One-step synthesis of circular RNA

Abstract

A method for preparing circular RNA is provided, the method comprising providing a template DNA containing a sequence encoding a precursor RNA in a reaction solution that enables in vitro transcription of the template DNA to synthesize the precursor RNA, and causing the precursor RNA to self-splice to produce a circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing (i.e., circularization) of the precursor RNA are carried out in the same reaction solution and under the same reaction conditions (e.g., the same reaction temperature). The method can be carried out in a single reaction vessel and does not require a step of purifying the precursor RNA before allowing the precursor RNA to self-splice.

Description

[Technical Field]

[0001] The present disclosure relates to circular RNAs and methods of making and using circular RNAs. [Background technology]

[0002] Messenger RNA (mRNA) is a type of single-stranded RNA involved in protein synthesis. Recently, in vitro transcribed (IVT) mRNA has attracted considerable attention as a novel drug with potential for therapeutic benefit. In particular, the successful use of mRNA vaccines against COVID-19 has demonstrated their in vivo safety and efficacy. However, the use of mRNA in non-vaccine therapies, such as protein supplementation, is limited by several factors, including mRNA stability, in vivo expression persistence, immunogenicity, and the type of cell in which it is expressed.

[0003] Circular RNAs (circRNAs) have emerged as therapeutic agents. CircRNAs are a type of single-stranded RNA that form a 3'-5' covalently closed loop. CircRNAs are created by a non-canonical splicing process called "backsplicing," in which the spliceosome fuses a splice donor site of a downstream exon with a splice acceptor site of an upstream exon. Although circRNAs are generally non-coding, several studies have provided evidence that some circRNAs can be translated into proteins. Unlike linear mRNAs, circRNAs do not require a 5' cap or 3' poly(A) tail for stability. Furthermore, circRNAs have beneficial features not found in mRNAs, such as reduced immunogenicity and a longer translation period. For these reasons, circRNAs have been investigated as therapeutic agents.

[0004] Researchers have developed several methods for ligating the ends of linear RNA precursors into closed circular RNAs in vitro. The most commonly used methods include enzymatic ligation and ribozyme ligation. Enzymatic ligation circularization typically requires complementary splints (DNA or RNA oligos) to bring the ends of the RNA molecule into close proximity. Ligation is catalyzed by enzymes from the bacteriophage T4, such as T4 DNA ligase, T4 RNA ligase 1, and T4 RNA ligase 2. However, none of these ligase-mediated circularization methods are sufficiently efficient, especially for large RNA molecules. Furthermore, the generation of intermolecular end-joining byproducts in the ligation reaction cannot be completely avoided, leading to complex system optimization and difficult scale-up for production.

[0005] Alternatively, circular RNAs can be generated by ribozyme ligation. Ribozyme-mediated RNA circularization is typically performed by the permuted intron and exon (PIE) method, which is based on a modified group I intron self-splicing system. Group I introns are large self-splicing ribozymes. Natural group I introns do not require the assistance of spliceosomes or other proteins for self-splicing; instead, they rely on magnesium and free guanosine nucleotides to initiate and complete the reaction. This process ligates the exon adjacent to the intron, resulting in internal intron circularization and generating intronic circRNAs. Previous studies have designed PIE systems using modified group I introns, such as by placing the 5' half of a group I intron at the end of an exon and moving the remaining 3' half to the beginning of the same exon (Puttaraju et al., 1992, "Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons," Nucleic Acids Res 20, 5357-5364; Wesselhoeft et al., 2018, "Engineering circular RNA for potent and stable translation in eukaryotic cells," Nature Communications 9, 2629). This method achieves RNA circularization through a standard group I intron self-splicing reaction, which involves two transesterifications at the defined splice site. Attack of free GTP at the 5' splice site releases the 3'-terminal sequence (5' half-intron) of the PIE construct (the first transesterification). The free 3'OH group of the newly generated 3' half-exon attacks the 3' splice site in a second transesterification reaction, releasing the circRNA and the 3' half-intron.

[0006] Compared to enzymatic ligation-based circularization, the PIE method can be used to circularize larger linear RNA precursors, does not require the addition of additional protein ligases, and is relatively easy to develop and optimize. Circular RNAs encoding foreign proteins synthesized by the PIE method have been validated both in vitro and in vivo and retain their low immunogenicity and long translation duration, expanding their applications (Wesselhoeft et al., 2019, "RNA circularization diminishes immunogenicity and can extend translation duration in vivo," Mol. Cell. 74, 508-520; Qu et al., 2022, "Circular RNA vaccines against SARS-CoV-2 and emerging variants," Cell 185, 1728-1744). Based on these advantages, the PIE system is currently the most studied and widely used method for RNA circularization.

[0007] In vitro generation of circRNA using the PIE method requires a construct containing the desired sequence flanked by the 3' and 5' introns of a substituted group I catalytic intron. The PIE sequence vector is linearized by single-enzyme digestion and then used as a template for in vitro transcription. Precursor RNA is obtained by a standard in vitro transcription (IVT) reaction using T7 polymerase and subsequent purification. The yield of the IVT reaction and the purity of the purified product must be optimized and guaranteed during the manufacturing process, and they are the technical core of nucleic acid drug manufacturing. To ensure efficient circularization, the purified precursor RNA must be pre-denatured at approximately 70°C before proceeding to the next reaction, followed by rapid cooling and renaturation. For industrial-scale production, a special pre-denaturation device may be required to achieve appropriate and uniform heating for large-scale reaction systems and ensure a rapid and accurate cooling process. The optimal temperature for circularization is approximately 55°C, and it must be performed in a specific buffer system (containing 2mM GTP, 50mM Tris HCl, 10mM MgCl2, 1mM DTT, pH 7.5). Furthermore, the precursor RNA concentration must be optimized for circularization. While low precursor RNA concentrations can be successfully circularized, the subsequent exponential increase in reaction volume during production scale-up poses significant challenges in both reaction temperature control and vessel selection. Increasing the precursor RNA concentration reduces the reaction volume, easing the pressure of scale-up production, but at the expense of circularization speed. According to established process flows, scale-up production requires at least two days from IVT of linearized plasmid to precursor purification, potentially resulting in RNA loss. Purified precursor RNA requires specialized denaturing and reaction equipment compatible with 55°C conditions for circularization, which is rapidly completed, followed by column chromatography purification. These steps take at least two days. Therefore, conventional processes require at least four days to obtain purified circRNA. Scaling up circRNA production using the conventional two-step process remains technically challenging and difficult.

[0008] There is a need for a ribozyme-mediated circularization process that is simpler, faster, and more efficient than conventional processes. Summary of the Invention

[0009] In one aspect, the present invention provides a method for preparing circular RNA (Method 1.0), comprising providing template DNA containing a sequence encoding a precursor RNA in a reaction solution that allows for in vitro transcription of the template DNA to synthesize the precursor RNA, and allowing the precursor RNA to self-splice to produce a circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing (i.e., circularization) of the precursor RNA are carried out in the same reaction solution and under the same reaction conditions (e.g., the same reaction temperature). This method can be carried out in a single step in a single reaction vessel and does not require a step of purifying the precursor RNA before allowing the precursor RNA to self-splice.

[0010] In some embodiments, the DNA template comprises the following elements operably connected to each other and arranged in the following order: an RNA polymerase promoter, optionally a 5' homology arm, a 3' Group I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insertion sequence, optionally a 3' spacer sequence, a 5' Group I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm. In some embodiments, the insertion sequence comprises a protein coding sequence, and optionally the insertion sequence comprises an IRES (internal ribosome entry site) sequence operably connected to the protein coding sequence.

[0011] In some embodiments, the reaction solution contains more than 26 mM Mg, e.g., more than 30 mM, or more than 35 mM. 2+ In certain embodiments, the solution contains Mg 2+ The concentration is 38 mM to 66 mM.

[0012] In some embodiments, the reaction solution contains pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, e.g., 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml.

[0013] In some embodiments, the reaction solution contains 38 to 66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na + or K + ) is included.

[0014] In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is performed at a temperature between 37° C. and 55° C. In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is performed at a temperature higher than 37° C., e.g., 39° C. to 55° C., 41° C. to 55° C., 43° C. to 55° C., 39° C. to 50° C., 41° C. to 50° C., 43° C. to 50° C., 39° C. to 47° C., 41° C. to 47° C., 43° C. to 47° C., 47° C. to 55° C., 50° C. to 55° C., 39° C. to 43° C., about 39° C., about 41° C., about 43° C., about 47° C., about 53° C., or about 55° C.

[0015] In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is carried out for at least 1 hour, e.g., at least 1.5 hours, at least 2.5 hours, at least 3 hours, 1 to 3 hours, 1.5 to 3 hours, 2 to 3 hours, or 2.5 to 3 hours. In certain embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is carried out for 2.5 to 3 hours.

[0016] In some embodiments, the method further comprises removing the DNA template after self-splicing of the RNA, optionally the DNA template is removed by adding DNase I, for example, at 37°C for 30 minutes.

[0017] In some embodiments, the method further comprises purifying the circular RNA after self-splicing of the RNA, or after removing the DNA template if the method includes removing the DNA template, in some embodiments, the purification step is selected from a precipitation step, a tangential flow filtration step, a chromatography step, and combinations thereof.

[0018] In another aspect, the present invention provides a method for treating ulcerative colitis by administering a leukemia renal failure vaccine containing ulcerative colitis at a concentration of more than 26 mM (e.g., 38 mM to 66 mM) of Mg 2+ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na + or K + ) a reaction solution for one-step circular RNA synthesis is provided.

[0019] Further scope of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. [Brief explanation of the drawings]

[0020] [Figure 1] A schematic diagram of the circRNA precursors used in the Examples is shown. [Figure 2] Schematics of the conventional procedure (left) and the one-step procedure (right) are shown. [Figure 3]Fragment analysis of the circularized product with and without pre-denaturation is shown. Peaks corresponding to intron sequences from both ends of the precursor, the circularized RNA (shown as circles), and the remaining precursor (shown as curved lines) are shown. The proportions of circular RNA and precursor are labeled. [Figure 4A] The effect of Mg2+ concentration on circularization efficiency (% total circRNA) in the IVT system is shown. The dotted line indicates a 40% circularization rate. [Figure 4B] Fragment analysis of IVT products at different Mg2+ concentrations is shown. Peaks corresponding to intron sequences, circularized RNA (shown as circles), and remaining precursor (shown as curved lines) are indicated. [Figure 4C] The effect of Mg2+ concentration on yield (total RNA) in the IVT system is shown. The dotted line indicates the yield at 200 μg. [Figure 5A] The figure shows the cyclization efficiency at different reaction times. The dotted line indicates a 40% cyclization rate. [Figure 5B] The yield (total RNA) in the IVT system at different reaction times is shown. The dotted line indicates the yield at 200 μg. [Figure 6A] 1 shows a Pareto plot of the normalized effect of various factors or specific factor combinations on yield (total RNA) in the IVT system. In the Pareto plot, bars above the baseline are statistically significant. [Figure 6B] Main effect plots of yield (total RNA) / reaction are shown. [Figure 7] Interaction plots showing the relationship between mean yield (total RNA) and temperature for the two T7 RNAP types are shown. [Figure 8A] 1 shows a Pareto plot of the normalized effect of various factors or specific factor combinations on circularization efficiency. In the Pareto plot, bars above the baseline are statistically significant. [Figure 8B] Main effect plots of cyclization efficiency / reaction are shown. [Figure 9] Interaction plots showing the mean circularization efficiency versus Mg2+ concentration for three pyrophosphatase concentrations are shown. [Figure 10]1 shows a line graph of the percentage of dsRNA in the one-step IVT reaction products at different temperatures and Mg2+ concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the disclosure, its application, or uses.

[0022] Ranges used throughout are used as a shorthand for describing all values ​​within a range. Any value within the range can be selected as the endpoint of the range. Furthermore, all references cited herein are incorporated by reference in their entirety. In the event of a discrepancy between the definitions in this disclosure and those in the references, this disclosure shall prevail.

[0023] The conventional PIE-based circularization process involves two separate steps: in vitro transcription (IVT) and circularization. Previous research (Wesselhoeft et al., (2018), "Engineering circular RNA for potent and stable translation in eukaryotic cells," Nature Communications 9, 2629) showed that IVT-derived precursor RNA circularization via the group I intron self-splicing process requires 2 mM GTP and 10 mM MgCl2, and the conventional IVT reaction solution contains 10 mM GTP and 26 mM MgCl2. It has been shown that precursor RNA is not efficiently circularized under the conventional IVT conditions (10 mM GTP and 26 mM MgCl2). Therefore, the conventional IVT conditions must be optimized for a one-step reaction system for circular RNA synthesis. In the present invention, MgCl2 is used. 2+Increasing the concentration from 26 mM to 36 mM not only promoted the circularization of circRNA precursors but also increased the IVT yield. In the present invention, the conditions of the one-step reaction system were further optimized. For example, adding pyrophosphatase to the reaction solution was shown to increase the IVT yield and circularization efficiency. This is believed to be due to the pyrophosphatase's role in maintaining a stable magnesium ion concentration throughout the IVT / circularization process. After multifactorial optimization, the one-step process achieved similar circularization efficiency to the conventional process while reducing the production of double-stranded RNA (dsRNA).

[0024] The one-step circular RNA synthesis system of the present invention has significant advantages over conventional circular RNA synthesis processes, including improved circularization efficiency, reduced production of potentially inflammatory by-products (e.g., dsRNA), and a shortened production cycle, which is beneficial for process scale-up and technology transfer. The synthesis of circRNA using conventional processes requires first generating precursors via IVT, and after purification, circularization of the purified precursors requires the addition of GTP and Mg to initiate circularization. 2+ Furthermore, a buffer containing α-methyl-2-methyl-1,3-dihydro-1,4-trimethyl ...

[0025] In one aspect, the present invention provides a method (Method 1.0) for preparing a circular RNA, the method comprising providing a template DNA comprising a sequence encoding a precursor RNA in a reaction solution that allows for the synthesis of the precursor RNA by in vitro transcription of the template DNA, and self-splicing the precursor RNA to produce a circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing (i.e., circularization) of the precursor RNA are carried out in the same reaction solution and under the same reaction conditions (e.g., the same reaction temperature). For example, the present invention includes the following: 1.1. Method 1.0, which does not include the step of purifying the precursor RNA prior to allowing the precursor RNA to self-splice. 1.2. The method of method 1.0 or 1.1, wherein the template DNA is circular, and optionally the circular template DNA is a DNA plasmid. 1.3. Method 1.0 or 1.1, wherein the template DNA is linear; optionally, the linear template DNA is prepared by linearizing a DNA plasmid, for example with a restriction enzyme. 1.4. Any of the preceding methods, wherein the DNA template comprises the following elements operably connected to each other and arranged in the following order: an RNA polymerase promoter, optionally a 5' homology arm, a 3' Group I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insertion sequence, optionally a 3' spacer sequence, a 5' Group I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm. 1.5. The method of 1.4, wherein the inserted sequence comprises a non-coding sequence having biological activity, and optionally the non-coding sequence is a microRNA or a lnc (long non-coding) RNA. 1.6. The method of 1.4, wherein the insert sequence comprises a protein coding sequence, and optionally, the insert sequence comprises an IRES sequence operably linked to the protein coding sequence. 1.7. The method of 1.6, wherein the protein coding sequence encodes an antibody. 1.8. The method of 1.6 to 1.7, wherein the IRES sequence is selected from the group consisting of taura syndrome virus, Triatomine bug virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stall enteric virus, Kashmir bee virus, human rhinovirus 2, human rhinovirus B, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, hepatitis GB virus, foot-and-mouth disease virus, human enterovirus 71, human enterovirus B, equine rhinitis virus, Ectropis obliqua picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, cruciferous tobamovirus, cricket paralysis virus, and bovine viral diarrhea virus. 1, black queen bee disease, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, hibiscus chlorotic ringspot virus, swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human The method further comprises using a gene encoding ... 1.9. The method of 1.8, wherein the IRES sequence is a Coxsackievirus B3 (CVB3) IRES sequence. 1.10. Any of the preceding methods, wherein the RNA polymerase promoter is a T7 viral RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter. 1.11. Any of the preceding methods, wherein the RNA polymerase promoter is a T7 viral RNA polymerase promoter. 1.12. Any of the preceding methods, wherein the 3' Group I intron fragment is a contiguous sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) homologous to the 3'-proximal fragment of a naturally occurring Group I intron. 1.13. Any of the preceding methods, wherein the 5' Group I intron fragment is a contiguous sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) homologous to the 5'-proximal fragment of a naturally occurring Group I intron. 1.14. Methods 1.12-1.13, wherein the native Group I intron is derived from the T4 bacteriophage gene td or the Cyanobacterium Anabaena sp. pre-tRNA-Leu gene. 1.15. Any of the preceding methods, wherein the 5' homologous arm and the 3' homologous arm are perfectly complementary to each other. 1.16. Any of Methods 1.0 to 1.14, wherein the 5' homologous arm and the 3' homologous arm are partially (e.g., at least 80%, at least 85%, at least 90%, or at least 95%) complementary to each other. 1.17. Any of the preceding methods, wherein the length of the 5' homology arm is 5 to 50 nucleotides, e.g., 9 to 19 nucleotides. 1.18. Any of the preceding methods, wherein the length of the 3' homology arm is 5 to 50 nucleotides, e.g., 9 to 19 nucleotides. 1.19. Any of the preceding methods, wherein the DNA template comprises a 5' spacer sequence between the 3' Group I intron fragment and the insert sequence, and optionally, the length of the 5' spacer sequence is 5 to 50 nucleotides, e.g., 10 to 20 nucleotides. 1.20. Any of the preceding methods, wherein the 5' spacer sequence is a polyA sequence or a polyAC sequence. 1.21. Any of the preceding methods, wherein the DNA template comprises a 3' spacer sequence between the insert sequence and the 5' Group I intron fragment, and optionally, the length of the 3' spacer sequence is 5 to 50 nucleotides, e.g., 10 to 20 nucleotides. 1.22. Any of the preceding methods, wherein the 3' spacer sequence is a polyA sequence or a polyAC sequence. 1.23. Any of the preceding methods, wherein the reaction solution contains more than 26 mM Mg, e.g., more than 30 mM, or more than 35 mM Mg. 2+ A method comprising: 1.24. In any of the preceding methods, Mg in solution 2+ is between 30 mM and 100 mM, for example between 30 mM and 90 mM, between 30 mM and 80 mM, between 30 mM and 70 mM, between 30 mM and 60 mM, between 30 mM and 50 mM, between 30 mM and 40 mM, between 35 mM and 100 mM, between 35 mM and 90 mM, between 35 mM and 80 mM, between 35 mM and 70 mM, between 35 mM and 60 mM, between 35 mM and 50 mM, between 35 mM and 40 mM, between 38 mM and 66 mM, for example about 38 mM, and optionally, Mg in the solution 2+ The concentration of is 38 mM to 66 mM. 1.25. Any of the preceding methods, wherein the reaction solution contains pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, e.g., 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml. 1.26. Any of the preceding methods, wherein the reaction solution contains an RNA polymerase, an RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and a monovalent cation (Na + or K + ). 1.27. Any of the preceding methods, wherein the reaction solution contains 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). 1.28. In any of the preceding methods, the reaction solution contains 38-66 mM Mg 2+ , optionally 1-4 U / ml of pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na + or K + ). 1.29. Any of the preceding methods, wherein the reaction solution contains 38 mM Mg 2+ 2 U / ml of pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na + or K + ). 1.30. In any of the preceding methods, the reaction solution contains 38-66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). 1.31. Any of the preceding methods, wherein the reaction solution contains 38 mM Mg 2+, 2 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). 1.32. Any of the preceding methods, wherein the reaction solution comprises a buffer. 1.33. Any of the preceding methods, wherein the pH of the reaction solution is 6 to 8, e.g., 7 to 8, or about 7.5. 1.34. Any of the preceding methods, wherein the reaction solution comprises an RNA polymerase selected from T7 viral RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase. 1.35. Any of the preceding methods, wherein the RNA polymerase promoter in the DNA template is a T7 viral RNA polymerase promoter, and the reaction solution comprises T7 viral RNA polymerase. 1.36. In any of the preceding methods, the in vitro transcription of the template DNA and circularization (i.e., self-splicing) of the precursor RNA is carried out at a temperature of 37°C to 55°C, e.g., 39°C to 55°C, 41°C to 55°C, 43°C to 55°C, 37°C to 50°C, 39°C to 50°C, 41°C to 50°C, 43°C to 50°C, 37°C to 47°C, 39°C to 47°C, 41°C to 47°C, 43°C to 47°C, 47°C to 55°C, 50°C to 55°C, 39°C to 43°C, about 37°C, about 39°C, about 41°C, about 43°C, about 47°C, about 53°C, or about 55°C, and optionally, in vitro transcription of the template DNA and circularization of the precursor RNA (i.e., self-splicing) are carried out at a temperature of 37°C to 55°C, e.g., 39°C to 55°C, 41°C to 55°C, 43°C to 55°C, 37°C to 47°C, 41°C to 47°C, about 53°C, or about 55°C. A method in which the in vitro transcription and circularization of the precursor RNA (i.e., self-splicing) is carried out at a temperature higher than 37°C, for example, 39°C to 55°C, 41°C to 55°C, 43°C to 55°C, 39°C to 50°C, 41°C to 50°C, 43°C to 50°C, 39°C to 47°C, 41°C to 47°C, 43°C to 47°C, 47°C to 55°C, 50°C to 55°C, 39°C to 43°C, about 39°C, about 41°C, about 43°C, about 47°C, about 53°C, or about 55°C. 1.37. Any of the preceding methods, wherein the in vitro transcription of the template DNA and circularization (i.e., self-splicing) of the precursor RNA is carried out at a temperature of 47°C to 55°C, e.g., 50°C to 55°C, about 47°C, about 53°C, or about 55°C, and the RNA polymerase is a thermostable polymerase (e.g., T7 Toyobo). 1.38. Any of the preceding methods, wherein the in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is carried out for at least 1 hour, e.g., at least 1.5 hours, at least 2.5 hours, at least 3 hours, 1 hour to 3 hours, 1.5 hours to 3 hours, 2 hours to 3 hours, or 2.5 hours to 3 hours, and optionally, the in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is carried out for 2.5 hours to 3 hours. 1.39. Any of the preceding methods, wherein the method further comprises removing the DNA template after synthesis of the precursor RNA, and optionally the DNA template is removed by adding DNase I, e.g., for 30 minutes at 37°C. 1.40. Any of the preceding methods, wherein if the method includes a step of removing the DNA template, the method further includes a step of purifying the circular RNA so synthesized, e.g., after the step of removing the DNA template. 1.41. The method of 1.40, wherein the purification step is selected from a precipitation step, a tangential flow filtration step, and a chromatography step, and combinations thereof. 1.42. The method of 1.41, wherein the precipitation step is an alcohol precipitation step or a LiCl precipitation step, optionally wherein the precipitation step is a LiCl precipitation step. 1.43. The method of 1.41, wherein the tangential flow filtration step is a diafiltration step using tangential flow filtration and / or a concentration step using tangential flow filtration. 1.44. The method of 1.41, wherein the chromatography step is selected from HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography, magnetic bead chromatography, and core bead chromatography, and optionally, the chromatography step is magnetic bead chromatography. 1.45. Any of the preceding methods, wherein the precursor RNA is unmodified, i.e., contains only naturally occurring nucleosides, including, for example, adenosine, guanosine, cytidine, and uridine. 1.46. Any of Methods 1.0 to 1.44, wherein the precursor RNA is partially or fully modified, i.e., contains nucleosides other than, or in addition to, adenosine, guanosine, cytidine, and uridine. 1.47. The method of method 1.46, wherein the precursor RNA comprises nucleosides selected from pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytidine, N6-methyladenosine, and combinations thereof. 1.48. Method 1.46, wherein some or all of the ribonucleoside triphosphates in the reaction solution include ribonucleoside triphosphates other than, or in addition to, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). 1.49. Method 1.46, wherein some or all of the ribonucleoside triphosphates in the reaction solution comprise modified nucleoside triphosphates, e.g., the modified nucleoside triphosphates are selected from pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, and combinations thereof. 1.50. Method 1.46, wherein the nucleosides in the precursor RNA do not include uridine, but include nucleosides selected from pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, and combinations thereof. 1.51. The method of method 1.46, wherein the nucleosides in the precursor RNA do not include cytidine, but do include 5-methylcytidine. 1.52. Any of the preceding methods, wherein the circularization efficiency is at least 70%. 1.53. Any of the preceding methods, wherein the ratio of dsRNA to total RNA in the final product is less than 1%, e.g., less than 0.1%. 1.54. Any of the preceding methods, wherein the reaction solution is a reaction solution according to Reaction Solution 2 et seq., described below.

[0026] In one embodiment, the reaction solution of the method according to the present disclosure contains Mg 2+ The reaction solution may further contain pyrophosphatase.

[0027] In a further embodiment, the reaction solution may include a nucleoside triphosphate.

[0028] In yet another embodiment, the reaction solution may include a reducing agent.

[0029] In yet another embodiment, the reaction solution may contain an RNA polymerase.

[0030] In another embodiment, the reaction solution may contain an RNase inhibitor.

[0031] In another embodiment, the reaction may include a monovalent cation.

[0032] The selection and / or concentration of each component is as described herein, and the concentrations are based on the reaction solution / mixture used in the method.

[0033] For example, Mg in solution 2+The concentration of is greater than 26 mM, specifically 38 to 66 mM, more specifically 38 mM. For example, the concentration of pyrophosphatase in the solution is 1 U / ml to 5 U / ml, specifically 1 U / ml to 4 U / ml, more specifically 2 U / ml. For example, the nucleoside triphosphates include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), each of which has a concentration of 10 mM. For example, the reducing agent is DTT, and its concentration is 10 mM. For example, the RNA polymerase is T7 viral polymerase, and its concentration is 5 U / μl. For example, the concentration of the RNase inhibitor is 1 U / μl. For example, the monovalent cation is Na + or K + and its concentration is 5 mM.

[0034] In one embodiment, the reaction solution of the method according to the present disclosure contains Mg 2+ , an RNA polymerase, an RNase inhibitor, a nucleoside triphosphate, a reducing agent, and a monovalent cation (Na+ or K+), and optionally a pyrophosphatase.

[0035] In one embodiment, the reaction conditions of the methods according to the present disclosure include a temperature for in vitro transcription of the template DNA and circularization (ie, self-splicing) of the precursor RNA.

[0036] In further embodiments, the reaction conditions of the methods according to the present disclosure include a time for in vitro transcription of the template DNA and circularization (ie, self-splicing) of the precursor RNA.

[0037] The conditions are selected as described herein. For example, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are carried out using a thermostable RNA polymerase (e.g., T7 Toyobo) at a temperature of 37°C to 55°C, particularly 39°C to 50°C or 47°C to 55°C. For example, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are carried out for 2.5 to 3 hours.

[0038] In one embodiment, the reaction conditions of the disclosed method include temperature and time for in vitro transcription of template DNA and circularization (ie, self-splicing) of precursor RNA.

[0039] In a most specific embodiment, the reaction solution of the method according to the present disclosure contains 38 mM Mg 2+ , 2 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ), in which in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is carried out at temperatures between 37°C and 55°C for 2.5 to 3 hours.

[0040] The present disclosure further provides a circular RNA obtained by any of Methods 1 below.

[0041] The present disclosure further provides a pharmaceutical composition comprising a circular RNA obtained by any of methods 1 below.

[0042] The present disclosure further provides a method for treating a cancer cell comprising administering to a subject ... 2+ , pyrophosphatase, RNA polymerase, RNase inhibitors, nucleoside triphosphates, reducing agents, and monovalent cations (e.g., Na + , K. +and combinations thereof), for example, 2.1. Reaction solution 2, wherein the reaction solution contains more than 30 mM or more than 35 mM Mg 2+ A reaction solution comprising: 2.2. Any of the preceding reaction solutions, wherein the Mg 2+ is 30 mM to 100 mM, for example, 30 mM to 90 mM, 30 mM to 80 mM, 30 mM to 70 mM, 30 mM to 60 mM, 30 mM to 50 mM, 30 mM to 40 mM, 35 mM to 100 mM, 35 mM to 90 mM, 35 mM to 80 mM, 35 mM to 70 mM, 35 mM to 60 mM, 35 mM to 50 mM, 35 mM to 40 mM, 38 to 66 mM, for example, about 38 mM, and optionally, Mg in the solution 2+ The concentration of the reaction solution is 38 mM to 66 mM. 2.3. Any of the preceding reaction solutions, comprising pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, e.g., 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml. 2.4. Any of the preceding reaction solutions, wherein the nucleoside triphosphate is selected from the group consisting of ATP, GTP, CTP, UTP, pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, and combinations thereof. 2.5. Any of the preceding reaction solutions, wherein the nucleoside triphosphates comprise ATP, GTP, CTP, and UTP. 2.6. Any of the preceding reaction solutions, comprising pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, e.g., 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml. 2.7. Any of the preceding reaction solutions, wherein the nucleoside triphosphates comprise pseudouridine-5'-triphosphate, ATP, GTP, and CTP. 2.8. Any of the preceding reaction solutions, wherein the nucleoside triphosphates comprise 1-methylpseudouridine-5'-triphosphate, ATP, GTP, and CTP. 2.9. Any of the preceding reaction solutions, wherein the reducing agent is DTT. 2.10. Any of the preceding reaction solutions, wherein the nucleoside triphosphate comprises pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, or a combination thereof. 2.11. Any of the preceding reaction solutions containing 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). 2.12. Any of the preceding reaction solutions containing 38-66 mM Mg 2+ , optionally 1-4 U / ml of pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na + or K + ). 2.13. Any of the preceding reaction solutions containing 38 mM Mg 2+ 2 U / ml of pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na + or K + ). 2.14. Any of the preceding reaction solutions containing 38-66 mM Mg 2+, optionally 1-4 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). 2.15. Any of the preceding reaction solutions containing 38 mM Mg 2+ , 2 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl RNase inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). 2.16. Any of the preceding reaction solutions, wherein the reaction solution comprises a buffer. 2.17. Any of the preceding reaction solutions, wherein the pH of the reaction solution is 6 to 8, e.g., 7 to 8, or about 7.5. 2.18. Any of the preceding reaction solutions, comprising an RNA polymerase selected from T7 viral RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase.

[0043] The present disclosure further provides the use of any of Reaction Solution 2, below, in a method for one-step circular RNA synthesis, for example, in a method according to any of Methods 1, below.

[0044] In the method of the present invention, precursor RNA is synthesized by in vitro transcription of template DNA. The DNA template contains a promoter upstream of the region encoding the precursor RNA. The promoter is recognized by an RNA polymerase, for example, a T7 promoter is recognized by T7 viral RNA polymerase. In some embodiments, the promoter is a T7 promoter and the RNA polymerase is a T7 viral RNA polymerase; alternatively, the promoter is a T6 promoter and the polymerase is a T6 viral RNA polymerase; alternatively, the promoter is an SP6 viral RNA polymerase promoter and the polymerase is an SP6 viral RNA polymerase; alternatively, the promoter is a T3 viral RNA polymerase promoter and the polymerase is a T3 viral RNA polymerase; or alternatively, the promoter is a T4 viral RNA polymerase promoter and the polymerase is a T4 viral RNA polymerase. In certain embodiments, the RNA polymerase promoter is a T7 viral RNA polymerase promoter and the polymerase is a T7 viral RNA polymerase.

[0045] The template DNA may be linear or circular. In some embodiments, the template DNA is prepared by linearizing a DNA plasmid, for example, with a restriction enzyme. In other embodiments, the template is circular (e.g., a DNA plasmid). The template DNA may include an RNA polymerase terminator sequence element downstream of the precursor RNA-encoding region, particularly when the template DNA is circular.

[0046] The template DNA contains a sequence encoding a precursor RNA. As used herein, "circular precursor RNA" or "precursor RNA" refers to a linear RNA molecule that can generate a circular RNA (circRNA) by self-splicing. In addition to the circRNA sequence, the precursor RNA contains splicing sequences (e.g., intron fragments and optional 5' and 3' homology arms) necessary to circularize the RNA. These splicing sequences are removed from the precursor RNA during circularization. The precursor RNA may be unmodified, partially modified, or fully modified. In some embodiments, the precursor RNA is unmodified, i.e., the nucleoside moieties in the precursor RNA are naturally occurring nucleosides such as adenosine, guanosine, cytidine, and uridine. In other embodiments, the precursor RNA is modified, i.e., the nucleoside moieties in the precursor RNA include nucleosides in addition to or in place of adenosine, guanosine, cytidine, and uridine, e.g., the nucleosides include pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytidine, N6-methyladenosine, or combinations thereof, e.g., uridine is replaced with pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, and / or cytidine is replaced with 5-methylcytidine and / or adenosine is replaced with N6-methyladenosine.

[0047] In some embodiments, the DNA template comprises the following elements operably linked to each other and arranged in the following order: a promoter recognized by an RNA polymerase, optionally a 5' homology arm, a 3' Group I intron fragment comprising a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insert sequence comprising a sequence of interest, optionally a 3' spacer sequence, a 5' Group I intron fragment comprising a 5' splice site dinucleotide, and optionally a 3' homology arm. As used herein, the phrase "operably linked" means that the elements are arranged on the DNA template such that precursor RNA can be synthesized by in vitro transcription of the template DNA, and the precursor RNA can then be circularized into circular RNA, using the methods disclosed herein.

[0048] In some embodiments, the 3' Group I intron fragment is a contiguous sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) homologous to the 3' proximal fragment of a naturally occurring Group I intron, comprising the 3' splice site dinucleotide and, optionally, at least 1 nucleotide in length (e.g., at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, or at least 50 nucleotides in length) of adjacent exon sequence.

[0049] In some embodiments, the 5' Group I intron fragment is a contiguous sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) homologous to the 5' proximal fragment of a naturally occurring Group I intron, including the 5' splice site dinucleotide and, optionally, at least 1 nucleotide in length (e.g., at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, or at least 50 nucleotides in length) of flanking exon sequence.

[0050] The naturally occurring Group I intron can be selected from any Group I intron known to self-splice. Examples of Group I introns include, but are not limited to, the Group I intron from the T4 bacteriophage gene td or the Cyanobacterium Anabaena sp. pre-tRNA-Leu gene.

[0051] In some embodiments, the template DNA comprises 5' and 3' homology arms at the 5' and 3' ends of the precursor RNA region. Adding 5' and 3' homology arms to the 5' and 3' ends of the precursor RNA region can improve circularization efficiency by bringing the 5' and 3' splice sites closer together, especially when the inserted sequence between the 3' group I intron fragment and the 5' group I intron fragment is long. In some embodiments, the 5' homology arms are 5 to 50 nucleotides long, e.g., 9 to 19 nucleotides long. In some embodiments, the 3' homology arms are 5 to 50 nucleotides long, e.g., 9 to 19 nucleotides long. In some embodiments, the 5' and 3' homology arms are fully complementary to each other. In other embodiments, the 5' and 3' homology arms are partially complementary to each other (e.g., at least 80%, at least 85%, at least 90%, or at least 95%).

[0052] Highly structured sequences, such as an internal ribosome entry site (IRES), between the 3' group I intron fragment and the 5' group I intron fragment can interfere with the folding of the splicing ribozyme through long-range contacts proximal to the 3' splice site or distal to the 5' splice site (Wesselhoeft et al., 2018). Adding a 5' spacer sequence between the 3' group I intron fragment and the insert sequence and / or between the insert sequence and the 5' group I intron fragment can improve circularization efficiency, especially when the insert sequence is highly structured. In some embodiments, the DNA template includes a 5' spacer sequence between the 3' group I intron fragment and the insert sequence. In some embodiments, the length of the 5' spacer sequence is 5 to 50 nucleotides. In some embodiments, the length of the 5' spacer sequence is 10 to 20 nucleotides. In certain embodiments, the 5' spacer sequence is a poly(A) sequence. In other embodiments, the 5' spacer sequence is a polyAC sequence. In some embodiments, the DNA template comprises a 3' spacer sequence between the insert sequence and the 5' group I intron fragment. In some embodiments, the 3' spacer sequence is 5 to 50 nucleotides in length. In some embodiments, the 3' spacer sequence is 10 to 20 nucleotides in length. In certain embodiments, the 3' spacer sequence is a polyA sequence. In other embodiments, the 3' spacer sequence is a polyAC sequence.

[0053] The insert sequence comprises a sequence of interest. The sequence of interest may be a protein-coding sequence or a non-coding sequence. In some embodiments, the insert sequence comprises a non-coding sequence with biological activity. Examples of non-coding sequences with biological activity include, but are not limited to, microRNAs and lnc (long non-coding) RNAs.

[0054] In some embodiments, the inserted sequence comprises a protein coding sequence. The protein coding sequence may encode any protein for therapeutic or diagnostic use. In some embodiments, the protein coding sequence encodes an antibody.

[0055] When the insertion sequence comprises a protein-coding sequence, the insertion sequence may further comprise a sequence required for translation, such as, for example, an internal ribosome entry site (IRES) sequence upstream of the protein-coding sequence. In some embodiments, the insertion sequence comprises an IRES sequence operably connected to the protein-coding sequence. As used herein, the phrase "operably connected" means that the IRES sequence is positioned upstream of the protein-coding sequence so that the protein-coding sequence can be translated into a protein in vivo (in eukaryotic cells, e.g., human cells) and / or in vitro. The IRES sequence may be any IRES sequence known in the art.In some embodiments, the IRES sequence is selected from the group consisting of Taura syndrome virus, Triatomine bug virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall enteric virus, Kashmir bee virus, Human rhinovirus 2, Human rhinovirus B, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Human enterovirus B, Equine rhinitis virus, Ectropis obliqua picorna-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black queen larvae disease, Aphid fatal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human The nucleic acid sequence is selected from BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1 alpha, human n-myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, salivirus, cosavirus, parechovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, human c-src, human FGF-1, monkey picornavirus, turnip crinkle virus, an aptamer against eIF4G, and an IRES sequence of coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1 / 2). In a particular embodiment, the IRES sequence is the IRES sequence of Coxsackievirus B3 (CVB3).

[0056] In the method of the present invention, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are performed in a single step. The method does not include a step of purifying precursor RNA before allowing the precursor RNA to self-splice. In other words, in vitro transcription and circularization occur in the same reaction solution and under the same reaction conditions (e.g., temperature). Therefore, the reaction solution and reaction conditions must be optimized for the efficiency of both in vitro transcription and circularization.

[0057] In some embodiments, the reaction solution contains more than 26 mM Mg, e.g., more than 30 mM, or more than 35 mM. 2+ In some embodiments, the solution contains Mg 2+ In certain embodiments, the concentration of Mg in the solution is 30 mM to 100 mM, e.g., 30 mM to 90 mM, 30 mM to 80 mM, 30 mM to 70 mM, 30 mM to 60 mM, 30 mM to 50 mM, 30 mM to 40 mM, 35 mM to 100 mM, 35 mM to 90 mM, 35 mM to 80 mM, 35 mM to 70 mM, 35 mM to 60 mM, 35 mM to 50 mM, 35 mM to 40 mM, 38 to 66 mM, e.g., about 38 mM. 2+ The concentration is 38 mM to 66 mM.

[0058] In some embodiments, the reaction solution contains pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, e.g., 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml. As used herein, 1 U (unit) of pyrophosphatase is defined as the amount of enzyme that produces 1 μmol of phosphate per minute from inorganic pyrophosphate under standard reaction conditions (20 mM Tris-HCl, pH 8.0, 2 mM MgCl, 2 mM PPi, 25°C for 10 minutes).

[0059] The reaction solution further comprises components necessary for in vitro transcription. In some embodiments, the reaction solution comprises an RNA polymerase, an RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and a monovalent cation (Na + or K + In certain embodiments, the reaction solution comprises about 5 U / μl of RNA polymerase, about 1 U / μl of RNase inhibitor, about 10 mM ATP, about 10 mM GTP, about 10 mM CTP, about 10 mM UTP, about 10 mM DTT, and 5 mM monovalent cations (Na + or K + The reaction solution may contain a buffer solution. The pH of the reaction solution may be 6 to 8, for example, 7 to 8, or about 7.5.

[0060] The precursor RNA may be unmodified, partially modified, or fully modified. In some embodiments, the precursor RNA is unmodified, i.e., contains only naturally occurring nucleotides. In other embodiments, the precursor RNA is partially modified or fully modified. To synthesize a partially or fully modified precursor RNA, some or all of the at least one ribonucleoside triphosphate in the reaction solution may be substituted with a modified nucleoside triphosphate. Examples of modified nucleoside triphosphates include, but are not limited to, pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, and 5-methylcytidine-5'-triphosphate.

[0061] The RNA polymerase used for in vitro transcription can be selected based on the RNA polymerase promoter in the DNA template. For example, if the RNA polymerase promoter in the DNA template is a T7 viral RNA polymerase promoter, the reaction solution can contain T7 RNA polymerase. In some embodiments, the reaction solution contains an RNA polymerase selected from T7 viral RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase. In certain embodiments, the RNA polymerase promoter in the DNA template is T7 viral RNA polymerase, and the reaction solution contains T7 viral RNA polymerase.

[0062] In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is performed at 37°C to 55°C, e.g., 39°C to 55°C, 41°C to 55°C, 43°C to 55°C, 37°C to 50°C, 39°C to 50°C, 41°C to 50°C, 43°C to 50°C, 37°C to 47°C, 39°C to 47°C, 41°C to 47°C, 43°C to 47°C, 47°C to 55°C, 50°C to 55°C, 39°C to 43°C, about 37°C, about 39°C, about 41°C, about 43°C, about 47°C, about 53°C, or about 55°C. The production of the major by-product, dsDNA, has been shown to decrease with increasing temperature. dsRNA is recognized by cytoplasmic sensors such as RIG-I and MDA5, which then activate the innate immune system (Wu et al., 2020, "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription," RNA 26, 345-360; Olejniczak, 2010, "Sequence-non-specific effects of RNA interference triggers and microRNA regulators," Nucleic Acids Res 38, 1-16). To minimize dsRNA production, a temperature of 37°C or higher is desirable. In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is performed at temperatures higher than 37°C, e.g., 39°C to 55°C, 41°C to 55°C, 43°C to 55°C, 39°C to 50°C, 41°C to 50°C, 43°C to 50°C, 39°C to 47°C, 41°C to 47°C, 43°C to 47°C, 47°C to 55°C, 50°C to 55°C, 39°C to 43°C, about 39°C, about 41°C, about 43°C, about 47°C, about 53°C, or about 55°C.

[0063] When in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are performed at elevated temperatures, recombinant RNA polymerases with improved thermostability (e.g., T7 Toyobo) may be preferred. In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are performed at temperatures between 47°C and 55°C, e.g., 50°C to 55°C, about 47°C, about 53°C, or about 55°C, and the RNA polymerase is a thermostable polymerase (e.g., T7 Toyobo).

[0064] In vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) can be carried out for at least 1 hour, e.g., at least 1.5 hours, at least 2.5 hours, at least 3 hours, 1 to 3 hours, 1.5 to 3 hours, 2 to 3 hours, or 2.5 to 3 hours. A reaction time of 1.5 hours or longer is desirable to ensure sufficient circularization. However, longer reaction times may increase the risk of side products. Therefore, the optimal reaction time for a one-step process may be 2.5 to 3 hours. In a preferred embodiment, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are carried out for 2.5 to 3 hours.

[0065] In some embodiments, the method further comprises removing the DNA template after self-splicing of the RNA. The DNA template may be removed by adding DNase I (e.g., at 37°C for 30 minutes).

[0066] In some embodiments, the method further comprises purifying the circular RNA after RNA self-splicing, or after removing the DNA template if the method includes removing the DNA template. In some embodiments, the purification step is selected from a precipitation step, a tangential flow filtration step, a chromatography step, and a combination thereof. The precipitation step may be an alcohol precipitation step or a LiCl precipitation step. The tangential flow filtration step may be a diafiltration step using tangential flow filtration and / or a concentration step using tangential flow filtration. The chromatography step may be selected from HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography, magnetic bead chromatography, and core bead chromatography. In some embodiments, the purification step comprises a precipitation step, e.g., LiCl precipitation. In other embodiments, the purification step comprises chromatography, e.g., magnetic bead chromatography. [Example]

[0067] Plasmid construction Plasmids containing circRNA precursor sequences were used as templates for in vitro transcription (IVT). The circRNA precursor sequences were designed based on the group I intron system described by Wesselhoeft et al. (Wesselhoeft et al., 2018, "Engineering circular RNA for potent and stable translation in eukaryotic cells," Nature Communications 9, 2629). The 3' and 5' introns of the replaced group I catalytic intron of the Anabaena pre-tRNA gene were placed on either side of an insert containing a coxsackievirus B3 (CVB3) IRES (internal ribosome entry site), a GFP sequence, and two short regions (E1 and E2) corresponding to exon fragments. The circRNA precursor sequence (SEQ ID NO: 1) was chemically synthesized and cloned into an expression vector containing a T7 polymerase promoter (Genscript). A schematic diagram of the circRNA precursor used in the experiments is shown in Figure 1.

[0068] Circular RNA synthesis by conventional procedures Linearized plasmid DNA is used as a template for in vitro transcription. A plasmid containing a circRNA precursor sequence is linearized by XbaI enzyme digestion, and the circRNA precursor is synthesized from the linearized plasmid DNA template by in vitro transcription using T7 RNA polymerase. Prepare the reaction mixture (20 µL total) as follows: 1 U / µL RNase inhibitor (Novoprotein E125), 6.67 mM ATP, 20 mM GTP, 6.67 mM CTP, 6.67 mM UTP, 1X transcription buffer (Novoprotein GMP-EB121 with 6 mM MgCl), 10 mM DTT (Sigma 43816), 4 U / mL inorganic pyrophosphatase (Novoprotein GMP-M036), 5 mM NaCl (Invitrogen AM9760G), 20 mM MgCl (Invitrogen M1028), 5 U / µL T7 RNA polymerase (Novoprotein GMP-E121), and 25 ng / µL linearized plasmid. In vitro transcription was carried out for 3 hours at 37°C, after which the reaction mixture was treated with DNase I (Novoprotein GMP-E127) for 30 minutes at 37°C to remove the DNA template. After DNase I treatment, the synthesized precursor RNA was purified by precipitation with 7.5 M LiCl.

[0069] Precursor RNA circularization was performed using a modified method described by Wesselhoeft et al. A 20 μL circularization reaction was performed by directly adding 6 μg of precursor RNA to the circularization reaction. Alternatively, the precursor mRNA was preheated at 70°C for 5 min and immediately placed on ice for 5 min. GTP was added to the precursor RNA to a final concentration of 2 mM along with a magnesium-containing buffer (50 mM Tris HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5). The reaction mixture was incubated at 55°C for 12 min, after which the RNA was purified on a column. The RNA product was evaluated using a fragment analyzer.

[0070] Precursor RNA is circularized by a self-splicing reaction requiring GTP and magnesium ions. This process depends on the structure of the group I intron. As shown in Figure 3, when precursor RNA is directly subjected to the circularization reaction without preheating, approximately 60% of the RNA is circularized. However, when precursor RNA is preheated to 70 °C and then circularized, approximately 70% circularization efficiency is achieved. This suggests that pre-denaturation of precursor RNA favors the formation of the structure required for self-splicing of the group I intron. These results indicate that in conventional procedures, LiCl-purified IVT products must be pre-denatured at high temperatures before circularization to promote precursor RNA circularization.

[0071] Circular RNA synthesis by a one-step process Linearized plasmid DNA is used as a template for in vitro transcription. The plasmid containing the circRNA precursor sequence is linearized by XbaI enzyme digestion. The one-step reaction mixture (20 μL total) is prepared as follows: 1 U / μL RNase inhibitor (Novoprotein E125), 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 1X transcription buffer (Novoprotein GMP-EB121, containing 6 mM MgCl), 10 mM DTT (Sigma 43816), 4 U / mL inorganic pyrophosphatase (Novoprotein GMP-M036), 5 mM monovalent cation (Na + or K + ), 20–80 mM MgCl2 (Invitrogen M1028), 5 U / μL T7 RNA polymerase (KactusBio GMP-T7P-EE101-12), and 25 ng / μL linearized plasmid. The reaction was carried out at 37°C for 3 hours, after which the reaction mixture was treated with DNase I (Novoprotein GMP-E127) for 30 minutes at 37°C to remove the DNA template. After DNase I treatment, the RNA was purified by precipitation with 7.5 M LiCl. The RNA product was evaluated using a fragment analyzer.

[0072] Previous research (Wesselhoeft et al., 2018) has shown that circularization of precursor RNA derived from IVT via the self-splicing process of group I introns requires 2 mM GTP and 10 mM MgCl2, whereas the conventional IVT system contains GTP (here 10 mM) and MgCl2 (here 26 mM). Figures 4A and 4B show that synthesized precursor RNA is not efficiently circularized under conventional IVT conditions (10 mM GTP and 26 mM MgCl2). Under these conditions, only 7.5% of the precursor RNA was circularized, suggesting that conventional IVT conditions need to be optimized for precursor RNA circularization.

[0073] Mg 2+ To investigate the effect of Mg concentration on the circularization of precursor RNA, we fixed other components in the one-step IVT / circularization system. 2+ The concentration of is varied (Table 1). [Table 1]

[0074] Tested final Mg 2+ The concentrations were 26 mM, 36 mM, 46 mM, 56 mM, 66 mM, and 86 mM, and the concentration of each NTP was adjusted to 10 mM (A:G:C:U=1:1:1:1). The RNA product was evaluated using a fragment analyzer. The results are shown in Figures 4A and 4B. 2+ We show that increasing the Mg concentration from 26 mM to 36 mM promotes circularization of circRNA precursors. 2+ At 36 mM Mg, only 7.5% of the precursor RNA is circularized. 2+ In the case of Mg, 41.8% of the precursor RNA is circularized. 2+ Further increase in concentration to 86 mM does not significantly change the efficiency of circularization.

[0075] The yield of total RNA was also 2+ Measurements were performed using a one-step IVT / cyclization system with varying concentrations of Mg (Figure 4C). 2+Increasing the Mg concentration from 26 mM to 36 mM not only promotes circularization of circRNA precursors but also increases RNA yield. 2+ Increasing the concentration to 86 mM significantly reduces RNA production.

[0076] In the one-step IVT / circularization process, circularization of precursor RNA can occur simultaneously with and / or after transcription. To address this issue, we investigate the efficiency and yield of circularization at different reaction time points. 2+ The IVT conditions were the same as in Table 1, except that the concentration was adjusted to 38 mM. The reaction was carried out at 37°C for 1, 1.5, 2, 2.5, 3, or 3.5 hours, after which the reaction mixture was treated with DNase I (Novoprotein GMP-E127) for 30 minutes at 37°C to remove the DNA template. After DNase I treatment, the RNA was purified by precipitation with 7.5 M LiCl.

[0077] The results of fragment analysis show that extending the reaction time up to 2.5 hours improves the efficiency of precursor RNA circularization (Figure 5A). However, the process is nearly complete at 2.5 hours. The circularization efficiency does not increase when the reaction time exceeds 2.5 hours. The effect of reaction time on total RNA yield is also investigated. The results show that the yield exceeds 200 μg / reaction when the reaction is performed for 1 hour, and further extension of the reaction time does not significantly improve the yield (Figure 5B). Longer reaction times may increase the production of by-products. Therefore, the optimal reaction time for the one-step process is 2.5 to 3 hours.

[0078] Optimization of the one-step cyclization system To comprehensively evaluate the factors affecting IVT and cyclization during the one-step process, we investigated the temperature, Mg 2+A multilevel crossover experiment was designed using software for four factors: concentration, pyrophosphatase concentration, and reaction temperature. The circularization efficiency was examined using a fragment analyzer, and the total RNA yield was calculated by determining the product concentration. The results are shown in Table 2. All data were entered into software for factorial analysis.

[0079] [Table 2-1] [Table 2-2] [Table 2-3]

[0080] Normalized effect analysis showed that magnesium ion concentration and temperature were the main factors affecting IVT yield, and the interaction between reaction temperature and the type of T7 RNAP also affected the yield (Figure 6A). As the temperature increased (above 37°C), the overall IVT yield decreased (Figure 6B). 2+ Increasing the concentration from 26 to 38 mM significantly improved the average IVT yield, but Mg 2+Further increases in the concentration do not increase the average yield under all test conditions (Figure 6B). Adding pyrophosphatase to the reaction increases the yield (Figure 6B). This is likely due to the contribution of pyrophosphatase in maintaining stable magnesium ion concentrations throughout the IVT process. The type of T7 RNAP used in IVT has little effect on the average yields of all tested groups, but the effect of temperature on IVT yields is highly dependent on the type of T7 RNAP used in IVT (Figure 7). When TOYOBO T7 RNAP (a thermostable T7 RNA polymerase) is used in IVT, the average IVT yield is lowest at 37°C, while the average IVT yields at higher temperatures (47, 53, or 55°C) are nearly identical. In contrast, when KACTUS T7 RNAP is used in IVT, the average IVT yield is highest at 37°C, while the average IVT yield at higher temperatures (47, 53, or 55°C) is significantly reduced, indicating that KACTUS T7 RNAP exhibits reduced enzymatic activity at higher temperatures.

[0081] For cyclization efficiency, Mg 2+ concentration, reaction temperature, pyrophosphatase concentration, and Mg 2+ The interaction of Mg with pyrophosphatase all influences the circularization of precursor RNA (Figure 8A). Increasing the temperature from 37 to 47°C promotes circularization, whereas higher temperatures (53 and 55°C) decrease the circularization efficiency (Figure 8B). 2+ Increasing the concentration from 26 mM to 66 mM promotes cyclization, but the higher Mg 2+ At higher concentrations (76 mM and 96 mM), circularization efficiency was not further increased (Figure 8B). Addition of pyrophosphatase to the IVT reaction promoted circularization, but the concentration of pyrophosphatase did not positively correlate with the average circularization efficiency, with the circularization efficiency being highest at 2 U / mL pyrophosphatase (Figure 8B). 2+ It is noteworthy that combining with pyrophosphatase further promotes cyclization (Figure 9). After multifactorial optimization, the one-step process can achieve cyclization efficiency similar to that of conventional processes. For example, Mg 2+When the concentration was adjusted to 38 mM, the pyrophosphatase concentration to 2 U / mL, and the reaction temperature to 47°C, the circularization efficiency increased to 74.9% (Table 2).

[0082] One of the major by-products identified in the one-step process is dsRNA, which is recognized by cytoplasmic sensors such as RIG-I and MDA5, and then activates the innate immune system. We investigated the amount of dsRNA generated under IVT conditions with various temperatures and magnesium concentrations. An antibody-dependent fluorescence resonance energy transfer (FRET) assay was used to detect dsRNA in IVT samples. The specific procedures were performed according to the kit manual (Cisbio 64RNAPEG). The results are shown in Table 3 and Figure 10. The results indicate that temperature is a factor that directly affects dsRNA generation (Table 3 and Figure 10). dsRNA generation was suppressed with increasing temperature, but increased with increasing magnesium. 2+ The concentration does not significantly affect the production of dsRNA. [Table 3]

[0083] While the present disclosure has been described in terms of specific examples, including presently preferred modes for carrying out the disclosure, those skilled in the art will recognize that there are numerous variations and permutations of the above-described systems and techniques. It is to be understood that other embodiments may be utilized, and structural and functional changes may be made, without departing from the scope of the present disclosure. Accordingly, the scope of the present disclosure should be construed broadly as set forth in the appended claims.

[0084] Sequence Listing SEQ ID NO: 1: precursor sequence

Claims

1. 1. A method for preparing circular RNA, comprising: providing a template DNA containing a sequence encoding a precursor RNA in a reaction solution that enables synthesis of the precursor RNA by in vitro transcription of the template DNA; and self-splicing the precursor RNA to produce circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing of the precursor RNA are carried out in the same reaction solution and under the same reaction conditions.

2. 2. The method of claim 1, which does not include a step of purifying the precursor RNA before allowing the precursor RNA to self-splice.

3. 3. The method of claim 1 or 2, wherein the DNA template comprises the following elements operably connected to each other and arranged in the following order: an RNA polymerase promoter, optionally a 5' homology arm, a 3' Group I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insertion sequence, optionally a 3' spacer sequence, a 5' Group I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm.

4. 4. The method of claim 3, wherein the insertion sequence comprises a protein coding sequence, and the insertion sequence comprises an IRES sequence operably connected to the protein coding sequence.

5. The reaction solution contains more than 26 mM of Mg ２＋ The method according to any one of claims 1 to 4, comprising:

6. The reaction solution contains more than 35 mM of Mg ２＋ and optionally, Mg in said solution ２＋ The method according to any one of claims 1 to 5, wherein the concentration of is between 38 mM and 66 mM.

7. 7. The method of claim 1, wherein the reaction solution comprises pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml.

8. The reaction solution contains 38 to 66 mM Mg ２＋ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, RNase inhibitor, ATP, GTP, CTP, UTP, DTT, and monovalent cations (Na ＋ or K ＋ 8. The method of claim 1, comprising:

9. 9. The method of claim 1, wherein the in vitro transcription of the template DNA and circularization (i.e., self-splicing) of the precursor RNA is carried out at a temperature between 37°C and 55°C.

10. 10. The method of any one of claims 1 to 9, wherein the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA is performed at a temperature greater than 37°C, and optionally, the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA is performed at a temperature between 39°C and 50°C.

11. 11. The method of any one of claims 1 to 10, wherein the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA is carried out for at least 1 hour, and optionally, the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA is carried out for 2.5 to 3 hours.

12. 12. The method of any one of claims 1 to 11, further comprising the step of removing the DNA template after synthesis of the precursor RNA, optionally the DNA template being removed by adding DNase I, e.g., at 37°C for 30 minutes.

13. 13. The method of claim 12, further comprising purifying the circular RNA after the step of removing the DNA template.

14. 14. The method of claim 13, wherein the purification step is selected from a precipitation step, a tangential flow filtration step, and a chromatography step, and combinations thereof.

15. A reaction solution for use in one-step circular RNA synthesis (e.g., for use in the method of any one of claims 1 to 14), comprising a Mg concentration of more than 26 mM. ２＋ , optionally pyrophosphatase (e.g., 1-4 U / ml pyrophosphatase), RNA polymerase, RNase inhibitor, nucleoside triphosphates (e.g., ATP, GTP, CTP, and UTP), reducing agent (e.g., DTT), and monovalent cations (e.g., Na ＋ , K. ＋ and combinations thereof).

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