspects of the present invention, an in vitro transcription template for preparing a circular RNA of interest in vitro by intron self-splicing, a vector comprising the template, or a product comprising the in vitro transcription template or vector is provided, wherein the template comprises: a 3′-intron and a 5′-intron, and a gene of interest (GOI) located therebetween; wherein the template has polyX at one or both ends or in the intron.
[0019] In some aspects of the present invention, a circular RNA or a product comprising a circular RNA or a precursor thereof, wherein the circular RNA is prepared using the method described herein or using the in vitro transcription template or vector described herein.
[0020] Those skilled in the art may arbitrarily combine the technical solutions and technical features described herein without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The present invention will be further described below with reference to the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention and are not intended to limit the scope of the present invention.
[0022] Figure 1: Schematic diagram of the principles of some embodiments of the present application.
[0023] Figure 2: Purification chromatograms using 0A template (Figure 2A), 1A template (Figure 2B), and 2A template (Figure 2C); FT = flow-through fraction; Wash = wash out fraction; Elute = elution fraction.
[0024] Figure 3: Agarose gel electrophoresis of each sample after chromatography. HMW represents high molecular weight polymers, including RNA concatemers formed between two RNA molecules.
[0025] Figure 4: Capillary gel electrophoresis (CGE) results of samples prepared using a template without polyA before and after purification.
[0026] Figure 5: Capillary gel electrophoresis (CGE) results of samples prepared using a polyA-terminated circular RNA template before and after purification.
[0027] Figure 6: Capillary gel electrophoresis (CGE) results of samples prepared using a polyA-terminated template before and after purification of circular RNA. DETAILED DESCRIPTION
[0028] During in vitro synthesis of linear mRNA, since such mRNAs typically have a polyA structure (i.e., a polyA tail) at their 3' end, they can be effectively isolated using affinity chromatography with oligo dT (e.g., POROS oligo dT25). This is then followed by ultrafiltration, concentration, and exchange of the buffer to yield a highly pure mRNA product. However, the addition of polynucleotide (polyX) purification tags during circular RNA production has not been disclosed in the art.
[0029] The inventors broke through the conventional thinking that circular RNA has no polyA structure and pioneered the design and attempt to add polyA to the ends (one or both ends) or the outer part of the intron (away from the GOI) of the in vitro transcription template during the process of circularization to prepare circular RNA by intron self-splicing. The results showed that the intron portion of the circular RNA will be removed during the circularization process after in vitro transcription, and the polyA portion added at the end can also be removed normally. This leaves the sequence of the target circular RNA unaffected (i.e., it does not contain the added polyA portion), while the uncircularized circular RNA precursor and the intron portion removed during the circularization process all have the polyA portion. The linear precursor and intron portion can then be easily and efficiently removed by oligo dT affinity chromatography (e.g., POROS oligo dT25). In addition, after in vitro transcription, polyA can be added to the ends of the linear RNA precursor and intron by methods such as polyA polymerase to achieve the same purpose as adding polyA to the template before transcription as described above. Similarly, using other polynucleotide tags in place of the polyA tag, along with binding moieties capable of binding to these polynucleotide tags, can also achieve similar effects as adding the polyA tag. Studies have demonstrated that the method of this application can address long-standing challenges such as the difficulty in purifying in vitro synthesized circular RNA, low purity, and low recovery rates.
[0030] All numerical ranges provided herein are intended to expressly include all values falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and any feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.
[0031] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”
[0032] The numerical ranges herein include their endpoints and each specific numerical point and sub-range within the numerical range. For example, 1 to 3 includes endpoints 1 and 3, the specific integer numerical point 2 and non-integer numerical point therein (for example, but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (for example, but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).
[0033] Template construction and linearization
[0034] One of the key raw materials for in vitro production of circular RNA is a designed template DNA and its vector (e.g., plasmid DNA, pDNA). The industrial production process for these templates and vectors is now mature, and production can be outsourced to suppliers or established in-house to obtain high-purity templates and vectors.
[0035] In some embodiments, the vector used is a plasmid, and its preparation process may include one or more steps, such as fermentation, bacterial harvesting, alkaline lysis, clarification, ultrafiltration concentration, and chromatography. For example, the chromatography process may include a three-step purification: 1. RNA removal using molecular sieves (e.g., Sepharose 6FF media); 2. Isolation of supercoiled plasmid using a thiophilic affinity media (e.g., Capto PlasmidSelect); and 3. Anion exchange chromatography (e.g., Capto Q ImpRes media) to remove trace impurities and endotoxins. This process has been validated over many years and is widely recognized as an efficient, versatile, and robust platform process.
[0036] The desired target gene can be inserted into a vector (such as a plasmid) as needed. The target genes that can be used herein include, but are not limited to, genes encoding therapeutic polypeptides, immunogenic peptides (such as the S protein or RBD region of a coronavirus), cytokines, transcription factors, immune checkpoint inhibitors, chimeric antigen receptors, T cell receptors, etc. The encoded polypeptide or protein can be a single polypeptide molecule, a concatenation or fusion of multiple or multiple polypeptides.
[0037] The full-length sequences of certain circular RNAs can be obtained from websites or data platforms (e.g., the circBase website) for use in template construction. In some embodiments, a conventional circular RNA template that does not contain a polyX-coding sequence is directly used, and in subsequent transcription and / or cyclization steps, the excised intron or uncircularized circularized mRNA precursor is tagged with a polyX tag. In other embodiments, a template is used that has a polynucleotide tag-coding sequence added to one or both ends, or to the end of the intron distal to the GOI.
[0038] In some preferred embodiments, a specially designed template containing a polyX corresponding sequence is used. As used herein, "polyX corresponding sequence" refers to a sequence fragment in a DNA template that can form a polyX tag in mRNA after transcription.
[0039] In some embodiments, the polyX corresponding sequence may correspond to a polyX polymerized from only one type of nucleotide molecule, such as a polynucleotide composed entirely of A, U, G, or C. In some embodiments, the polyX corresponding sequence may correspond to a polyX polymerized from primarily one type of nucleotide molecule, such as primarily composed of A, U, G, or C, but also containing a small amount of other types of nucleotide molecules, such as 1 to 20 non-major nucleotides of other types. The polyX corresponding sequence may be located in the template at one or more positions selected from the group consisting of: one end of the template, both ends of the template, and the outer end of an intron (away from the target gene).
[0040] The vector can be linearized using methods known in the art, such as cleavage with a restriction endonuclease. After the reaction is complete, the enzyme can be removed (e.g., by chromatography) and the buffer can be replaced with a reaction solution suitable for in vitro transcription.
[0041] In vitro transcription, circularization, and circular RNA enrichment
[0042] After obtaining the vector comprising the template, methods known in the art can be used to perform in vitro transcription. For example, T7 RNA polymerase can be used to perform in vitro transcription to obtain single-stranded RNA. Preferably, the DNA template is removed after in vitro transcription, for example, by using DNA enzyme.
[0043] The cyclization in this application is performed by in vitro intron self-splicing, which can be done by type I intron or type II intron self-splicing as needed.
[0044] In some embodiments, transcription is performed using a transcription template comprising a sequence corresponding to a polyX tag as described herein to obtain a transcription product comprising a polyX tag, and then in vitro intron self-splicing is performed to obtain a cleaved linear intron with a polyX tag at the corresponding position and / or a non-circularized linear RNA precursor with a polyX tag. These linear RNAs are subsequently removed by binding to the polyX tag to achieve enrichment of circular RNA.
[0045] In some embodiments, the transcript is polyX-tagged so that one or both ends of the transcript contain a polyX tag. This allows the linear introns and / or linear circular RNA precursors produced after intron self-splicing in vitro to be polyX-tagged, allowing for subsequent removal of these linear RNAs and enrichment of circular RNAs. PolyX-tagged transcripts can be added using any suitable method, such as using a polynucleotide polymerase or a polynucleotide phosphorylase to add a polyX tag to the termini of the transcript.
[0046] In some embodiments, after the cyclization reaction, the linear introns and / or linear circular RNA precursors produced after intron self-splicing in vitro are labeled with a polyX tag at one or both ends, which facilitates the subsequent removal of these linear RNAs and the enrichment of circular RNAs. PolyX-tag addition of the linear RNA molecules in the cyclization product can be performed using any suitable method, such as using a polynucleotide polymerase or a polynucleotide phosphorylase to add a polyX tag to the termini of the linear RNA.
[0047] After obtaining a mixture of polyX-tagged linear RNA and target circular RNA, the polyX-tagged linear RNA (including linear intron fragments and / or linear circular RNA precursors) can be removed by binding of the polyX tag to its specific binding substance. The method for removing undesired linear RNA molecules comprises: contacting the polyX-tagged linear RNA (e.g., linear introns and linear circular RNA precursors) with a substance that specifically binds to polyX under conditions suitable for binding of polyX to its specific binding substance to form a binding complex, and separating the binding complex from the circularization reaction mixture.
[0048] Optionally, the reaction system can be exchanged after the circularization step, for example, using a tangential flow (TFF) method. Optionally, RNase R can be added after the circularization step to remove some linear RNA.
[0049] In some embodiments, the methods described herein include the steps of:
[0050] 1. Construct a circular RNA (type I or type II intron self-splicing circularization) in vitro transcription template, the ends of which (one or both ends) contain the sequence corresponding to the polyX tag;
[0051] 2. RNA in vitro transcription (e.g., 37°C for 4 hours);
[0052] 3. Intron self-splicing and circularization;
[0053] 4. Optionally, perform a liquid exchange (e.g., TFF chromatography liquid exchange);
[0054] 5. Perform binding to polyX and separation of the bound products, such as affinity chromatography (e.g., using POROS oligo dT25 for polyA tags);
[0055] 6. Optionally, perform a TFF chromatography buffer exchange.
[0056] In some embodiments, the methods described herein include the steps of:
[0057] 1′. Construction of circular RNA (type I or type II intron self-splicing circularization) in vitro transcription template;
[0058] 2′. RNA in vitro transcription (e.g., 37°C for 4 h);
[0059] 3′. Intron self-splicing and circularization;
[0060] 4′. Add polyX polymerase to add a polyX tag to the end of the linear RNA (such as the excised intron, uncircularized circular RNA precursor);
[0061] 5'. Optionally, perform a liquid exchange (eg, TFF chromatography liquid exchange);
[0062] 6'. Perform binding to polyX and separation of the binding product, such as affinity chromatography (such as using POROS oligo dT25 for the polyA tag);
[0063] 7'. Optionally, perform TFF chromatography buffer exchange.
[0064] Products and Applications
[0065] The in vitro transcription template, vector and method of the present application can be used to prepare various target circular RNAs and products containing target circular RNAs in vitro through intron self-splicing.
[0066] In some embodiments, provided are in vitro transcription templates for circular RNA, vectors comprising the templates, and products (e.g., kits) comprising the templates or vectors. These templates, vectors, and products can be used in the methods of the present application to prepare high-purity circular RNA of interest with high recovery rates.
[0067] In some embodiments, provided are circular RNAs of interest and products comprising such circular RNAs, prepared using the methods, templates, and vectors of the present application. In some embodiments, the uses of the circular RNAs of interest or products include, but are not limited to: translation into functional polypeptides or proteins (e.g., for therapeutic or preventive purposes); use as molecular sponges for miRNAs; regulation of gene expression; regulation of alternative splicing of parental genes; and formation of circular RNA-protein complexes to regulate signaling pathways. In some embodiments, the products comprise highly purified circular RNAs of interest.
[0068] In some embodiments, the purity of the circular RNA of interest is not less than 70%, not less than 75%, not less than 80%, not less than 85%, not less than 90%, not less than 92%, not less than 95%, not less than 98%, not less than 99%, or any value or range of values therein.
[0069] In summary, by adding poly A to the ends of in vitro transcription templates, the circular RNA production process is simplified, improving the recovery rate and purity of circular RNA purification. Testing and analysis have shown that the methods and products disclosed herein can achieve circular RNA purity of approximately 90% and an overall recovery rate exceeding 50%, meeting GMP production requirements and making them suitable for widespread application.
[0070] Exemplary embodiments
[0071] The present application provides the following embodiments. It should be understood that those skilled in the art may make appropriate modifications, changes, and combinations to the present invention, and these modifications, changes, and combinations are all within the scope of the present invention.
[0072] 1. A method for preparing a target circular RNA in vitro by intron self-splicing, the method comprising:
[0073] (a) providing a circular RNA in vitro transcription template, wherein the template comprises: a 3′-intron and a 5′-intron, and a target gene (GOI) located therebetween;
[0074] (b) performing in vitro transcription using the template to form an in vitro transcription product;
[0075] (c) allowing the in vitro transcription product to self-splice and circularize to form an in vitro circularized product;
[0076] In any one or more steps of steps (a) to (c), the introns removed from the in vitro cyclization product and / or the uncyclized linear RNA precursor are poly-X-dylated, thereby carrying a poly-X (polyX) tag, wherein X is selected from the group consisting of adenylate (A), uridine (U), cytidylate (C) and guanylate (G);
[0077] (d) contacting the in vitro cyclized product with a poly(X)-specific binding substance to remove the excised intron with a poly(X) tag and the uncyclized linear RNA precursor with a poly(X) tag in the product to obtain the target circular RNA.
[0078] 2a. The method of embodiment 1, wherein the intron is a type I intron or a type II intron.
[0079] 2b. The method according to embodiment 1, wherein the polyX tag is located at one or both ends of the uncircularized linear RNA precursor or at the outer end of the intron away from the GOI.
[0080] 2c. The method according to embodiment 1, wherein the polyX tag is directly linked to an intron or an uncircularized linear RNA precursor or is linked via a linker.
[0081] 2d. The method of embodiment 2c, wherein the length of the linker is less than 10 bases.
[0082] 3. The method according to embodiment 1, wherein the method comprises one or more steps selected from the group consisting of:
[0083] One or both ends of the in vitro transcription template in step (a) or the outer end of the intron away from the GOI is provided with a sequence corresponding to a polyX tag; and / or
[0084] In step (b), polyX polymerase or polynucleotide phosphorylase and corresponding nucleotide monomers are added so that the ends of the in vitro transcription products are tagged with polyX tags; and / or
[0085] In step (c), polyX polymerase or polynucleotide phosphorylase and corresponding nucleotide monomers are added.
[0086] 4a. The method according to embodiment 3, wherein the in vitro transcription template in step (a) is provided with a sequence corresponding to a polyX tag at one or both ends or in an intron, wherein
[0087] (I) The intron is a type I intron, and the circular RNA in vitro transcription template comprises the following elements: 3′-P1→PI1-I1→E2→GOI→E1→I2-PI2→P2-5′ (A)
[0088] in:
[0089] I1 is the 3′-end group I intron; E2 is exon 2; GOI is the target gene; E1 is exon 1; I2 is the 5′-end group I intron;
[0090] 3'-P1 is the sequence corresponding to the polyX tag at the 3' end or it does not exist; P2-5' is the sequence corresponding to the polyX tag at the 5' end or it does not exist; PI1 is the sequence corresponding to the polyX tag contained in intron I1 away from the GOI side or it does not exist; PI2 is the sequence corresponding to the polyX tag contained in intron I2 away from the GOI side or it does not exist.
[0091] The condition is that at least one (e.g., 1, 2, 3, or 4) of 3'-P1, P2-5', PI1, and PI2 is a sequence corresponding to a polyX tag.
[0092] 4b. The method of embodiment 3, wherein the in vitro transcription template in step (a) is provided with a polyX tag corresponding sequence at one or both ends or in an intron, wherein
[0093] (II) The intron is a type II intron, and the circular RNA in vitro transcription template comprises the following elements: 3′-P′1→PI1′-I1′→GOI′→I2′-PI2′→P′2-5′ (A)
[0094] in:
[0095] I1′ is the 3′-end group II intron; GOI is the target gene; I2′ is the 5′-end group II intron;
[0096] 3′-P′1 is the sequence corresponding to the polyX tag at the 3′ end or it does not exist; P′2-5′ is the sequence corresponding to the polyX tag at the 5′ end or it does not exist; PI1′ is the sequence corresponding to the polyX tag contained in intron I1′ away from the GOI side or it does not exist; PI2′ is the sequence corresponding to the polyX tag contained in intron I2′ away from the GOI side or it does not exist.
[0097] The condition is that at least one (e.g., 1, 2, 3, or 4) of 3′-P′1, P′2-5′, PI1′, and PI2′ is a sequence corresponding to a polyX tag.
[0098] 5a. The method of embodiment 4a or 4b, wherein the template further comprises an element selected from the group consisting of an internal ribosome entry site (IRES), a transcriptional regulatory region (such as a promoter and / or enhancer), a spacer, and a linker.
[0099] 5b. The method according to embodiment 4a or 4b, wherein the in vitro transcription template in step (a) is provided with a sequence corresponding to a polyX tag at one or both ends or in an intron.
[0100] 5c. The method according to embodiment 4a or 4b, wherein the sequence corresponding to the polyX tag is directly linked to the end of the template and / or the intron, or is linked via a linker (eg, a linker with a length of 10 bases or less).
[0101] 6a. The method according to embodiment 1, wherein the polyX tag comprises 6 or more X nucleotides, for example, 6 to 250, 8 to 200, 10 to 100, 15 to 80, or 20 to 60 X nucleotides.
[0102] 6b. The method of embodiment 1, wherein the target gene encodes one or more polypeptides or proteins or fusion peptides selected from the following groups: therapeutic polypeptides, immunogenic peptides, cytokines, transcription factors, immune checkpoint inhibitors, chimeric antigen receptors, and T cell receptors.
[0103] 7a. A method as described in embodiment 1, wherein the polyA-specific binding substance is selected from: Oligo dT (e.g., POROS oligo dT25), polyA binding protein (PABP); the polyU-specific binding substance is selected from: poly(A), oligo(A), polyU binding protein; the polyC-specific binding substance is selected from: poly(Gu), oligo(Gu), polyC binding protein; the polyG-specific binding substance is selected from: poly(C), oligo(C), polyG binding protein.
[0104] 7b. The method according to embodiment 1, wherein the specific binding substance is immobilized, for example, on magnetic beads, columns, or chips.
[0105] 8a. The method of embodiment 1, further comprising one or more steps selected from the group consisting of: PCR amplification, plasmid fermentation, plasmid extraction, and plasmid linearization; and precipitation, degradation, or isolation of uncircularized RNA after in vitro transcription and cyclization, such as precipitation of uncircularized RNA with lithium chloride, enzymatic degradation of uncircularized RNA with RNase R, and tangential flow filtration for liquid exchange.
[0106] 8b. The method of embodiment 1, wherein the method does not include the use of RNase R enzyme.
[0107] 9. An in vitro transcription template for preparing a target circular RNA by intron self-splicing in vitro, a vector comprising the template, or a product comprising the in vitro transcription template or vector: the template comprises: a 3′-intron and a 5′-intron, and a target gene (GOI) located therebetween; wherein the template has polyX at one or both ends or in the intron.
[0108] 10a. The in vitro transcription template or vector according to embodiment 9, wherein the template molecule is as defined in embodiments 2 to 6.
[0109] 10b. The in vitro transcription template or vector as described in embodiment 9, wherein the vector is selected from: a plasmid (e.g., a bacterial plasmid, a yeast plasmid), a phage, a viral vector (e.g., a lentivirus, adenovirus, adeno-associated virus vector), and a PCR product.
[0110] 11. A circular RNA or a product comprising a circular RNA or a precursor thereof, wherein the circular RNA is prepared using the method of any one of embodiments 1-8 or using the in vitro transcription template or vector of embodiment 9 or 10.
[0111] 12a. The product of embodiment 11, wherein the circular RNA or product is selected from circular RNA or products for the following applications: for translation into functional polypeptides or proteins (e.g., for therapeutic or preventive purposes); for use as a molecular sponge for miRNA; for regulation of gene expression; for regulation of selective splicing of parental genes; for formation of circular RNA-protein complexes to regulate signaling pathways.
[0112] 12b. The product of embodiment 11, wherein the product is selected from the group consisting of: a drug, a vaccine, a test, or a diagnostic kit.
[0113] 12c. The product according to embodiment 11, wherein the circular RNA preparation precursor is selected from: a circular RNA in vitro transcription template, in vitro transcription product, and / or in vitro cyclization product as defined in any one of embodiments 1-8; and optionally, a poly(X)-nucleotide-specific binding substance.
[0114] Example
[0115] The present application will be further described below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art may make appropriate modifications and variations to the present invention, and these modifications and variations are all within the scope of the present invention.
[0116] All reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, where specific conditions are not specified, generally follow conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, 4th edition, by Michael R. Green et al., Cold Spring Harbor Laboratory Press, New York, 2017, or according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.
[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to this application. The preferred embodiments and materials described herein are for illustrative purposes only.
[0118] Example 1. Construction of in vitro transcription templates for circular RNA containing or without polyA at the end
[0119] Prepare a circular RNA in vitro transcription template with the following structure:
[0120] Template 0A
[0121] An in vitro transcription template 0A for preparing circular RNA by group I intron self-splicing cyclization was constructed. The template expressed the GFP gene and did not contain polyA. The entire sequence was synthesized according to SEQ ID NO: 1, and the template plasmid had a total length of approximately 3900 bp.
[0122] The in vitro transcription template contains the following elements from 3' to 5':
[0123] 3′-intron → E2 → IRES → GOI → E1 → 5′-intron
[0124] in:
[0125] The 3′-intron is a type I intron;
[0126] E2 = exon 2;
[0127] IRES = internal ribosome entry site;
[0128] GOI = gene of interest, specifically GFP gene;
[0129] E1 = exon 1;
[0130] The 5′-intron is a type I intron.
[0131] Template 1A
[0132] An in vitro transcription template containing polyA at one end for the production of circular RNA by group I intron self-splicing circularization was constructed, with a 60A polyA addition. Template 1A is identical to Template 0A, except for the addition of a 60A polyA at one end. The entire sequence was synthesized according to SEQ ID NO: 2. The template plasmid is approximately 3960 bp in length.
[0133] Template 1A includes the following elements from 3' to 5':
[0134] 3′-intron → E2 → IRES → GOI → E1 → 5′-intron → 5′-polyA
[0135] Template 2A
[0136] An in vitro transcription template containing polyA at both ends for the production of circular RNA by group I intron self-splicing circularization was constructed. 60A residues were added to each end of Template 2A. Template 2A was identical to Template 0A, except for the addition of 60A residues at each end. The entire sequence was synthesized according to SEQ ID NO: 3. The template plasmid was approximately 4020 bp in length.
[0137] Template 2A includes the following elements from 3' to 5':
[0138] 3′-polyA→3′-intron→E2→IRES→GOI→E1→5′-intron→5′-polyA
[0139] Example 2. Preparation and purification of circular RNA
[0140] Circular RNAs were prepared by in vitro transcription using template 0A, template 1A, and template 2A prepared in Example 1, respectively, and then purified.
[0141] 1. Plasmid Amplification, Plasmid Linearization, and Linearized Plasmid Purification
[0142] The plasmid was amplified by bacterial fermentation (30 or 37°C, approximately 16 hours), extracted using a plasmid extraction kit, linearized with restriction endonuclease BspQ I (37°C, 2 hours), and purified by ultrafiltration.
[0143] 2. In vitro transcription
[0144] RNA was transcribed in vitro using templates 0A, 1A, and 2A. The in vitro transcription conditions were 37°C for 4 hours. The reaction composition was as follows:
[0145] T7 RNA polymerase: 7500 U / mL;
[0146] Mouse RNase inhibitor: 2000 U / mL;
[0147] Pyrophosphatase (Inorganic (yeast)): 5 U / mL;
[0148] NTP solution (ATP, UTP, CTP, GTP): 7.5 mM (each);
[0149] Template (linearized plasmid or PCR product): 50 μg / mL;
[0150] In vitro transcription reaction buffer: 1×
[0151] 2. Precipitate RNA with lithium chloride and dissolve it in RNase-free water. For larger reaction volumes and GMP production, perform liquid exchange via tangential flow filtration (TFF).
[0152] 3. Affinity chromatography was performed using POROS oligo dT25, with 0.25 M NaCl and 1xTE loading, and washing under the same conditions. The flow-through (FT) and wash fractions were collected as purified samples, and the eluent was 1xTE.
[0153] 4. Purify the sample (i.e., the flow-through and wash fraction in step 3) by exchanging the buffer to 1xTE using TFF chromatography.
[0154] 5. Detect circular RNA (cirRNA), RNA precursor (Precursor) and intron (Intron) contained in the sample solution, flow-through part, clean part and eluate by agarose gel electrophoresis.
[0155] The chromatogram is shown in Figure 2, and the agarose gel electrophoresis results of each sample after chromatography are shown in Figure 3. The results show that the desired circular RNA was produced using templates 0A, 1A, and 2A in Example 1. Furthermore, compared with template 0A, which does not contain polyA, templates 1A and 2A, which contain polyA at one or both ends, can more effectively separate impurities such as RNA precursors and introns, thereby obtaining the target circular RNA of higher purity.
[0156] Example 3. Analysis of circular RNA purity by capillary gel electrophoresis (CGE-LIF)
[0157] The samples before and after affinity chromatography purification in Example 2 were analyzed using capillary gel electrophoresis (CGE-LIF).
[0158] The CGE results for samples obtained using templates 0A, 1A, and 2A before and after purification are shown in Figures 4, 5, and 6, respectively. The results demonstrate that the addition of polyA can produce the target circular RNA and significantly improves the purity of the target RNA after purification, almost completely removing introns and high molecular weight (HMW) impurities from the product.
[0159] Table 1. CGE results: proportion of each component
[0160] Table 2. CGE statistical results: *In the table above: load = sample load; FT = flow-through fraction; Wash = washout fraction; Elute = eluted fraction; HMW = high molecular weight impurities; Oligo dT25 = POROS™ Oligo(dT)25 affinity purification medium (Thermo Fisher Scientific, containing 25-mer poly-dT);
[0161] The CGE analysis above demonstrates that the present method can achieve a circular RNA purity of approximately 90%, resolving the primary technical issue of low circular RNA purity in the prior art. Furthermore, the overall recovery rate of the present method exceeds 50%, meeting GMP production requirements. In summary, the present method is suitable for widespread application.
[0162] Example 4. Preparation and purification of circular RNA with polyA added to the ends of linear RNA precursors and introns after in vitro transcription
[0163] A circular RNA template 0A′ without polyA was constructed as described in Example 1. PolyA was added to the uncircularized linear RNA precursor and the 3′ segment of the intron excised after circularization using polyA polymerase after in vitro transcription or RNA circularization.
[0164] In vitro transcription was performed using the method described in Example 2. Poly A was added to the ends of the linear RNA precursor and / or introns using poly A polymerase after filtration with lithium chloride or ultrafiltration. Affinity chromatography was performed using POROS oligo dT25, and the sample was then purified (Example 2, Steps 3-4).
[0165] The samples were tested by agarose gel electrophoresis and CGE. The results showed that adding polyA to the ends of linear RNA precursors and introns after in vitro transcription and then purification could also obtain high-purity circular RNA with high recovery rate.
[0166] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.
[0167] Appendix: Sequence Information
[0168] Contains the full-length sequence of in vitro transcription template 0A (SEQ ID NO: 1)
[0169] Contains the full-length sequence of in vitro transcription template 1A (SEQ ID NO: 2)
[0170] Contains the full-length sequence of in vitro transcription template 2A (SEQ ID NO: 3):
Claims
1. A method for preparing a target circular RNA in vitro by intron self-splicing, the method comprising: (a) providing a circular RNA in vitro transcription template, wherein the template comprises: a 3′-intron and a 5′-intron, and a target gene (GOI) located therebetween; (b) performing in vitro transcription using the template to form an in vitro transcription product; (c) allowing the in vitro transcription product to self-splice and circularize to form an in vitro circularized product; In any one or more steps of steps (a) to (c), the introns removed from the in vitro cyclization product and / or the uncyclized linear RNA precursor are poly-X-dylated, thereby carrying a poly-X (polyX) tag, wherein X is selected from the group consisting of adenylate (A), uridine (U), cytidylate (C) and guanylate (G); (d) contacting the in vitro cyclized product with a poly(X)-specific binding substance to remove the excised intron with a poly(X) tag and the uncyclized linear RNA precursor with a poly(X) tag in the product to obtain the target circular RNA.
2. The method according to claim 1, wherein The intron is a group I intron or a group II intron; and / or The polyX tag is located at one or both ends of the uncircularized linear RNA precursor or at the outer end of the intron away from the GOI; and / or The polyX tag is directly linked to the intron or the uncircularized linear RNA precursor or is linked via a linker (eg, a linker with a length of 10 bases or less).
3. The method according to claim 1, wherein The method comprises one or more selected from the following group, so that the ends of the cleaved introns and / or the uncircularized linear circular RNA precursors in the in vitro circularization product are provided with polyX tags: One or both ends of the in vitro transcription template in step (a) or the outer end of the intron away from the GOI is provided with a sequence corresponding to a polyX tag; In step (b), polyX polymerase or polynucleotide phosphorylase and corresponding nucleotide monomers are added so that the ends of the in vitro transcription products are tagged with polyX tags; and / or In step (c), polyX polymerase or polynucleotide phosphorylase and corresponding nucleotide monomers are added.
4. The method according to claim 3, wherein: The in vitro transcription template in step (a) is provided with a polyX tag corresponding sequence at one end or both ends or in an intron, wherein (I) The intron is a type I intron, and the circular RNA in vitro transcription template comprises the following elements: 3′-P1→PI1-I1→E2→GOI--→E1→I2-PI2→P2-5′ (A) in: I1 is the 3′-end group I intron; E2 is exon 2; GOI is the target gene; E1 is exon 1; I2 is the 5′-end group I intron; 3'-P1 is the sequence corresponding to the polyX tag at the 3' end or it does not exist; P2-5' is the sequence corresponding to the polyX tag at the 5' end or it does not exist; PI1 is the sequence corresponding to the polyX tag contained in intron I1 away from the GOI side or it does not exist; PI2 is the sequence corresponding to the polyX tag contained in intron I2 away from the GOI side or it does not exist. The condition is that at least one of 3′-P1, P2-5′, PI1 and PI2 (e.g., 1, 2, 3 or 4) is a sequence corresponding to a polyX tag; or (II) The intron is a type II intron, and the circular RNA in vitro transcription template comprises the following elements: 3′-P′1→PI1′-I1′→GOI′→I2′-PI2′→P′2-5′ (A) in: I1′ is the 3′-end group II intron; GOI is the target gene; I2′ is the 5′-end group II intron; 3′-P′1 is the sequence corresponding to the polyX tag at the 3′ end or it does not exist; P′2-5′ is the sequence corresponding to the polyX tag at the 5′ end or it does not exist; PI1′ is the sequence corresponding to the polyX tag contained in intron I1′ away from the GOI side or it does not exist; PI2′ is the sequence corresponding to the polyX tag contained in intron I2′ away from the GOI side or it does not exist. The condition is that at least one (e.g., 1, 2, 3, or 4) of 3′-P′1, P′2-5′, PI1′, and PI2′ is a sequence corresponding to a polyX tag.
5. The method according to claim 4, wherein: The template further comprises an element selected from the group consisting of an internal ribosome entry site (IRES), a transcriptional regulatory region (such as a promoter and / or enhancer), a spacer, a linker; and / or The polyX tag corresponding sequence is directly connected to the end of the template and / or the intron, or connected through a linker (e.g., a linker with a length of less than 10 bases); For example, the template comprises the sequence shown in SEQ ID NO: 1, but the GFP gene sequence therein is replaced with a selected target gene.
6. The method of claim 1, wherein: The polyX tag contains more than 6 X nucleotides, such as 6 to 250, 8 to 200, 10 to 100, 15 to 80, or 20 to 60 X nucleotides; and / or The target gene encodes one or more polypeptides or proteins or fusion peptides selected from the following groups: therapeutic polypeptides, immunogenic peptides, cytokines, transcription factors, immune checkpoint inhibitors, chimeric antigen receptors, and T cell receptors.
7. The method of claim 1, wherein: The polyA-specific binding substance is selected from the group consisting of: Oligo dT (e.g., POROS oligo dT25), polyA binding protein (PABP); The polyU-specific binding substance is selected from the group consisting of: polyadenylic acid, oligoadenylic acid, and polyU-binding protein; The polyC-specific binding substance is selected from the group consisting of: polyguanylate, oligoguanylate, and polyC-binding protein; The polyG-specific binding substance is selected from the group consisting of: polycytidylic acid, oligocytidylic acid, and polyG-binding protein; Preferably, the specific binding substance is immobilized, for example, immobilized on magnetic beads, columns, or chips.
8. The method of claim 1, wherein: The method further comprises one or more steps selected from the group consisting of: PCR amplification, plasmid fermentation, plasmid extraction, and plasmid linearization; after in vitro transcription and cyclization, precipitating, degrading, or isolating the uncyclized RNA, such as precipitating the uncyclized RNA with lithium chloride, enzymatically degrading the uncyclized RNA with RNase R, or performing tangential flow filtration and liquid exchange; Alternatively, the method does not include the use of RNase R enzyme.
9. An in vitro transcription template for preparing a target circular RNA in vitro by intron self-splicing, a vector comprising the template, or a product comprising the in vitro transcription template or vector: The template includes: 3′-intron and 5′-intron, and the gene of interest (GOI) located therebetween; Wherein, the template has polyX at one end or both ends or in the intron.
10. The in vitro transcription template or vector according to claim 9, wherein The template molecule is as defined in claims 2 to 6; and / or The vector is selected from the group consisting of: plasmid (eg, bacterial plasmid, yeast plasmid, such as pUC57), phage, viral vector (eg, lentivirus, adenovirus, adeno-associated virus vector), and PCR product.
11. A circular RNA or a product comprising a circular RNA or a precursor thereof, wherein: The circular RNA is prepared using the method of any one of claims 1 to 8 or using the in vitro transcription template or vector of claim 9 or 10.
12. The product of claim 11, wherein the circular RNA or product is selected from the group consisting of: for translation into functional polypeptides or proteins (e.g., for therapeutic or preventive purposes); for use as a molecular sponge for miRNA; for regulation of gene expression; for regulation of alternative splicing of parental genes; for formation of circular RNA-protein complexes to regulate signaling pathways; and / or For example, the product is selected from: a drug, a vaccine, a test or diagnostic kit; and / or The circular RNA preparation precursor is selected from: a circular RNA in vitro transcription template, in vitro transcription product and / or in vitro cyclization product as defined in any one of claims 1 to 8; and optionally, a poly (X) nucleotide-specific binding substance.