WO2024131232A1 - 环状rna分离和纯化方法 - Google Patents

环状rna分离和纯化方法 Download PDF

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WO2024131232A1
WO2024131232A1 PCT/CN2023/124875 CN2023124875W WO2024131232A1 WO 2024131232 A1 WO2024131232 A1 WO 2024131232A1 CN 2023124875 W CN2023124875 W CN 2023124875W WO 2024131232 A1 WO2024131232 A1 WO 2024131232A1
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intron
polyx
tag
template
rna
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French (fr)
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董小明
高荧荧
沈晨
陈小冰
李涛
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杭州明德生物医药技术有限公司
杭州明德生物新技术开发有限公司
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • This article belongs to the field of biotechnology and biopharmaceutical or vaccine production. Specifically, this application relates to the production and purification of circular RNA (e.g., mRNA vaccine), and achieves efficient isolation of 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.
  • circular RNA e.g., mRNA vaccine
  • mRNA vaccines have become the most promising and effective platform to fight a variety of diseases, including the COVID-19 pandemic.
  • mRNA vaccines currently include three main categories: traditional mRNA, self-amplifying RNA, and circular RNA (circRNA).
  • traditional mRNA traditional mRNA
  • self-amplifying RNA self-amplifying RNA
  • circular RNA circular RNA
  • circular RNA is increasingly favored by researchers and has great potential.
  • 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 doses. 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.
  • Circular RNA technology has obvious advantages in replacing mRNA, and its industrialization prospects are bright. How to achieve artificial preparation and purification of circular RNA is the key to the function of circular RNA.
  • RNA ligase method has disadvantages such as low reaction concentration, low ligation efficiency, and easy intermolecular connection during the cyclization reaction; while the ribozyme self-splicing method mainly realizes RNA cyclization through intron splicing, which currently mainly includes type I and type II intron splicing cyclization methods.
  • Type I introns are a type of intron with enzymatic catalytic function. After being transcribed into RNA, they can self-splice. This self-splicing of type I introns is catalyzed by the activity of nucleases of specific RNA sequences. In vitro, this self-splicing does not require the catalysis of any protein enzymes, but in vivo, it requires proteins to help fold into secondary structures.
  • RNA circularization by self-splicing of type I/II introns can achieve a very high circularization rate, a small amount of linear RNA still cannot be completely circularized during this process, and self-splicing is also generated.
  • the intron part of the RNA needs to be purified to remove the residual linear RNA precursor and the intron part spliced during the circularization process.
  • the most successful purification method reported in the literature is to first specifically digest the linear RNA with RNase R, and then further separate and purify the circular RNA through size exclusion chromatography (SEC). Since the molecular weight difference between the circular RNA product and its precursor RNA is small, 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 has a large demand for RNase R, which will have a huge cost burden, and the removal of RNase R also requires additional consideration.
  • SEC size exclusion chromatography
  • This article provides an efficient separation and purification method for in vitro synthesis of circular RNA molecules.
  • the method in this article solves the problems of difficult purification, low purity and low recovery rate of in vitro synthesized circular RNA.
  • a method for preparing a circular RNA of interest in vitro by intron self-splicing comprising:
  • a method for preparing a target circular RNA in vitro by intron self-splicing 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;
  • the introns cut out of the in vitro cyclization product and/or the uncyclized linear RNA precursor are poly-X-dysinated, thereby carrying a poly-X-nucleotide (polyX) tag, wherein the poly-X-nucleotide is selected from the group consisting of poly-adenylic acid (polyA), poly-uridylic acid (polyU), poly-cytidylic acid (polyC) and poly-guanylic acid (polyG);
  • 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
  • the template includes: a 3'-intron and a 5'-intron, and a target gene (GOI) located therebetween; wherein, one end or both ends of the template or the intron carries polyX.
  • GOI target gene
  • FIG. 1 Schematic diagram of the principles of some embodiments of the present application.
  • HMW is a high molecular weight polymer, including RNA concatemers formed between two RNA molecules.
  • FIG. 4 Capillary gel electrophoresis (CGE) results of samples prepared using a template without polyA before and after purification.
  • FIG. 5 Capillary gel electrophoresis (CGE) results of samples prepared using a template with polyA at one end before and after purification.
  • FIG. 6 Capillary gel electrophoresis (CGE) results of samples prepared using a template with polyA at both ends before and after purification.
  • the mRNA can be effectively separated by affinity chromatography oligo dT (e.g., POROS oligo dT25), and then further ultrafiltration concentration and liquid exchange to obtain a high-purity mRNA product.
  • affinity chromatography oligo dT e.g., POROS oligo dT25
  • further ultrafiltration concentration and liquid exchange to obtain a high-purity mRNA product.
  • polyX polynucleotide
  • the intron part of the circular RNA will be removed during the circularization process after in vitro transcription, and the polyA part added to the end can also be removed normally, so that the sequence of the target circular RNA is not affected (i.e., it does not contain the added polyA part), while the uncircularized circular RNA precursor and the intron part removed during the circularization process all have the polyA part, and then the linear precursor and the intron part can be easily and efficiently removed by oligo dT affinity chromatography (such as POROS oligo dT25).
  • polyA can be added to the ends of the linear RNA precursor and intron after in vitro transcription by polyA polymerase and the like to achieve the same purpose as adding polyA to the template before transcription as described above.
  • polynucleotide tags instead of polyA tags and using binding parts that can bind to these polynucleotide tags can also achieve similar effects as adding polyA tags.
  • the method of the present application can solve long-standing problems such as the difficulty in purifying in vitro synthesized circular RNA, low purity, and low recovery rate.
  • containing,” “having,” or “including” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”
  • the numerical range herein includes its endpoints and each specific numerical point and sub-range within the numerical range.
  • 1 to 3 includes endpoints 1 and 3, 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.).
  • One of the important raw materials for preparing circular RNA in vitro is the designed template DNA and its vector (e.g. plasmid DNA, pDNA).
  • the industrial production process of templates and their vectors has become mature, and suppliers can be outsourced or internal platforms can be established for preparation to obtain high-purity templates and vectors.
  • the vector used is a plasmid
  • its preparation process may include one or more steps such as fermentation, bacterial harvesting, alkaline lysis, clarification, ultrafiltration concentration, chromatography, etc.
  • chromatography may include Three-step purification: 1. Use molecular sieves (such as Sepharose 6FF fillers) to remove RNA; 2. Use thiophilic affinity fillers (such as Capto PlasmidSelect) to separate supercoiled plasmids; 3. Anion exchange chromatography (such as Capto Q ImpRes fillers) to remove trace impurities and endotoxins. This process has been verified for many years and is widely recognized as an efficient, universal and robust platform process route.
  • molecular sieves such as Sepharose 6FF fillers
  • thiophilic affinity fillers such as Capto PlasmidSelect
  • Anion exchange chromatography such as Capto Q ImpRes fillers
  • 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, for encoding therapeutic polypeptides, immunogenic peptides (such as the S protein or RBD region of coronaviruses), 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 connector or fusion of multiple or multiple polypeptides.
  • the full-length sequence of some circular RNA can be obtained from a website or data platform (e.g., circBase website) for use in constructing templates.
  • a conventional circular RNA template that does not contain a polyX corresponding sequence is directly used, and in the subsequent transcription and/or cyclization steps, the excised intron or the uncyclized circularized mRNA precursor is provided with a polyX tag.
  • a template is used that has a polynucleotide tag corresponding sequence added to one or both ends or the intron away from the GOI end.
  • polyX corresponding sequence refers to a sequence fragment in a DNA template that can form a polyX tag in mRNA after transcription.
  • 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 mainly one type of nucleotide molecule, such as mainly composed of A, U, G or C, but also containing a small amount of other types of nucleotide molecules, such as other types of nucleotides containing 1 to 20 non-main nucleotides.
  • the polyX corresponding sequence may be located in the template at one or more positions selected from the following group: one end of the template, both ends of the template, and the outer end of the intron (away from the target gene).
  • the vector can be linearized by methods known in the art, such as cutting with a restriction endonuclease.
  • the reaction enzyme can be removed (eg, by chromatography), and the buffer can be replaced with a reaction solution suitable for in vitro transcription.
  • the vector containing the template After obtaining the vector containing the template, it can be transcribed in vitro using methods known in the art. For example, T7 RNA polymerase can be used for in vitro transcription to obtain single-stranded RNA. After exogenous transcription, the DNA template is removed, for example using DNase.
  • the cyclization in the present application is carried out by in vitro intron self-splicing process.
  • Type I intron or type II intron self-splicing can be used as required.
  • 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 an uncircularized linear RNA precursor with a polyX tag, and subsequently these linear RNAs are removed by binding to the polyX tag to achieve enrichment of circular RNA.
  • the transcription product is polyX-tagged so that one or both ends of the transcription product contain a polyX tag, thereby making the linear intron and/or linear circular RNA precursor produced after intron self-splicing in vitro carry a polyX tag for subsequent removal of these linear RNAs to achieve enrichment of circular RNAs.
  • the polyX-tagged transcription product can be performed in an appropriate manner, such as using a polynucleotide polymerase or a polynucleotide phosphorylase to add a polyX tag to the end of the transcription product.
  • the linear intron and/or linear circular RNA precursor produced after in vitro intron self-splicing has a polyX tag at one or both ends for subsequent removal of these linear RNAs to achieve enrichment of circular RNAs.
  • the polyX-ization of the linear RNA molecules in the cyclization product can be performed in an appropriate manner, such as using a polynucleotide polymerase or a polynucleotide phosphorylase to add a polyX tag to the end of the linear 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 intron and linear circular RNA precursor) with a substance that specifically binds to polyX under conditions suitable for the binding of polyX to its specific binding substance to form a binding complex, and separating the binding complex from the cyclization reaction mixture.
  • the reaction system can be exchanged after the circularization step, for example using a tangential flow exchange (TFF) method.
  • RNase R can be added after the circularization step to remove some linear RNA.
  • the methods described herein include the steps of:
  • RNA in vitro transcription e.g., 37°C for 4 hours
  • a fluid exchange process e.g., TFF chromatography fluid exchange
  • the methods described herein include the steps of:
  • RNA in vitro transcription e.g., 37°C for 4 h
  • 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.
  • an in vitro transcription template of a circular RNA a vector comprising the template, and a product (e.g., a kit) comprising the template or the vector are provided.
  • a product e.g., a kit
  • These templates, vectors, and products can be used in the method of the present application to prepare a high-purity circular RNA of interest with a high recovery rate.
  • a circular RNA of interest and a product comprising the circular RNA of interest are provided, which are prepared using the methods, templates and vectors of the present application.
  • the uses of the circular RNA of interest or the product include, but are not limited to: for translation into functional polypeptides or proteins (such as for therapeutic or preventive purposes); for use as a molecular sponge for miRNA; for gene expression regulation; for regulating the selective splicing of parental genes; for forming a circular RNA-protein complex to regulate signaling pathways.
  • the product comprises a highly purified circular RNA of interest.
  • 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.
  • the circular RNA production process can be simplified by adding poly A to the end of the in vitro transcription template.
  • the method and product disclosed in this article can make the purity of circular RNA reach about 90%, and the total recovery rate exceeds 50%, which meets the GMP production requirements and is suitable for promotion and application.
  • a method for preparing a target circular RNA in vitro by intron self-splicing 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;
  • the introns cut out of the in vitro cyclization product and/or the uncyclized linear RNA precursor are poly-X-dysinated, thereby carrying a poly-X-nucleotide (polyX) tag, wherein X is selected from the group consisting of adenylic acid (A), uridylic acid (U), cytidylic acid (C) and guanylic acid (G);
  • 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;
  • step (b) polyX polymerase or polynucleotide phosphorylase and corresponding nucleotide monomers are added so that the end of the in vitro transcription product has a polyX tag;
  • step (c) polyX polymerase or polynucleotide phosphorylase and corresponding nucleotide monomers are added.
  • the intron is a type I intron, and the circular RNA in vitro transcription template comprises the following elements: 3′-P 1 ⁇ PI1-I1 ⁇ E2 ⁇ GOI ⁇ E1 ⁇ I2-PI2 ⁇ P 2 -5′ (A)
  • 3′-P 1 is the sequence corresponding to the polyX tag at the 3′ end or does not exist
  • P 2 -5′ is the sequence corresponding to the polyX tag at the 5′ end or does not exist
  • PI1 is the sequence corresponding to the polyX tag at the side away from the GOI contained in intron I1 or does not exist
  • PI2 is the sequence corresponding to the polyX tag at the side away from the GOI contained in intron I2 or does not exist.
  • the condition is that at least one (eg, 1, 2, 3 or 4) of 3′-P 1 , P 2 -5′, PI1 and PI2 is a sequence corresponding to a polyX tag.
  • 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)
  • I1′ is the type II intron at the 3′ end;
  • GOI is the target gene;
  • I2′ is the type II intron at the 5′ end;
  • 3′-P′ 1 is the sequence corresponding to the polyX tag at the 3′ end or does not exist
  • P′ 2 -5′ is the sequence corresponding to the polyX tag at the 5′ end or does not exist
  • PI1′ is the sequence corresponding to the polyX tag contained in intron I1′ away from the GOI side or does not exist
  • PI2′ is the sequence corresponding to the polyX tag contained in intron I2′ away from the GOI side or does not exist
  • the condition is that at least one (eg, 1, 2, 3 or 4) of 3′-P′ 1 , P′ 2 -5′, PI1′ and PI2′ is a sequence corresponding to a polyX tag.
  • IRS internal ribosome entry site
  • transcriptional regulatory region such as a promoter and/or enhancer
  • spacer such as a spacer and/or linker
  • step (a) The method of embodiment 4a or 4b, wherein the in vitro transcription template in step (a) has a sequence corresponding to a polyX tag at one or both ends or in an intron.
  • a linker e.g., a linker with a length of less than 10 bases
  • polyX tag comprises more than 6 X nucleotides, for example, 6 to 250, 8 to 200, 10 to 100, 15 to 80, or 20 to 60 X nucleotides.
  • 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.
  • polyA specific binding substance is selected from: Oligo dT (e.g., POROS oligo dT25), polyA binding protein (PABP);
  • PABP polyA binding protein
  • 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.
  • 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 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.
  • a target gene GOI
  • a plasmid e.g., a bacterial plasmid, a yeast plasmid
  • a viral vector e.g., a lentivirus, adenovirus, adeno-associated virus vector
  • PCR product e.g., a PCR product
  • RNA or product used for translation into functional polypeptides or proteins (such as for therapeutic or preventive purposes); used as a molecular sponge for miRNA; used for gene expression regulation; used for regulating the alternative splicing of parental genes; used for forming circular RNA-protein complexes to regulate signaling pathways.
  • RNA preparation precursor is selected from: a circular RNA in vitro transcription template, an in vitro transcription product and/or an in vitro cyclization product as defined in any one of embodiments 1 to 8; and optionally, a poly-X-nucleotide-specific binding substance.
  • the reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods.
  • the experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Michael R. Green et al., fourth edition, Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2017), or according to conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.
  • Example 1 Construction of in vitro transcription templates of circular RNA without or with polyA at the end
  • An in vitro transcription template 0A was constructed for preparing circular RNA by the type I intron self-splicing cyclization method.
  • the template expresses the GFP gene and does not contain polyA.
  • the entire sequence was synthesized according to SEQ ID NO: 1, and the total length of the template plasmid was approximately 3900bp.
  • the in vitro transcription template contains the following elements from 3' to 5':
  • the 3′-intron is a type I intron
  • IRES internal ribosome entry site
  • GOI target gene, specifically GFP gene
  • the 5′-intron is a type I intron.
  • Template 1A has the same structure as template 0A, except that 60A polyA is added at one end.
  • the entire sequence is synthesized according to SEQ ID NO: 2, and the total length of the template plasmid is about 3960bp.
  • Template 1A includes the following elements from 3' to 5':
  • Template 2A has the same structure as template 0A, except that 60A polyA is added at both ends.
  • the entire sequence is synthesized according to SEQ ID NO: 3, and the total length of the template plasmid is about 4020bp.
  • Template 2A includes the following elements from 3' to 5':
  • the template 0A, template 1A and template 2A prepared in Example 1 were respectively used to prepare circular RNA by in vitro transcription and purified.
  • the plasmid was amplified by bacterial fermentation (30 or 37°C, about 16 hours), and the plasmid was extracted using a plasmid extraction kit.
  • the plasmid was linearized by restriction endonuclease BspQ I (37°C, 2 hours), and the linearized plasmid was purified by ultrafiltration.
  • T7 RNA polymerase 7500U/mL
  • Pyrophosphatase Inorganic (yeast): 5 U/mL;
  • NTP solution (ATP, UTP, CTP, GTP): 7.5 mM (each);
  • Template linearized plasmid or PCR product: 50 ⁇ g/mL;
  • RNA circular RNA
  • Precursor RNA precursor
  • Intron intron
  • 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 prepared using the templates 0A, 1A and 2A in Example 1; and compared with the template 0A without polyA, the templates 1A and 2A containing polyA at one or both ends can more effectively separate impurities such as RNA precursors and introns, thereby obtaining a higher purity target circular RNA.
  • CGE-LIF Capillary gel electrophoresis
  • the CGE results of the samples obtained using templates 0A, 1A and 2A before and after purification are shown in Figures 4, 5 and 6, respectively.
  • the results show that the target circular RNA can be obtained after adding polyA, and the purity of the target RNA can be significantly improved after purification, and introns and high molecular weight (HMW) impurities in the product are almost completely removed.
  • HMW high molecular weight
  • the above CGE detection analysis shows that the method of the present application can make the purity of circular RNA reach about 90%.
  • the main technical problem of low purity of circular RNA in the prior art in this field is solved.
  • the total recovery rate of the method of this application has exceeded 50%, meeting the GMP production requirements.
  • the method of this application is suitable for promotion and application.
  • a circular RNA template 0A′ without polyA was constructed as described in Example 1, and polyA was added to the uncircularized linear RNA precursor and the 3′ segment of the intron excised after circularization using a polyA polymerase after in vitro transcription or after RNA circularization.
  • Example 2 In vitro transcription was performed using the method described in Example 2, and polyA was added to the ends of the linear RNA precursor and/or introns by polyA polymerase after filtration through lithium chloride or ultrafiltration. Affinity chromatography was performed using POROS oligo dT25, and the sample was then purified (Example 2 Steps 3-4).
  • the full-length sequence of the in vitro transcription template 2A (SEQ ID NO: 3) is as follows:

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Abstract

本公开提供了环状RNA分离和纯化方法。具体涉及通过内含子自剪接体外制备目的环状RNA的方法，其包括：(a)提供环状RNA体外转录模板；(b)用所述模板进行体外转录，形成体外转录产物；(c)使体外转录产物自剪接环化，形成体外环化产物；在步骤(a)～(c)中的任一个或多个步骤中，使体外环化产物中被切割下的内含子和/或未环化的线性RNA前体能够被聚X苷酸化，从而带有聚X苷酸(polyX，X＝A、U、C或G)标签；(d)使体外环化产物与聚X苷酸特异性结合物质接触，去除产物中带有polyX标签的被切割下的内含子和带有polyX标签的未环化的线性RNA前体，获得目的环状RNA。还提供了相应的产品及应用。

Description

环状RNA分离和纯化方法 技术领域
本文属于生物技术和生物药或疫苗生产领域。具体而言，本申请涉及环状RNA(例如mRNA疫苗)的生产纯化，通过在环状RNA生产过程中对体外转录模板、体外转录后的内含子和/或环状RNA前体添加polyA序列实现高效分离环状RNA。
背景技术
近年来，mRNA疫苗已成为对抗包括COVID-19大流行在内的多种疾病的最有希望和最有效的平台。随着对RNA的研究，目前mRNA疫苗主要包括三类：传统mRNA、自扩增RNA和环状RNA(circRNA)。其中，环状RNA越来越受到研究人员的青睐，具有极大的潜力。
环状RNA是一类特殊的非编码RNA分子，其在自然界中也有存在，更是RNA领域最新的研究热点。与传统的线性RNA(linear RNA，含5′和3′两个末端)不同，环状分子呈封闭环状结构，不易受RNA外切酶影响，表达更稳定，不易降解。并且，环状RNA还具有免疫原性低，所需剂量低等优点。另外，环状RNA疫苗在合成过程中不需要额外的加帽、碱基修饰等步骤，这也使得环状RNA生产成本更低。
环状RNA技术替代mRNA优势明显，产业化前景光明，如何实现环状RNA的人工制备和纯化是环状RNA发挥作用的关键。目前环状RNA体外合成方案主要包括两类：RNA连接酶法和核酶自剪接法。其中，通过RNA连接酶环化的方式在环化反应过程中存在反应浓度低、连接效率低、易产生分子间连接等缺点；而核酶自剪接的方式主要是通过内含子的剪接方式实现RNA的环化，目前主要包括I型和II型内含子剪接环化方式。
I型内含子是一类具有酶催化功能的内含子，转录成RNA后，可自我剪接。I型内含子这种自我剪接由RNA的特定序列的核酸内切酶的活性所催化，在体外该自我剪接不需要任何蛋白质酶的催化，而在体内则需要蛋白质帮助折叠成二级结构。
通过I/II型内含子自剪接完成方式进行RNA环化虽然能够实现非常高的环化率，但是此过程中仍有少量线性RNA无法完全环化，同时也会产生自剪接下 的内含子部分，由此需要通过纯化手段去除残余的线性RNA前体以及环化过程中被剪接下的内含子部分。
目前文献报道比较成功的纯化方式是先通过RNase R特异性消化线性RNA，再通过分子排阻色谱(SEC)进一步分离纯化环状RNA。由于环状RNA产物与其前体RNA分子量差异较小，分子排阻色谱的分离效果有限。其次，据了解，目前还未有GMP级别的RNase R，无法满足环状RNA生产的GMP要求；而且RNase R也会一定程度的降解目标环状RNA，造成环状RNA收率偏低。另外，此部分操作对RNase R的需求量较大会有极大的成本负担，同时RNase R的去除也是需要额外考虑。
综上所述，本领域中急需开发出能够用于环状RNA分子体外合成的高效分离纯化方法。
发明内容
本文中正是提供了一种可用于环状RNA分子体外合成的高效分离纯化方法。本文的方法解决体外合成环状RNA纯化困难、纯度低、回收率低等问题。
在本文的第一方面中，提供了一种通过内含子自剪接体外制备目的环状RNA的方法，所述方法包括：
一种通过内含子自剪接体外制备目的环状RNA的方法，所述方法包括：
(a)提供环状RNA体外转录模板，所述模板包括：3′-内含子和5′-内含子，以及位于其间的目的基因(GOI)；
(b)用所述模板进行体外转录，形成体外转录产物；
(c)使体外转录产物自剪接环化，形成体外环化产物；
在步骤(a)～(c)中的任一个或多个步骤中，使体外环化产物中被切割下的内含子和/或未环化的线性RNA前体能够被聚X苷酸化，从而带有聚X苷酸(polyX)标签，其中所述聚X苷酸选自：聚腺苷酸(polyA)、聚尿苷酸(polyU)、聚胞苷酸(polyC)和聚鸟苷酸(polyG)；
(d)使体外环化产物与聚X苷酸特异性结合物质接触，去除产物中带有polyX标签的被切割下的内含子和带有polyX标签的未环化的线性RNA前体，获得目的环状RNA。
在本文的一些方面中，提供了用于内含子自剪接体外制备目的环状RNA的体外转录模板、包含所述模板的载体、或包含所述体外转录模板或载体的产品，其中 所述模板包括：3′-内含子和5′-内含子，以及位于其间的目的基因(GOI)；其中，所述模板的一端或两端或内含子中带有polyX。
在本文的一些方面中，一种环状RNA或包含环状RNA或其制备前体的产品，其中，所述环状RNA其采用本文所述的方法制备或采用本文所述的体外转录模板或载体制备。
本领域的技术人员可对本文所述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本发明作进一步说明，其中这些显示仅为了图示说明本发明的实施方案，而不是为了局限本发明的范围。
图1：本申请一些实施方式的原理示意图。
图2：采用0A模板(图2A)、lA模板(图2B)和2A模板(图2C)的纯化色谱图；其中，FT＝流穿部分；Wash＝洗出部分；Elute＝洗脱部分。
图3：层析后各样品琼脂糖凝胶电泳图。其中，HMW为高分子量聚合物，包括由两个RNA分子间形成的RNA串联体。
图4：采用未添加polyA的模板制备环状RNA纯化前后的样品毛细管凝胶电泳(CGE)结果。
图5：采用一端加polyA的模板制备环状RNA纯化前后的样品毛细管凝胶电泳(CGE)结果。
图6：采用两端加polyA的模板制备环状RNA纯化前后的样品毛细管凝胶电泳(CGE)结果。
具体实施方式
在体外合成线性mRNA过程中，由于此类mRNA的3′端通常具有polyA结构(即polyA尾)，可以通过亲和层析oligo dT(例如POROS oligo dT25)有效分离mRNA，然后进一步经过超滤浓缩换液得到纯度较高的mRNA产品。然而本领域中从未公开在环状RNA生产过程中添加多聚核苷酸(polyX)纯化标签。
发明人突破常规思维中环状RNA无polyA结构，首创性地设计并尝试了在通过内含子自剪接方式环化来制备环状RNA的过程中，在体外转录模板末端(一 端或两端)或内含子外侧(远离GOI)部分添加polyA。结果显示环状RNA在体外转录后环化的过程中内含子部分会被切除，同时添加在末端的polyA部分也可以被正常切除，这样使得目标环状RNA的序列不受影响(即不含有添加的polyA部分)，而未环化的环状RNA前体以及环化过程中被切除的内含子部分均具有polyA部分，然后通过oligo dT亲和层析(例如POROS oligo dT25)即可轻松高效地去除线性前体和内含子部分。此外，还可在体外转录后，通过polyA聚合酶等方式在线性RNA前体和内含子末端加入polyA来实现与如前所述在转录前模板中加入polyA一样的目的。类似地，采用其他多聚核苷酸标签来代替polyA标签，并采用能够结合这些多聚核苷酸标签的结合部分，也能够实现与添加polyA标签类似的效果。研究证明了采用本申请的方法可解决体外合成环状RNA纯化困难、纯度低、回收率低等长期有待解决的问题。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。
本文中的数值范围包括其端点以及该数值范围内的各具体数值点和子范围。例如，1～3包括端点1和3、其中的具体整数数值点2和非整数数值点(例如但不限于：1.2、1.5、1.8、2.1、2.3、2.4、2.8等，以及其子范围(例如但不限于：1～2、2～3、1～1.2、1.5～1.8等)。
模板构建和线性化
体外制备环状RNA的重要原料之一是经设计的模板DNA及其载体(例如质粒DNA，pDNA)。模板及其载体的工业化生产工艺目前已经趋于成熟，可选择供应商外包或者建立内部平台进行制备，以得到高纯度模板和载体。
在一些实施方式中，所用载体为质粒，其制备工艺过程可包括发酵、菌体收获、碱裂解、澄清、超滤浓缩、层析等一个或多个步骤。例如，层析可包含 三步纯化：1.用分子筛(例如包含Sepharose 6FF填料)除去RNA；2.采用嗜硫亲和填料(例如Capto PlasmidSelect)分离超螺旋的质粒；3.阴离子交换层析(例如采用Capto Q ImpRes填料)去除痕量杂质和内毒素。该工艺经多年验证，得到普遍认可，是高效、通用、稳健的平台化工艺路线。
可根据需要在载体(如质粒)中插入所需的目的基因。可用于本文中的目的基因包括但不限于：用于编码治疗性多肽、免疫原性肽(例如冠状病毒的S蛋白或RBD区)、细胞因子、转录因子、免疫检查点抑制剂、嵌合抗原受体、T细胞受体等。所编码的多肽或蛋白质可以是单一的多肽分子、多个或多种多肽的连接体或融合体。
某些环状RNA的全长序列可从网站或数据平台(例如circBase网站)获得，以用于构建模板。在一些实施方式中，直接采用常规的不包含polyX对应序列的环状RNA模板，而在后续的转录和/或环化步骤中使得切割下的内含子或未环化的环化mRNA前体带有polyX标签。在另一些实施方式中，采用在其一端或两端或内含子远离GOI端增加了多聚核苷酸标签对应序列的模板。
在一些优选实施方式中，采用了包含polyX对应序列的特殊设计的模板。如本文所用，“polyX对应序列”是指DNA模板中经转录可在mRNA中形成polyX标签的序列片段。
在一些实施方式中，polyX对应序列可对应于仅由一种核苷酸分子聚合而成的polyX，例如完全由A、U、G或C组成的多聚核苷酸。在一些实施方式中，polyX对应序列可对应于主要由一种核苷酸分子聚合而成的polyX，例如主要由A、U、G或C组成，但也包含少量其他类型的核苷酸分子，例如包含1～20个非主要核苷酸的其他类型核苷酸。polyX对应序列可位于模板中选自下组中的一个或多个位置：模板的一个末端、模板的两个末端、内含子(远离目的基因)外侧端。
可采用本领域中已知的方法对载体进行线性化，例如采用限制性内切酶切割。待反应完成后，可去除反应酶(例如通过层析)，并将缓冲液置换为适于体外转录的反应溶液。
体外转录、环化与环状RNA富集
在获得了包含模板的载体后可采用本领域已知的方法对其进行体外转录。例如，可采用T7 RNA聚合酶进行体外转录，以得到单链RNA。优选地，在体 外转录后去除DNA模板，例如采用DNA酶。
本申请中的环化通过体外内含子自剪接过程进行。可根据需要采用I型内含子或II型内含子自剪接方式。
在一些实施方式中，采用如本文所述包含polyX标签对应序列的转录模板进行转录，以获得包含polyX标签的转录产物，并进而在体外内含子自剪接后获得在相应位置带有polyX标签的被切割下的线性内含子和/或带有polyX标签的未环化线性RNA前体，后续通过与polyX标签的结合来去除这些线性RNA，实现对环状RNA的富集。
在一些实施方式中，对转录产物进行polyX化，使转录产物的一端或两端包含polyX标签，进而使得在体外内含子自剪接后产生的线性内含子和/或线性的环状RNA前体带有polyX标签，用于后续去除这些线性RNA，实现对环状RNA的富集。转录产物的polyX化可采用适当的方式进行，例如采用多核苷酸聚合酶或采用多核苷酸磷酸化酶在转录产物末端添加polyX标签。
在一些实施方式中，在环化反应后，使得在体外内含子自剪接后产生的线性内含子和/或线性的环状RNA前体的一端或两端带有polyX标签，用于后续去除这些线性RNA，实现对环状RNA的富集。环化产物中线性RNA分子的polyX化可采用适当的方式进行，例如采用多核苷酸聚合酶或采用多核苷酸磷酸化酶在线性RNA的末端添加polyX标签。
获得带有polyX标签的线性RNA与目标环状RNA的混合物后，可通过polyX标签与其特异性结合物的结合来去除带有polyX标签的线性RNA(包括线性内含子片段和/或线性环状RNA前体)。去除不为所需线性RNA分子的方法包括：在适于polyX与其特异性结合物质结合的条件下，使带有polyX标签的线性RNA(例如线性内含子和线性的环状RNA前体)与特异性结合polyX的物质接触形成结合复合物，并将所述结合复合物从环化反应混合物中分离。
可任选地，可在环化步骤后可对反应体系进行换液，例如采用切向流换液(TFF)法。可任选地，可在环化步骤后加入RNase R以去除部分线性RNA。
在一些实施方式中，本文所述的方法包括如下步骤：
1.构建环状RNA(I型或II型内含子自剪接环化)体外转录模板，其末端(一端或两端)包含polyX标签对应序列；
2.RNA体外转录(例如，37℃反应4h)；
3.内含子自剪接环化；
4.可任选地，进行换液处理(例如TFF层析换液)；
5.进行针对polyX的结合和结合产物分离，例如亲和层析(如针对polyA标签采用POROS oligo dT25)；
6.可任选地，进行TFF层析换液。
在一些实施方式中，本文所述的方法包括如下步骤：
1′.构建环状RNA(I型或II型内含子自剪接环化)体外转录模板；
2′.RNA体外转录(例如，37℃反应4h)；
3′.内含子自剪接环化；
4′.加入polyX聚合酶，以在线性RNA(如剪切下的内含子、未环化的环状RNA前体)末端加上polyX标签；
5′.可任选地，进行换液处理(例如TFF层析换液)；
6′.进行针对polyX的结合和结合产物分离，例如亲和层析(如针对polyA标签采用POROS oligo dT25)；
7′.可任选地，TFF层析换液。
产品及应用
本申请的体外转录模板、载体和方法可用于通过内含子自剪接在体外制备各种目的环状RNA以及包含目的环状RNA的产品。
在一些实施方式中，提供了环状RNA的体外转录模板、包含所述模板的载体，以及包含所述模板或载体的产品(例如试剂盒)。这些模板、载体和产品可用于本申请的方法中以高回收率制备高纯度目的环状RNA。
在一些实施方式中，提供了目的环状RNA以及包含目的环状RNA的产品，其采用本申请的方法、模板和载体制备。在一些实施方式中，目的环状RNA或产品的用途包括但不限于：用于翻译成功能多肽或蛋白质(如用于治疗或预防目的)；用于作为miRNA的分子海绵；用于基因表达调控；用于调控亲本基因的选择性剪接；用于形成环形RNA-蛋白质复合体调控信号通路。在一些实施方式中，所述产品包含高纯度的目的环状RNA。
在一些实施方式中，目的环状RNA的纯度不低于70％、不低于75％、不低于80％、不低于85％、不低于90％、不低于92％、不低于95％、不低于98％、不低于99％，或为其中的任意数值或数值范围。
综上，通过在体外转录模板末端添加poly A(多聚A)，简化环状RNA生产工 艺，提高环状RNA纯化回收率和纯度。经检测分析，本文所公开的方法和产品可以使环状RNA纯度达到90％左右，总回收率超过50％，满足GMP生产要求，适宜推广应用。
示例性实施方式
本申请中提供了如下的实施方式。应理解，本领域技术人员可对本发明做出适当的修改、变动、组合，这些修改、变动和组合都在本发明的范围之内。
1.一种通过内含子自剪接体外制备目的环状RNA的方法，所述方法包括：
(a)提供环状RNA体外转录模板，所述模板包括：3′-内含子和5′-内含子，以及位于其间的目的基因(GOI)；
(b)用所述模板进行体外转录，形成体外转录产物；
(c)使体外转录产物自剪接环化，形成体外环化产物；
在步骤(a)～(c)中的任一个或多个步骤中，使体外环化产物中被切割下的内含子和/或未环化的线性RNA前体能够被聚X苷酸化，从而带有聚X苷酸(polyX)标签，其中X选自：腺苷酸(A)、尿苷酸(U)、胞苷酸(C)和鸟苷酸(G)；
(d)使体外环化产物与聚X苷酸特异性结合物质接触，去除产物中带有polyX标签的被切割下的内含子和带有polyX标签的未环化的线性RNA前体，获得目的环状RNA。
2a.如实施方式1所述的方法，其中，所述内含子为I型内含子或II型内含子。
2b.如实施方式1所述的方法，其中，所述polyX标签位于未环化的线性RNA前体的一端或两端或位于内含子远离GOI的外侧端。
2c.如实施方式1所述的方法，其中，所述polyX标签与内含子或未环化的线性RNA前体直接连接或通过连接子连接。
2d.如实施方式2c所述的方法，其中，所述连接子的长度为10个碱基以下。
3.如实施方式1所述的方法，其中，所述方法包括选自下组的一种或多种，以使得体外环化产物中被切割的内含子和/或未环化的线性环状RNA前体的末端带有polyX标签：
使步骤(a)中的体外转录模板的一端或两端或者内含子远离GOI的外侧端带有polyX标签对应序列；和/或
在步骤(b)中加入polyX聚合酶或多核苷酸磷酸化酶以及相应的核苷酸单体，使得所述体外转录产物的末端带有polyX标签；和/或
在步骤(c)中加入polyX聚合酶或多核苷酸磷酸化酶以及相应的核苷酸单体。
4a.如实施方式3所述的方法，其中，使步骤(a)中的体外转录模板的一端或两端或者内含子中带有polyX标签对应序列，其中
(I)所述内含子为I型内含子，且所述环状RNA体外转录模板包含如下元件：
3′-P1→PI1-I1→E2→GOI→E1→I2-PI2→P2-5′    (A)
其中：
I1为3′端的I型内含子；E2为外显子2；GOI为目的基因；E1为外显子1；I2为5′端的I型内含子；
3′-P1为3′末端的polyX标签对应序列或不存在；P2-5′为5′末端的polyX标签对应序列或不存在；PI1为内含子I1所含的远离GOI侧的polyX标签对应序列或不存在；PI2为内含子I2所含的远离GOI侧的polyX标签对应序列或不存在，
条件是：3′-P1、P2-5′、PI1和PI2中的至少一个(例如1、2、3或4个为polyX标签对应序列。
4b.如实施方式3所述的方法，其中，使步骤(a)中的体外转录模板的一端或两端或者内含子中带有polyX标签对应序列，其中
(II)所述内含子为II型内含子，且所述环状RNA体外转录模板包含如下元件：
3′-P′1→PI1′-I1′→GOI′→I2′-PI2′→P′2-5′    (A)
其中：
I1′为3′端的II型内含子；GOI为目的基因；I2′为5′端的II型内含子；
3′-P′1为3′末端的polyX标签对应序列或不存在；P′2-5′为5′末端的polyX标签对应序列或不存在；PI1′为内含子I1′所含的远离GOI侧的polyX标签对应序列或不存在；PI2′为内含子I2′所含的远离GOI侧的polyX标签对应序列或不存在，
条件是：3′-P′1、P′2-5′、PI1′和PI2′中的至少一个(例如1、2、3或4个为polyX标签对应序列。
5a.如实施方式4a或4b所述的方法，其中，所述模板还包含选自下组的元件：内部核糖体进入位点(IRES)、转录调控区(如启动子和/或增强子)、间隔子、接头。
5b.如实施方式4a或4b所述的方法，其中，使步骤(a)中的体外转录模板的一端或两端或者内含子中带有polyX标签对应序列。
5c.如实施方式4a或4b所述的方法，其中，所述polyX标签对应序列与模板的末端和/或内含子直接连接，或通过接头(例如长度为10个碱基以下的接头)连 接。
6a.如实施方式1所述的方法，其中，所述polyX标签包含6个以上X苷酸，例如6～250个、8～200个、10～100个、15-80个、20-60个X苷酸。
6b.如实施方式1所述的方法，其中，所述目的基因编码选自下组的一种或多种多肽或蛋白质或融合肽：治疗性多肽、免疫原性肽、细胞因子、转录因子、免疫检查点抑制剂、嵌合抗原受体、T细胞受体。
7a.如实施方式1所述的方法，其中，polyA特异性结合物质选自：Oligo dT(例如POROS oligo dT25)、polyA结合蛋白(PABP)；polyU特异性结合物质选自：多聚腺苷酸、寡聚腺苷酸、polyU结合蛋白；polyC特异性结合物质选自：多聚鸟苷酸、寡聚鸟苷酸、polyC结合蛋白；polyG特异性结合物质选自：多聚胞苷酸、寡聚胞苷酸、polyG结合蛋白。
7b.如实施方式1所述的方法，其中，所述特异性结合物质是固定化的，例如固定于磁珠、柱、芯片。
8a.如实施方式1所述的方法，其中，所述方法还包括选自下组的一个或多个步骤：PCR扩增、质粒发酵、质粒提取、质粒线性化；体外转录和环化后，沉淀、降解或分离未环化的RNA，例如用氯化锂沉淀未环化的RNA、用RNase R酶促降解未环化的RNA、进行切向流过滤换液。
8b.如实施方式1所述的方法，其中，所述方法中不包括使用RNase R酶。
9.一种用于内含子自剪接体外制备目的环状RNA的体外转录模板、包含所述模板的载体、或包含所述体外转录模板或载体的产品：所述模板包括：3′-内含子和5′-内含子，以及位于其间的目的基因(GOI)；其中，所述模板的一端或两端或内含子中带有polyX。
10a.如实施方式9所述的体外转录模板或载体，其中，所述模板分子如实施方式2～6中所限定。
10b.如实施方式9所述的体外转录模板或载体，其中，所述载体选自：质粒(例如细菌质粒、酵母质粒)、噬菌体、病毒载体(例如慢病毒、腺病毒、腺相关病毒载体)、PCR产物。
11.一种环状RNA或包含环状RNA或其制备前体的产品，其中，所述环状RNA采用实施方式1-8中任一项所述的方法制备或采用实施方式9或10所述的体外转录模板或载体制备。
12a.如实施方式11所述的产品，所述环状RNA或产品选自用于如下应用环状 RNA或产品：用于翻译成功能多肽或蛋白质(如用于治疗或预防目的)；用于作为miRNA的分子海绵；用于基因表达调控；用于调控亲本基因的选择性剪接；用于形成环形RNA-蛋白质复合体调控信号通路。
12b.如实施方式11所述的产品，其中，所述产品选自：药物、疫苗、检测或诊断试剂盒。
12c.如实施方式11所述的产品，其中所述环状RNA制备前体选自：如实施方式1-8中任一项中所限定的环状RNA体外转录模板、体外转录产物和/或体外环化产物；以及可任选地，聚X苷酸特异性结合物质。
实施例
下面结合具体实施例和附图，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
本发明所用试剂和原料均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Michael R.Green等人第四版，纽约，冷泉港实验室出版社《分子克隆：实验室指南》(New York：Cold Spring Harbor Laboratory Press，2017)中所述的条件，或按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本申请中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1.末端不含或包含polyA的环状RNA体外转录模板的构建
制备具有如下结构的环状RNA体外转录模板：
模板0A
构建用于I型内含子自剪接环化法制备环状RNA的体外转录模板0A，该模板表达GFP基因，不含polyA；根据SEQ ID NO：1进行全序列合成，模板质粒全长约3900bp。
该体外转录模板从3′至5′包含如下元件：
3′-内含子→E2→IRES→GOI→E1→5′-内含子
其中：
3′-内含子为I型内含子；
E2＝外显子2；
IRES＝内部核糖体进入位点；
GOI＝目的基因，具体为GFP基因；
E1＝外显子1；
5′-内含子为I型内含子。
模板1A
构建一端包含polyA的、用于I型内含子自剪接环化法制备环状RNA的体外转录模板，加60A。模板1A与模板0A结构相同，除了在一端添加了60A的polyA。根据SEQ ID NO：2进行全序列合成，模板质粒全长约3960bp。
模板1A从3′至5′包含如下元件：
3′-内含子→E2→IRES→GOI→E1→5′-内含子→5′-polyA
模板2A
构建两端均包含polyA的、用于I型内含子自剪接环化法制备环状RNA的体外转录模板，两端各加60A。模板2A与模板0A结构相同，除了在两端添加了各60个A的polyA。根据SEQ ID NO：3进行全序列合成，模板质粒全长约4020bp。
模板2A从3′至5′包含如下元件：
3′-polyA→3′-内含子→E2→IRES→GOI→E1→5′-内含子→5′-polyA
实施例2.环状RNA的制备与纯化
分别采用实施例1中制备的模板0A、模板1A和模板2A，通过体外转录制备环状RNA，并对其进行纯化。
1.质粒扩增、质粒线性化和线性化质粒纯化
通过细菌发酵扩增质粒(30或37℃，约16小时)，通过质粒提取试剂盒提取质粒。通过限制性内切酶BspQ I线性化质粒(37℃，2小时)，线性化质粒通过超滤进行纯化。
2.体外转录
分别采用模板0A、1A和2A，进行RNA体外转录。体外转录的条件为37℃下反应4小时。反应组合物如下：
T7 RNA聚合酶：7500U/mL；
鼠RNase抑制剂：2000U/mL；
焦磷酸酶(Pyrophosphatase，Inorganic(yeast))：5U/mL；
NTP溶液(ATP、UTP、CTP、GTP)：7.5mM(每种)；
模板(线性化质粒或PCR产物)：50μg/mL；
体外转录反应缓冲液：1×
2.通过氯化锂沉淀RNA，使用RNase Free水溶解RNA，反应体积较大以及GMP生产时通过切向流过滤TFF换液完成；
3.采用POROS oligo dT25进行亲和层析，以0.25M NaCl和1xTE上样，相同条件清洗，收集流穿(FT)部分和洗出(Wash)部分作为纯化样品，洗脱液为1xTE。
4.纯化样品(即步骤3中的流穿和清洗部分)，用TFF层析换液至1xTE。
5.琼脂糖凝胶电泳检测上样液、流穿部分、清洁部分以及洗脱液中所含的环状RNA(cirRNA)、RNA前体(Precursor)和内含子(Intron)。
层析图谱如图2所示、层析后各样品的琼脂糖凝胶电泳结果如图3所示。结果显示，采用实施例1中的模板0A、1A和2A均制得了所需的环状RNA；并且，与不含polyA的模板0A相比，采用一端或两端包含polyA的模板1A和2A可更有效地分离RNA前体和内含子等杂质，获得更高纯度的目标环状RNA。
实施例3.毛细管凝胶电泳法(CGE-LIF)分析环状RNA的纯度
采用毛细管凝胶电泳法CGE-LIF对实施例2中亲和层析纯化前、后的样品进行分析。
采用模板0A、1A和2A所得样品在纯化前、后的CGE结果分别如图4、图5和图6所示。结果显示：添加polyA后可制得目标环状RNA，且在纯化后能显著提供目标RNA的纯度，几乎完全去除了产物中的内含子和高分子量(HMW)杂质。
表1.CGE结果：各组分占比

表2.CGE统计结果：

*上表中：load＝上样样品；FT＝流穿部分；Wash＝洗出部分；Elute＝洗脱部分；HMW＝
高分子量杂质；Oligo dT25＝POROSTM Oligo(dT)25亲和纯化填料(赛默飞，含25-mer poly-dT)；
以上CGE检测分析显示：采用本申请的方法可使环状RNA纯度达到90％左 右，解决了本领域现有技术中环状RNA纯度低的主要技术问题。并且，本申请方法的总回收率已超过50％，满足GMP生产要求。综上，本申请的方法适宜推广应用。
实施例4.体外转录后在线性RNA前体和内含子末端加入polyA的环状 RNA制备与纯化
如实施例1中所述构建不带polyA的环状RNA模板0A′，在体外转录后或RNA环化后使用polyA聚合酶在未环化线性RNA前体和环化后切除的内含子3’段添加polyA。
采用实施例2中所述方法进行体外转录，经氯化锂或超滤渗滤后通过polyA聚合酶在线性RNA前体和/或内含子末端加入polyA。用POROS oligo dT25进行亲和层析，然后纯化样品(实施例2步骤3-4)。
采用琼脂糖凝胶电泳和CGE对所得样品进行检测，结果显示体外转录后在线性RNA前体和内含子末端加入polyA然后进行纯化也能以高回收率获得高纯度的环状RNA。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
附录：序列信息
包含体外转录模板0A的全长序列(SEQ ID NO：1)


包含体外转录模板1A的全长序列(SEQ ID NO：2)

包含体外转录模板2A的全长序列(SEQ ID NO：3)：


Claims (12)

  1. 一种通过内含子自剪接体外制备目的环状RNA的方法，所述方法包括：
    (a)提供环状RNA体外转录模板，所述模板包括：3′-内含子和5′-内含子，以及位于其间的目的基因(GOI)；
    (b)用所述模板进行体外转录，形成体外转录产物；
    (c)使体外转录产物自剪接环化，形成体外环化产物；
    在步骤(a)～(c)中的任一个或多个步骤中，使体外环化产物中被切割下的内含子和/或未环化的线性RNA前体能够被聚X苷酸化，从而带有聚X苷酸(polyX)标签，其中X选自：腺苷酸(A)、尿苷酸(U)、胞苷酸(C)和鸟苷酸(G)；
    (d)使体外环化产物与聚X苷酸特异性结合物质接触，去除产物中带有polyX标签的被切割下的内含子和带有polyX标签的未环化的线性RNA前体，获得目的环状RNA。
  2. 如权利要求1所述的方法，其中，所述内含子为I型内含子或II型内含子；和/或
    所述polyX标签位于未环化的线性RNA前体的一端或两端或位于内含子远离GOI的外侧端；和/或
    所述polyX标签与内含子或未环化的线性RNA前体直接连接或通过连接子(例如长度为10个碱基以下的接头)连接。
  3. 如权利要求1所述的方法，其中，所述方法包括选自下组的一种或多种，以使得体外环化产物中被切割的内含子和/或未环化的线性环状RNA前体的末端带有polyX标签：
    使步骤(a)中的体外转录模板的一端或两端或者内含子远离GOI的外侧端带有polyX标签对应序列；
    在步骤(b)中加入polyX聚合酶或多核苷酸磷酸化酶以及相应的核苷酸单体，使得所述体外转录产物的末端带有polyX标签；和/或
    在步骤(c)中加入polyX聚合酶或多核苷酸磷酸化酶以及相应的核苷酸单体。
  4. 如权利要求3所述的方法，其中，使步骤(a)中的体外转录模板的一端或两 端或者内含子中带有polyX标签对应序列，其中
    (I)所述内含子为I型内含子，且所述环状RNA体外转录模板包含如下元件：
    3′-P1→PI1-I1→E2→GOI--→E1→I2-PI2→P2-5′    (A)
    其中：
    I1为3′端的I型内含子；E2为外显子2；GOI为目的基因；E1为外显子1；I2为5′端的I型内含子；
    3′-P1为3′末端的polyX标签对应序列或不存在；P2-5′为5′末端的polyX标签对应序列或不存在；PI1为内含子I1所含的远离GOI侧的polyX标签对应序列或不存在；PI2为内含子I2所含的远离GOI侧的polyX标签对应序列或不存在，
    条件是：3′-P1、P2-5′、PI1和PI2中的至少一个(例如1、2、3或4个为polyX标签对应序列；或者
    (II)所述内含子为II型内含子，且所述环状RNA体外转录模板包含如下元件：
    3′-P′1→PI1′-I1′→GOI′→I2′-PI2′→P′2-5′    (A)
    其中：
    I1′为3′端的II型内含子；GOI为目的基因；I2′为5′端的II型内含子；
    3′-P′1为3′末端的polyX标签对应序列或不存在；P′2-5′为5′末端的polyX标签对应序列或不存在；PI1′为内含子I1′所含的远离GOI侧的polyX标签对应序列或不存在；PI2′为内含子I2′所含的远离GOI侧的polyX标签对应序列或不存在，
    条件是：3′-P′1、P′2-5′、PI1′和PI2′中的至少一个(例如1、2、3或4个为polyX标签对应序列。
  5. 如权利要求4所述的方法，其中，所述模板还包含选自下组的元件：内部核糖体进入位点(IRES)、转录调控区(如启动子和/或增强子)、间隔子、接头；和/或
    所述polyX标签对应序列与模板的末端和/或内含子直接连接，或通过接头(例如长度为10个碱基以下的接头)连接；
    例如，所述模板包含SEQ ID NO：1所示的序列，但其中的GFP基因序列用所选的目的基因替代。
  6. 如权利要求1所述的方法，其中，所述polyX标签包含6个以上X苷酸，例如6～250个、8～200个、10～100个、15-80个、20-60个X苷酸；和/或
    所述目的基因编码选自下组的一种或多种多肽或蛋白质或融合肽：治疗性多肽、免疫原性肽、细胞因子、转录因子、免疫检查点抑制剂、嵌合抗原受体、T细胞受体。
  7. 如权利要求1所述的方法，其中，polyA特异性结合物质选自：Oligo dT(例如POROS oligo dT25)、polyA结合蛋白(PABP)；
    polyU特异性结合物质选自：多聚腺苷酸、寡聚腺苷酸、polyU结合蛋白；
    polyC特异性结合物质选自：多聚鸟苷酸、寡聚鸟苷酸、polyC结合蛋白；
    polyG特异性结合物质选自：多聚胞苷酸、寡聚胞苷酸、polyG结合蛋白；
    优选，所述特异性结合物质是固定化的，例如固定于磁珠、柱、芯片。
  8. 如权利要求1所述的方法，其中，所述方法还包括选自下组的一个或多个步骤：PCR扩增、质粒发酵、质粒提取、质粒线性化；体外转录和环化后，沉淀、降解或分离未环化的RNA，例如用氯化锂沉淀未环化的RNA、用RNase R酶促降解未环化的RNA、进行切向流过滤换液；
    或者，所述方法中不包括使用RNase R酶。
  9. 一种用于内含子自剪接体外制备目的环状RNA的体外转录模板、包含所述模板的载体、或包含所述体外转录模板或载体的产品：
    所述模板包括：3′-内含子和5′-内含子，以及位于其间的目的基因(GOI)；
    其中，所述模板的一端或两端或内含子中带有polyX。
  10. 如权利要求9所述的体外转录模板或载体，其中，所述模板分子如权利要求2～6中所限定；和/或
    所述载体选自：质粒(例如细菌质粒、酵母质粒，如pUC57)、噬菌体、病毒载体(例如慢病毒、腺病毒、腺相关病毒载体)、PCR产物。
  11. 一种环状RNA或包含环状RNA或其制备前体的产品，其中，所述环状RNA采用权利要求1-8中任一项所述的方法制备或采用权利要求9或10所述的体外转录模板或载体制备。
  12. 如权利要求11所述的产品，所述环状RNA或产品选自用于如下应用环状RNA或产品：用于翻译成功能多肽或蛋白质(如用于治疗或预防目的)；用于作为miRNA的分子海绵；用于基因表达调控；用于调控亲本基因的选择性剪接；用于形成环形RNA-蛋白质复合体调控信号通路；和/或
    例如，所述产品选自：药物、疫苗、检测或诊断试剂盒；和/或
    所述环状RNA制备前体选自：如权利要求1-8中任一项中所限定的环状RNA体外转录模板、体外转录产物和/或体外环化产物；以及可任选地，聚X苷酸特异性结合物质。
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