Methods for making and isolating circular RNA and circular RNA compositions

Abstract

Provided herein are methods for making circular RNA, methods for isolating circular RNA, and compositions comprising circular RNA.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 481,944, filed January 27, 2023, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing The Sequence Listing submitted on January 26, 2024, was created on January 25, 2024, is entitled "10063-079WO1_ST26.xml", is in .XML format, has a file size of 100,751 bytes, and is incorporated by reference herein pursuant to 37 C.FR § 1.52(e)(5). [Background technology]

[0003] Circular RNA (circRNA) is a promising platform for RNA-based therapeutics. Like linear RNA, circRNA can also be translated into therapeutic proteins or vaccines. However, because circRNAs lack termini, they are less susceptible to degradation by most RNAse enzymes. Therefore, they are more stable than linear RNA, maintaining longer durations per RNA unit and enabling greater protein production. Prolonging the duration of protein expression not only promotes the efficacy of RNA vaccines but is particularly important for diseases requiring long-term therapeutic effects. Other advantages of circRNA include: (a) unlike linear RNA, circRNA does not require expensive modified nucleosides; (b) circRNA avoids cellular detection of exogenous RNA, which is known to induce immune responses; and (c) circRNA is known to be more heat-resistant than linear RNA, making circRNA storage more cost-effective.

[0004] Traditionally, circRNAs have been generated using an exon-intron reordering method called PIE, which involves chemical splicing under harsh conditions. These conditions pose two major limitations: (a) inefficient splicing and (b) the accumulation of contaminant by-products that create "nicks" within the circRNA, leading to linear "open" circles. Therefore, improved methods for generating circRNAs are needed. Summary of the Invention

[0005] Included herein is a method of making a circular RNA comprising: transcribing a vector to form a precursor RNA, wherein the vector comprises elements operably linked in the following order: a 3' Group I or 3' Group II intron sequence comprising a 3' splice site dinucleotide, a non-coding sequence, and a corresponding 5' Group I or 5' Group II intron sequence comprising a 5' splice site dinucleotide, excluding complementary sequences; and contacting the precursor RNA with a paired Group I or Group II maturase polypeptide to allow formation of the circular RNA. In some embodiments, the vector further comprises an internal ribosome entry site (IRES), as well as a protein-coding sequence and a second non-coding sequence.

[0006] Also included herein is a method of making a circular RNA comprising: transcribing a vector to form a precursor RNA, wherein the vector comprises the following elements operably linked in the following order: a 5' complementary sequence, a 3' Group I intron sequence or a 3' Group II intron sequence comprising a 3' splice site dinucleotide, a non-coding sequence, a corresponding 5' Group I intron sequence or a 5' Group II intron sequence comprising a 5' splice site dinucleotide, and the 3' complementary sequence; and contacting the precursor RNA with a paired Group I or Group II maturase polypeptide to allow formation of the circular RNA. In some embodiments, the vector further comprises an internal ribosome entry site (IRES), a protein-coding sequence, and a second non-coding sequence, in that order.

[0007] In certain embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature between 20° C. and 45° C. and a magnesium concentration between 100 micromolar and 25 millimolar.

[0008] In some embodiments, the vector further comprises a second IRES, a second protein-coding sequence, and a third non-coding sequence between the second non-coding sequence and the 5' Group II intron sequence comprising the 5' splice site dinucleotide.

[0009] In some embodiments, the vector further comprises a third IRES, a third protein-coding sequence, and a fourth non-coding sequence between the third non-coding sequence and the 5' Group II intron sequence comprising the 5' splice site dinucleotide.

[0010] In certain embodiments, the group I intron is an Aspergillus nidulans COB1 intron. The Aspergillus nidulans I-AniI maturase can pair with the Aspergillus nidulans COB1 intron. In some embodiments, the Aspergillus nidulans I-AniI maturase has reduced DNA endonuclease activity and / or increased splicing activity. The Aspergillus nidulans I-AniI maturase can also perform N-terminal truncations.

[0011] In some embodiments, the Group II intron is selected from the Oryza sativa trnK gene, tRNAs (V-UAC, I-GAU, A-UGC, and K-UUU), ribosomal proteins (rpl2 and rps12), and chloroplast ATPase (atpF). These Group II introns can pair with Oryza sativa maturase K.

[0012] In some embodiments, the group II intron is a Lactococcus lactis LtrA intron, which is capable of pairing with a Lactococcus lactis LtrA maturase.

[0013] In some embodiments, the group II intron is the Lactococcus lactis LtrB intron, which is capable of pairing with the Lactococcus lactis LtrB maturase.

[0014] In some embodiments, the maturase is cyt-19 from Neurospora crassa and the paired intron is selected from aI5γ and bI1 group II introns. In some embodiments, the maturase is cyt-19 from Saccharomyces cerevisiae and the paired intron is IIA intron aI2. In some embodiments, the maturase is Mss116p from Saccharomyces cerevisiae and the paired intron is IIA intron aI2. In some embodiments, the maturase is MatR from a Brassicaceae plant and the paired intron is nad1 i4. In some embodiments, the intron and paired maturase are as described in Table 2.

[0015] In certain embodiments, one or more of the non-coding sequences is between approximately 10 and 50 nucleotides, or between approximately 20 and 30 nucleotides. In certain embodiments, one or more of the non-coding sequences comprises a poly(A) sequence. In other embodiments, one or more of the non-coding sequences comprises only A and C nucleotides.

[0016] The IRES elements of the vectors are an aptamer for eIF4G, Homo sapiens cDNA FLJ43058, acute bee paralysis virus IRES, aphid lethal paralysis virus IRES, avian encephalomyelitis virus IRES, bovine viral diarrhea virus type 1 IRES, canine camper IRES, classical swine fever virus IRES, cosavirus, coxsackievirus type A (CVB1 / 2) IRES, coxsackievirus type B3 (CVB3) IRES, cricket paralysis virus IRES, crucifer tobamovirus IRES, Drosophila antennapedia IRES, Diresapivirus B1 IRES, Drosophila type C virus IRES, Drosophila hairless IRES, Drosophila reaper IRES, and Drosophila Ubx. IRES, Tea Grassworm-like Picorna-like Virus IRES, Encephalomyocarditis Virus (EMCV) IRES, Equine Rhinitis Virus IRES, Foot-and-Mouth Disease Virus IRES, Guangxi Discolored Lizard Picornavirus Type 2 IRES, Hepatitis A Virus IRES, Hepatitis C Virus IRES, Hepatitis GB Virus IRES, Hibiscus Yellow Ring Spot Virus IRES, Himetobi P Virus IRES, Homalodisca Coagulata Virus Type 1 IRES, Human AML1 / RUNX1 Gene IRES, Human Human AQP4 gene IRES, human AT1R gene IRES, human BAG1 gene IRES, human BCL2 gene IRES, human BiP gene IRES, human c-IAP1 gene IRES, human c-myc gene IRES, human c-src gene IRES, human eIF4G gene IRES, human enterovirus type 71 IRES, human FGF-1 gene IRES, human FGF2 gene IRES, human immunodeficiency virus type 1 IRES, human LEF1 gene IRES, human n.myc gene IRES, human p27kipl gene IRES, human p53 gene IRES, human papillomavirus IRES, human PDGF2 / c-sis gene IRES, human Pim-1 gene IRES, human rhinovirus 2 IRES, human SFTPAl gene IRES, human UNR gene IRES, human VEGF-A gene IRES, human XIAP gene IRES, human ELG1 IRES, human caspase 8-related protein 2 (CASP8AP2), Kashmir bee virus IRES, mouse eukaryotic translation initiation factor 1A domain-containing 14 (Eif1ad14) gene IRES, mouse Gtx gene IRES, mouse HIF1 alpha gene IRES, mouse NDST4L gene IRES, mouse Rbm3 gene IRES, mouse UtrA gene IRES, mouse LINE-1 ORF1 The IRES may have a sequence selected from IRESs, parechovirus IRESs, pink-eared duck picornavirus IRESs, brown marmorated stink bug enteric virus IRESs, poliovirus type 1 IRESs, reticuloendotheliosis virus IRESs, wheat aphid IRESs, rus sarcoma virus IRESs, Saccharomyces cerevisiae TFIID gene IRESs, Saccharomyces cerevisiae YAP1 gene IRESs, salivirus IRESs, simian picornavirus IRESs, simian virus 40 IRESs, red fire ant virus type 1 IRESs, taura syndrome virus IRESs, Theiler's encephalomyelitis virus IRESs, Triatomine bug virus IRESs, California sea slug ELH gene IRESs, synthetic IRESs, or novel synthetic IRESs. In some embodiments, the synthetic IRES is generated by creating a chimera by adding a fragment of a known IRES to the IRES. In some embodiments, the synthetic IRES is PPT19. In some embodiments, the synthetic IRES is KMI1.

[0017] In some embodiments, the protein coding sequence encodes a viral protein, a eukaryotic protein, or a prokaryotic protein. The eukaryotic protein can be a human protein. In some embodiments, the protein coding sequence encodes an antibody, such as a bispecific antibody or a monoclonal antibody. In some embodiments, the protein coding sequence encodes a viral antigen or a bacterial antigen. In other embodiments, the protein coding sequence encodes a fungal antigen or a protozoan antigen.

[0018] Also included herein are circular RNAs made according to the methods described herein, in some embodiments, the circular RNAs contain between 300 and 12,000 nucleotides.

[0019] Further included herein is a pharmaceutical composition comprising a circular RNA as described herein and a pharmaceutically acceptable nanocarrier selected from the group consisting of a lipid nanoparticle, a lipid, a lipid polymer, a lipopolymer hybrid, an exosome, and a leucosome.

[0020] Still further included herein are methods for isolating circular RNAs described herein. In some embodiments, the methods include obtaining a mixture of linear RNA and circular RNAs produced by the methods described herein, contacting the mixture with one or more immobilized 5'-binding proteins and one or more immobilized 3'-binding proteins, and isolating the circular RNAs. In some embodiments, the 5'-binding protein is a DXO 5'-binding protein from Saccharomyces cerevisiae, or a homolog thereof. In other embodiments, the method for isolating circular RNAs includes obtaining a mixture of linear RNA and circular RNAs produced by the methods described herein and isolating the circular RNAs using size exclusion chromatography. In other embodiments, polymerase A, RtcB RNA ligase, or a polynucleotide specific for the circularization junction is used for isolation. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram showing maturase-assisted circular RNA production. [Figure 2] This shows that expression of the reporter protein GFP (green fluorescent protein) from circular RNA continues to increase even 48 hours after transfection, whereas protein expression from linear RNA begins to decrease after 16 hours. [Figure 3] Chemical splicing of AnCob1 linear RNA (full size (mid-size Cob)) and truncated size (small intron) is shown over time. [Figure 4] AB show the purification of I-aniI maturase (A) and expression of I-aniI maturase from total bacterial lysate (B). [Figure 5] The test conditions for I-aniI maturase expression in E. coli are shown using an SDS-page gel, with the arrow indicating the overexpressed recombinant maturase. [Figure 6] 1 shows SDS-page gels for each step for the purification of I-aniI maturase to obtain pure recombinant I-aniI protein. [Figure 7] Diagram of the general mechanism of intron splicing in permuted intron-exon (PIE) constructs to generate circRNAs. [Figure 8] Based on the predictions of the RNAfold web server, we show that the structure of the PIE (permutation-intron-exon) version of the AnCob1 intron retains the same structure as the original one. [Figure 9] The designs of PIE constructs (based on AnCob1 introns) A to E are shown with their corresponding predicted structures using the RNAfold web server. [Figure 10]Shown is an agarose gel (e-gel) showing the initial chemical splicing reaction in tested constructs A, C, and E. Some splicing occurs to the 5' intron (225 bases). [Figure 11] Shown is an agarose gel (e-gel) of chemical splicing of construct D at different temperatures, incubation times and buffers. In condition 6, construct D appears to splice more efficiently. [Figure 12] Agarose gel (e-gel) showing chemical splicing of construct F in generating circRNA and showing an increase in splicing intermediates over time. [Figure 13] FIG. 1 is a schematic diagram showing a method for isolating circular RNA using 5′ and 3′ RNA binding proteins. [Figure 14] Figures A-B show successful recombinant protein expression for DXO, cleaved I-aniI (NusA) (A), and I-aniI (OEC-1 and OEC-2) (B). Bands of the expected size (in kD) were observed for high protein expression. [Figure 15] We show that the N-terminal truncation of I-aniI purification has been optimized to prevent protein cleavage during the bacterial lysis step. [Figure 16-1]Figures A–E show that maturase-assisted circularization of the designed constructs in the presence of I-aniI resulted in the production of mostly circRNAs, consistent with the structural prediction. To characterize construct B, which is more efficient at producing circRNAs than the other constructs, we performed extended experiments. The newly generated RNA material was resistant to RNAse R treatment, confirming its high likelihood of being circular RNA. (A) RNA obtained by in vitro transcription of construct B was incubated without maturase I-aniI or with maturase I-aniI at a molar ratio (RNA to maturase) approaching 1:1 at 37°C for 1 hour. Only in the presence of maturase did a novel band corresponding to the circRNA appear. (B) CircRNA production using the construct B ribozyme and increasing the dose of I-aniI showed improved circRNA production (intensified circRNA band). When treated with RNAse R, which degrades linear RNA but not circRNA, this novel band was observed to be resistant to RNAse R, thereby confirming its identity as circRNA material. [Figure 16-2]Figures A–E show that maturase-assisted circularization of the designed constructs in the presence of I-aniI resulted in the production of mostly circRNAs, consistent with the structural prediction. To characterize construct B, which is more efficient at producing circRNAs than the other constructs, extended experiments were performed. The newly generated RNA material was resistant to RNAse R treatment, confirming its high likelihood of being circular RNA. (C) Constructs A, B, C, D, and E were all spliced ​​with I-aniI at a 1:1 molar ratio for 1 hour and 30 minutes at 37°C. CircRNA bands were observed for A, B, and C. Construct E (negative control) did not produce circRNA, and construct D spliced, but the size of the circRNA band was not visible on the gel. Spliced ​​constructs F, G, and H were not spliced ​​at a 1:1 molar ratio. While some splicing was present, the reaction was not optimized. (D) Construct B was incubated in the presence or absence of I-aniI maturase for the indicated times. In the presence of maturase, circRNAs were clearly produced, and the reaction reached greater completion after 160 min. [Figure 16-3] (A–E) show that maturase-assisted circularization of the designed constructs in the presence of I-aniI resulted in the production of mostly circRNAs, consistent with the structural prediction. To characterize construct B, which is more efficient at producing circRNAs than the other constructs, we extended the experiment. The newly generated RNA material was resistant to RNAse R treatment, confirming its high likelihood of being circular RNA. (E) Incubation of circRNA or precursor RNA derived from construct B with RNAse R resulted in the degradation of most of the precursor (-I-aniI+R) and the circRNA becoming resistant to degradation (+I-aniI+R), suggesting that the RNA material produced is circular RNA. [Figure 17]Using RT-PCR and Sanger sequencing, we demonstrate that circRNAs are produced using maturase-assisted RNA circularization. The unique junction sequence linking the 5'- and 3'-truncated exons is only possible when the RNA is circular. [Figure 18-1] Figures A–C show that the newly designed circRNA sequence (A) can form circRNAs in vitro (B). In this figure, "placeholder" refers to the "coding sequence." (C) Predicted schematic diagrams of all ribozyme structures tested. Each structure was constructed using the RNAfold web server with standard parameters. Figures A, B, and C show conservation of splicing configurations enabled by homologous arms (gray), while in Figure E, homologous arms are absent, distorting the ribozyme structure. The predicted structure consists of an intron, exon 1, exon 2, homologous arms, IRES, Nluc ORF, and MCS. [Figure 18-2] Figures A–C show that the newly designed circRNA sequence (A) can form circRNAs in vitro (B). In this figure, "placeholder" refers to the "coding sequence." (C) Predicted schematic diagrams of all ribozyme structures tested. Each structure was constructed using the RNAfold web server with standard parameters. Figures A, B, and C show conservation of splicing configurations enabled by homologous arms (gray), while in Figure E, homologous arms are absent, distorting the ribozyme structure. The predicted structure consists of an intron, exon 1, exon 2, homologous arms, IRES, Nluc ORF, and MCS. [Figure 19-1]Panels A–C show a series of optimization experiments to increase circularization efficiency. Constructs A, B, and C were successfully used to produce circRNA. (A) Constructs A, B, and C were incubated with I-AniI maturase for 3 hours at 37°C. (B) Construct B was incubated with I-AniI maturase for 20 and 45 minutes at temperatures ranging from room temperature to 65°C, using buffer r3.1 (NEB: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml recombinant albumin, pH 7.9) or buffer TKM (TK + 25 mM MgCl2, 50 mM Tris pH 7, 1 mM DTT), all in the presence of 1 mM GTP. [Figure 19-2] A-C show a series of optimization experiments to increase circularization efficiency. Constructs A, B, and C successfully produced circRNA. (C) Incubation of construct B with I-AniI maturase in TKM buffer: 2x the amount of maturase (molar ratio) of construct B for 4 hours at 37°C (lanes 3 and 4), 3x the amount of maturase (molar ratio) of construct B for 3 hours at 37°C (lanes 5 and 6), and 2x the amount of maturase (molar ratio) of construct B for 3 hours at 42°C (lanes 7 and 8). [Figure 20] We demonstrate that circRNAs generated by maturase (I-AniI)-assisted circularization can be translated into proteins. Nanoluciferase activity was detected from circRNAs encoding nanoluciferase reporter proteins. [Figure 21] It has been shown that maturase K has been cloned and expressed. [Figure 22] A-B show recombinant LtrA maturase expression in E. coli as seen on an unstained SDS-PAGE gel and by Western blot analysis using an anti-V5 antibody (A). Recombinant LtrA maturase was purified from E. coli cell lysates (B). The arrow indicates the expected LtrA fusion protein band. [Figure 23-1] Figures A–C show the self-splicing and maturase-assisted RNA circularization profiles for P2.1 (working control) and P2.2 (A). The P2.1 construct is P2 with a 15-nucleotide extension on the 5' exon (to create a long non-coding sequence) to facilitate RNA circularization. The P2.2 construct is P2.1 with a 15-nucleotide extension on the 3' exon (to create a long non-coding sequence) to facilitate RNA circularization. Figures B show the RNA circularization profiles for P2.1 and P2.2 after adjusting the incubation time and assaying the RNA at a 1:4 ratio with LtrA maturase, as seen on a 1.2% FlashGel™ RNA cassette. The double asterisk indicates the circRNA. Detection of novel circRNA-specific sequence junctions using reverse transcriptase PCR followed by Sanger sequencing demonstrates that the circRNA is fully circular (C). [Figure 23-2] Figures A–C show the self-splicing and maturase-assisted RNA circularization profiles for P2.1 (working control) and P2.2 (A). The P2.1 construct is P2 with a 15-nucleotide extension on the 5' exon (to create a long non-coding sequence) to facilitate RNA circularization. The P2.2 construct is P2.1 with a 15-nucleotide extension on the 3' exon (to create a long non-coding sequence) to facilitate RNA circularization. Figures B show the RNA circularization profiles for P2.1 and P2.2 after adjusting the incubation time and assaying the RNA at a 1:4 ratio with LtrA maturase, as seen on a 1.2% FlashGel™ RNA cassette. The double asterisk indicates the circRNA. Detection of novel circRNA-specific sequence junctions using reverse transcriptase PCR followed by Sanger sequencing demonstrates that the circRNA is fully circular (C). [Figure 24-1]A–D show different sequence designs for circRNAs using the LtrA maturase, with "D" referring to the RNA domain of the LtrB intron (A). (B–C) RNA structure predictions generated by RNAfold for both the linear control RNA (Lin1) and the RNA circularization constructs during the initial design, validation, and optimization phases of the project. The designations D1, D2, D3, D4, D5, and D6 in (A) correspond to the RNA domains numbered I, II, III, IV, V, and VI, respectively, shown in the schematic diagram provided in (D). [Figure 24-2] A–D show different sequence designs for circRNAs using the LtrA maturase, with "D" referring to the RNA domain of the LtrB intron (A). (B–C) RNA structure predictions generated by RNAfold for both the linear control RNA (Lin1) and the RNA circularization constructs during the initial design, validation, and optimization phases of the project. The designations D1, D2, D3, D4, D5, and D6 in (A) correspond to the RNA domains numbered I, II, III, IV, V, and VI, respectively, shown in the schematic diagram provided in (D). [Figure 24-3] A–D show different sequence designs for circRNAs using the LtrA maturase, with "D" referring to the RNA domain of the LtrB intron (A). (B–C) RNA structure predictions generated by RNAfold for both the linear control RNA (Lin1) and the RNA circularization constructs during the initial design, validation, and optimization phases of the project. The designations D1, D2, D3, D4, D5, and D6 in (A) correspond to the RNA domains numbered I, II, III, IV, V, and VI, respectively, shown in the schematic diagram provided in (D). [Figure 25]A-B show the RNA circularization profile for P2 and the splicing profile for Lin1 after adjusting the incubation time and assaying a 1:2 ratio of RNA to LtrA maturase (A). The maturase-assisted generation of circRNAs using an optimized circularization sequence (P2.1 construct has an extension to facilitate RNA circularization) and the splicing profile for Lin1 linear RNA control (B). The double asterisk indicates the circRNA. [Figure 26] Nanoluciferase reporter activity in U2OS-K294A cells transfected with LtrA maturase-assisted circRNA (circ5.1Nluc) and reference circRNA (circNluc), demonstrating that LtrA maturase-assisted circular RNAs can express encoded proteins. [Figure 27-1] A-C show recombinant MatR maturase expression in E. coli as seen on an unstained SDS-PAGE gel and by Western blot analysis using an anti-V5 antibody (A). Various conditions are evaluated to optimize MatR expression in E. coli (B). Recombinant MatR maturase is purified from E. coli cell lysates as seen on an unstained SDS-PAGE gel (C). The arrow indicates the predicted MatR maturase protein. [Figure 27-2] A-C show recombinant MatR maturase expression in E. coli as seen on an unstained SDS-PAGE gel and by Western blot analysis using an anti-V5 antibody (A). Various conditions are evaluated to optimize MatR expression in E. coli (B). Recombinant MatR maturase is purified from E. coli cell lysates as seen on an unstained SDS-PAGE gel (C). The arrow indicates the predicted MatR maturase protein. [Figure 28] A–B show that the Cyt-19 DEAD box protein was highly expressed in bacteria (A) and successfully purified from E. coli lysate (B). [Figure 29] This shows that in the circularization reaction using circRNA construct B (described in Figures 17-19), increasing the amount of cyt19 protein did not have a positive effect on promoting circRNA formation. [Figure 30] Using reverse transcriptase PCR followed by Sanger sequencing to detect sequence junctions specific to the novel circRNA, we demonstrate that the circRNA is perfectly circular. This particular circRNA design uses a non-coding RNA sequence to increase the efficiency of circRNA production. [Figure 31-1] Figures A–C show a schematic diagram of the plasmid backbone for generating a pooled internal ribosome entry site (IRES) testing library (up to 108 unique 150-nt random sequences). Each 150-nt random sequence precedes the mCherry reporter protein coding sequence (A). [Figure 31-2] A-C show a schematic diagram of the plasmid backbone for generating an internal ribosome entry site (IRES) test pooled library (up to 108 unique 150-base random sequences). CircRNAs were generated using the linearized plasmid library as a template (B). A micrograph shows that a small number of cells can express the mCherry reporter protein from the transfected circRNAs. Only circRNAs with a functional IRES sequence should be able to produce mCherry protein in cells (C). [Figure 32] Isolation of pure circRNA using the circRNA capture method is shown. A biotinylated locked nucleic acid oligonucleotide (TT177) specific to the RNA circularization junction was hybridized to the circRNA to separate and purify the intact circRNA (indicated by the oval) from the linear RNA (bar). A 2-hour incubation resulted in good recovery of the circRNA using streptavidin beads, and the circRNA was intact. [Figure 33]A-B show the isolation of pure circRNA (linear RNA capture method #1) using RNA ligase and desthiobiotin, followed by recovery using streptavidin beads, to separate and purify intact circRNA (indicated by ovals) from linear RNA contaminants (bars). Experiments were performed using both crude circRNA preparations (A) and pure circRNA preparations (B). Little enrichment of circRNAs was observed. [Figure 34] We demonstrate the isolation of pure circRNA (Linear RNA Capture Method #2) using RtcB protein to bind and capture linear RNA by-products. This circRNA passes through the procedure unmodified, albeit with some loss of abundance (Sample 5 in different lanes of two gels). Results clearly demonstrate a reduction in the major linear RNA species (faint band in Sample 1 on the bottom gel), but the ability to capture linear RNA by-products was not quantitatively assessed. [Figure 35] A-B show the first part of the isolation of pure circRNA using DXO protein to bind and capture linear RNA by-products (linear RNA capture method #3.1 and #3.2). Expression (A) and purification (B) of wild-type and exonuclease mutant DXO proteins from E. coli cell lysates. Arrows point to the expected WT and D236A / E253A mutant DXO protein bands (expected 54 kDa). [Figure 36]Figures A-B show the second part of the isolation of pure circRNAs (linear RNA capture methods #3.1 and #3.2) using DXO protein to bind and capture linear RNA byproducts. Exoribonuclease assays were performed on two different circRNAs using wild-type and D236A / E253A (nuclease-inactive) mutant DXO. Purified WT DXO possesses nuclease activity, as indicated by the RNA cleavage products (brackets) in two different RNA substrates treated with two different methods: circESAT6 IVT RNA (A) and circGFP-3XFL IVT RNA (B). These cleavage products were not observed after mutant DXO treatment, suggesting that nuclease activity is abolished in the D236A / E253A mutant DXO produced. [Figure 37-1] A-B show the isolation of pure circRNA (linear RNA capture method #4), which uses poly(A) polymerase to extend linear RNA by-products with biotinylated adenosines, which are then used to bind and capture the linear RNA by-products. The products after poly(A) extension of a mixed pool of circRNA and linear RNA substrates (A: short arrows indicate linear by-products, while long color-matched arrows indicate polyadenylated linear RNA by-products after the procedure). The resulting samples were assayed using a TapeStation chromatogram (B). The intensities of the samples separated in each lane of the chromatogram in (B) are shown in (C-H). [Figure 37-2] A-B show the isolation of pure circRNA (linear RNA capture method #4), which uses poly(A) polymerase to extend linear RNA by-products with biotinylated adenosines, which are then used to bind and capture the linear RNA by-products. The products after poly(A) extension of a mixed pool of circRNA and linear RNA substrates (A: short arrows indicate linear by-products, while long color-matched arrows indicate polyadenylated linear RNA by-products after the procedure). The resulting samples were assayed using a TapeStation chromatogram (B). The intensities of the samples separated in each lane of the chromatogram in (B) are shown in (C-H). [Figure 37-3] A-B show the isolation of pure circRNA (linear RNA capture method #4), which uses poly(A) polymerase to extend linear RNA by-products with biotinylated adenosines, which are then used to bind and capture the linear RNA by-products. The products after poly(A) extension of a mixed pool of circRNA and linear RNA substrates (A: short arrows indicate linear by-products, while long color-matched arrows indicate polyadenylated linear RNA by-products after the procedure). The resulting samples were assayed using a TapeStation chromatogram (B). The intensities of the samples separated in each lane of the chromatogram in (B) are shown in (C-H). [Figure 37-4] A-B show the isolation of pure circRNA (linear RNA capture method #4), which uses poly(A) polymerase to extend linear RNA by-products with biotinylated adenosines, which are then used to bind and capture the linear RNA by-products. The products after poly(A) extension of a mixed pool of circRNA and linear RNA substrates (A: short arrows indicate linear by-products, while long color-matched arrows indicate polyadenylated linear RNA by-products after the procedure). The resulting samples were assayed using a TapeStation chromatogram (B). The intensities of the samples separated in each lane of the chromatogram in (B) are shown in (C-H). [Figure 37-5] A-B show the isolation of pure circRNA (linear RNA capture method #4), which uses poly(A) polymerase to extend linear RNA by-products with biotinylated adenosines, which are then used to bind and capture the linear RNA by-products. The products after poly(A) extension of a mixed pool of circRNA and linear RNA substrates (A: short arrows indicate linear by-products, while long color-matched arrows indicate polyadenylated linear RNA by-products after the procedure). The resulting samples were assayed using a TapeStation chromatogram (B). The intensities of the samples separated in each lane of the chromatogram in (B) are shown in (C-H). DETAILED DESCRIPTION OF THE INVENTION

[0022] Provided herein is a method for producing circular RNA, which is a significant improvement over methods in the prior art. In some embodiments, the method is more efficient and effective than methods in the prior art. Also included herein are the methods for isolating circular RNA described herein, circular RNA compositions, and pharmaceutical compositions comprising circular RNA compositions. The methods provided herein utilize maturase, more specifically, maturase-intron pairs, which allows for the use of milder reaction conditions and improved circRNA yields.

[0023] Terms used throughout this application should be interpreted with their ordinary and typical meanings to those of ordinary skill in the art. However, applicant wishes to give specific definitions to the following terms, as defined below.

[0024] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0025] The terms "about" and "approximately" are defined as "approximate to" as understood by one of ordinary skill in the art. In one non-limiting embodiment, these terms are defined to be within 10%. In another non-limiting embodiment, these terms are defined to be within 5%. In yet another non-limiting embodiment, these terms are defined to be within 1%.

[0026] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L chains) and two identical heavy chains (H chains). Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end.

[0027] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. The phrase "functional fragment or analog" of an antibody refers to a compound that has qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that is capable of binding to IgE immunoglobulins in a manner that prevents or substantially reduces the ability of such molecules to bind to the high-affinity receptor, FcεRI. As used herein, "functional fragment" with respect to antibodies refers to Fv, F(ab) and F(ab')2 fragments. An "Fv" fragment is the smallest antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer (V) of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. H -V L In this configuration, the three CDRs of each variable domain interact to form a V H -V LThe six CDRs define a target-binding site on the surface of the dimer. Collectively, the six CDRs confer target-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three target-specific CDRs) has the ability to recognize and bind to a target, albeit with lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the V of an antibody. H and V L Fv polypeptides generally contain V domains, and these domains are present in a single polypeptide chain. H Domains and V L It further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for target binding.

[0028] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, and the like. "Consisting of" shall mean excluding more than trace elements of other ingredients, and substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0029] A "control" is a substitute subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0030] "Control sequences" refer to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader can be operably linked to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous.

[0031] "Reduction" or "decrease" can refer to any change that results in a lesser amount of a symptom, disease, composition, condition, or activity. A reduction or decrease can be a statistically significant amount of any individual, median, or mean decrease in a condition, symptom, activity, or composition. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease, as long as the decrease is statistically significant.

[0032] "Homologs" are defined herein as two polynucleotides or two polypeptides that share a degree of identity. Homologues include allelic variants, orthologs, and paralogs that have the same related function (e.g., the ability to function as a maturase). In some embodiments, homologs have about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identity. In other embodiments, homologs have about 80% or about 85% identity. In other embodiments, homologs have about 50% identity. In some embodiments, homologs have about 50%, 60%, 70%, or 80% identity.

[0033] The term "identity" shall be interpreted as meaning the percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical to the bases or residues of the corresponding sequence being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence; conservative substitutions are not considered part of sequence identity. Neither N- nor C-terminal extensions or insertions shall be interpreted as a reduction in identity or homology. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that that percentage of bases (or amino acids) match when the two sequences are compared and aligned over their entire length. It should be understood that alignment over "their entire length" applies not only to full-length polynucleotides but also to polynucleotide regions. Such polynucleotide regions are referred to herein as "conserved regions." This alignment and percent sequence identity can be calculated using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment, the BLAST program is used with default parameters. In one embodiment, the BLAST programs BLASTN and BLASTP are used with the following default parameters: genetic code=standard, filter=none, strand=both, cutoff=60, expectation=10, matrix=BLOSUM62, number of descriptions=50 sequences, sort criteria=highest score, database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR.

[0034] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or mean increase in a condition, symptom, activity, or composition by a statistically significant amount. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, as long as the increase is statistically significant.

[0035] The term "maturase" is used herein to refer to a protein that can assist or facilitate intron splicing. In some embodiments, the maturase is an RNA chaperone such as cyt-19.

[0036] "Non-coding sequence" as used herein refers to an amino acid sequence that does not encode an amino acid.

[0037] A "pharmaceutical composition" is intended to include a combination with a pharmaceutically acceptable carrier, inert or active, that makes the composition suitable for diagnostic or therapeutic use in vivo or in vitro.

[0038] The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable nanocarrier" generally refer to a carrier or excipient that is safe and nontoxic and useful in preparing pharmaceutical compositions, including carriers suitable for veterinary and / or human pharmaceutical use. As used herein, the term "pharmaceutically acceptable carrier" encompasses standard pharmaceutical carriers such as phosphate-buffered saline, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations, as well as materials further described below. Pharmaceutical compositions may also include preservatives. As used herein and in the claims, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable nanocarrier" encompasses both one and more than one such carrier. In some embodiments, the pharmaceutically acceptable nanocarrier is a lipid nanoparticle, a lipid, a lipid polymer, a lipopolymer hybrid, an exosome, or a leucosome.

[0039] As used herein, the term "protein coding sequence" refers to a polynucleotide sequence that encodes a polypeptide, including fragments of known functional polypeptides. Similarly, the term "protein" includes complete proteins and fragments of proteins.

[0040] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one aspect, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or an animal under veterinary care. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0041] The term "vector" refers to a vehicle that carries a polynucleotide into a cell for expression within the cell. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, may be integrated into the genome itself. In some embodiments, a vector is a DNA construct that contains a DNA sequence operably linked to suitable control sequences that are capable of affecting the expression of DNA in a suitable host cell. In some embodiments, such control sequences may include a promoter for initiating transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control the termination of transcription and translation.

[0042] explanation As described above, provided herein are methods for generating circular RNAs that significantly improve upon prior art methods. Also included herein are methods for isolating circular RNAs, circular RNA compositions, and pharmaceutical compositions containing the circular RNA compositions described herein. Among other things, it is a surprising discovery that the use of maturase-assisted intron pairs increases splicing efficiency and reduces by-product formation. Figure 1 provides a schematic diagram of the maturase-assisted process for creating circular RNAs.

[0043] In some embodiments, the method of generating a circular RNA comprises: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' Group I intron or a 3' Group II intron sequence including a 3' splice site dinucleotide; ii. a non-coding sequence; iii. the corresponding 5' Group I intron or 5' Group II intron sequence, including the 5' splice site dinucleotide; and excluding the complementary sequence; b. contacting the precursor RNA with a pair of Group I or Group II maturase polypeptides to allow formation of a circular RNA; It should be understood that for all embodiments described herein, the transferring and contacting steps can occur simultaneously or sequentially, and in some embodiments, the contacting step begins before the transfer is complete.

[0044] In some embodiments, the method of making a circular RNA comprises: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' Group I intron or a 3' Group II intron sequence including a 3' splice site dinucleotide; ii. a first non-coding sequence; and iii. a protein coding sequence; iv. a second non-coding sequence; and v. the corresponding 5' Group I intron or 5' Group II intron sequence including the 5' splice site dinucleotide; and and excluding the complementary sequence; b. contacting the precursor RNA with a paired Group I or Group II maturase polypeptide to allow formation of a circular RNA; Includes.

[0045] In some methods, the vector further comprises an internal ribosome entry site (IRES), a protein coding sequence, and a second non-coding sequence, in that order, between elements a.ii. and a.iii. Thus, included herein are: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' Group I intron sequence or a 3' Group II intron sequence including a 3' splice site dinucleotide; ii. a first non-coding sequence; and iii. an internal ribosome entry site (IRES); iv. a protein coding sequence; v. a second non-coding sequence; and vi. the corresponding 5' Group I intron sequence or 5' Group II intron sequence containing a 5' splice site dinucleotide; and excluding homologous sequences; b. contacting the precursor RNA with a paired group I maturase or a paired group II maturase polypeptide to allow formation of the circular RNA.

[0046] As used herein, "group I intron" refers to a self-splicing ribozyme capable of catalyzing self-cleavage from precursor RNA. Group I introns generally consist of nine paired regions, P1-P9, and often fold into two domains: the P4-P6 domain, formed by stacking helices P5, P4, P6, and P6a, and the P3-P9 domain, formed by helices P8, P3, P7, and P9. "Group II intron" also refers to a self-splicing ribozyme capable of catalyzing self-cleavage from precursor RNA. However, group II introns differ from group I in that excision can occur even in the absence of GTP. Group II introns generally have a secondary structure of six stem-loop structures, D1-D6, with the domains radiating from a central core that brings the 5' and 3' splice junctions into close proximity.

[0047] The term "3' Group I intron sequence" refers to any DNA sequence encoding a Group I intron RNA sequence that includes the 3' end of the Group I intron but not the 5' end of the Group I intron. The term "3' Group II intron sequence" refers to any DNA sequence encoding a Group II intron RNA sequence that includes the 3' end of the Group II intron but not the 5' end of the Group II intron. The term "5' Group I intron sequence" refers to any DNA sequence encoding a Group I intron RNA sequence that includes the 5' end of the Group I intron but not the 3' end of the Group I intron. The term "5' Group II intron sequence" refers to any DNA sequence encoding a Group II intron RNA sequence that includes the 5' end of the Group II intron but not the 3' end of the Group II intron. In some embodiments, the 5' or 3' Group I or II sequence contains a deletion. An example of such a deletion is within the "minimal D2 and D3" element in construct P2. In some embodiments, the 5' or 3' Group I or II sequence is approximately 100 to 750 nucleotides in length. "Corresponding 5' Group I or 5' Group II intron sequence" refers to a 5' Group I or Group II intron sequence derived from a Group I or Group II intron that is identical to the aforementioned 3' Group I or Group II intron sequence, respectively. In some embodiments, the 3' and 5' intron sequences are approximately half of a Group I or Group II intron, respectively. In some embodiments, the 3' and 5' intron sequences are the result of differential cleavage of a Group I or Group II intron. The 3' and 5' splice site dinucleotides can be any dinucleotide that is a splice site for a Group I or Group II intron. In some embodiments, the 5' splice site dinucleotide is GT.

[0048] The 3' Group I intron sequence comprises SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:32, or SEQ ID NO:34. Thus, in some embodiments, the 3' Group I intron sequence comprises or is SEQ ID NO:27. In some embodiments, the 3' Group I intron sequence comprises or is SEQ ID NO:30. In some embodiments, the 3' Group I intron sequence comprises or is SEQ ID NO:32. In some embodiments, the 3' Group I intron sequence comprises or is SEQ ID NO:34. In some aspects, the 5' Group I intron sequence comprises or is SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35. Thus, in some embodiments, the 5' Group I intron sequence comprises or is SEQ ID NO:29. In some embodiments, the 5' Group I intron sequence comprises or is SEQ ID NO:31. In some embodiments, the 5' Group I intron sequence comprises or is SEQ ID NO: 33. In some embodiments, the 5' Group I intron sequence comprises or is SEQ ID NO: 35.

[0049] Thus, in some embodiments, a method for generating a circular RNA comprises: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' group I intron sequence including a 3' splice site dinucleotide; and ii. a non-coding sequence; iii. the corresponding 5' Group I intron sequence, including the 5' splice site dinucleotide; and and excluding the complementary sequence; b. contacting the precursor RNA with a paired Group II maturase polypeptide to allow formation of a circular RNA; Includes.

[0050] In another embodiment, the method for generating circular RNA comprises: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' group II intron sequence including a 3' splice site dinucleotide; ii. a non-coding sequence; iii. the corresponding 5' Group II intron sequence including the 5' splice site dinucleotide; and and excluding the complementary sequence; b. contacting the precursor RNA with a paired Group II maturase polypeptide to allow formation of a circular RNA; Includes.

[0051] In some methods, the vector further comprises an internal ribosome entry site (IRES), a protein coding sequence, and a second non-coding sequence, in that order, between elements a.ii. and a.iii.

[0052] Thus, included herein are: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' group I intron sequence including a 3' splice site dinucleotide; and ii. a first non-coding sequence; and iii. an internal ribosome entry site (IRES); iv. a protein coding sequence; v. a second non-coding sequence; and vi. the corresponding 5' Group I intron sequence including the 5' splice site dinucleotide; and and excluding the complementary sequence; b. contacting the precursor RNA with a pair of Group I maturase polypeptides to allow formation of a circular RNA, wherein the 3' splice site dinucleotide and the 5' splice site dinucleotide are splice sites for the Group I intron; A method for producing circular RNA, comprising:

[0053] In another embodiment, the method for generating circular RNA comprises: a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 3' group II intron sequence including a 3' splice site dinucleotide; ii. a first non-coding sequence; and iii. an internal ribosome entry site (IRES); iv. a protein coding sequence; v. a second non-coding sequence; and vi. the corresponding 5' Group II intron sequence including the 5' splice site dinucleotide; and excluding the complementary sequence; b. contacting the precursor RNA with a pair of Group II maturase polypeptides to allow the formation of a circular RNA, wherein the 3' splice site dinucleotide and the 5' splice site dinucleotide are splice sites for the Group II intron; Includes.

[0054] A non-coding sequence of a vector is any DNA sequence that does not encode an amino acid. In some embodiments, the non-coding sequence is between about 15-50 nucleotides in length or between about 20-30 nucleotides in length. In some aspects, the non-coding sequence includes a poly(A) sequence. In other or further embodiments, the non-coding sequence includes only A and C nucleotides. In some embodiments, each non-coding sequence is identical. In other embodiments, each non-coding sequence is different. "Non-coding" sequences are also referred to herein as "exons." For example, each "exon" in FIG. 9 is or includes a non-coding sequence. In some embodiments, a "short" non-coding sequence or exon is about 15-25 nucleotides in length or 18-22 nucleotides in length. In other or further embodiments, a "long" non-coding sequence or exon is about 39 nucleotides in length. In some embodiments, the first non-coding sequence is or includes SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the second non-coding sequence is or comprises SEQ ID NO:42 or SEQ ID NO:43.

[0055] A "protein coding sequence" of a vector is a DNA sequence that encodes one or more amino acids. Therefore, the term "protein coding sequence" should be understood to include DNA sequences that encode complete protein fragments or portions. In some embodiments, the protein coding sequence encodes a viral protein, a eukaryotic protein, a prokaryotic protein, a fungal protein, or a protozoan protein, including viral antigens, bacterial antigens, fungal antigens, and protozoan antigens. In some embodiments, the protein coding sequence encodes a human protein. In some aspects, the protein coding sequence encodes an antibody. The antibody can be any antibody, including as that term is defined herein, and in some aspects, the antibody is bispecific. In some embodiments, the protein coding sequence encodes one or more proteins or protein fragments.

[0056] The present disclosure includes vectors comprising multiple protein-coding sequences. In some embodiments, the vector comprises one, two, or three coding sequences. Accordingly, included herein is a method of generating a circular RNA, wherein the vector further comprises a second IRES, a second protein-coding sequence, and a third non-coding sequence between the second non-coding sequence and the 5' Group I or Group II intron sequence comprising a 5' splice site dinucleotide. In even further embodiments, the vector comprises three IRESs, a third protein-coding sequence, and a fourth non-coding sequence between the third non-coding sequence and the 5' Group I or Group II intron sequence comprising a 5' splice site dinucleotide.

[0057] An internal ribosome entry site (IRES) is an RNA sequence that allows translation initiation. In some embodiments, the IRES is a sequence of an aptamer for eIF4G, Homo sapiens cDNA FLJ43058, acute honeybee paralysis virus IRES, aphid lethal paralysis virus IRES, avian encephalomyelitis virus IRES, bovine viral diarrhea virus type 1 IRES, canine scamper IRES, classical swine fever virus IRES, cosavirus, coxsackievirus A (CVB1 / 2) IRES, coxsackievirus B3 (CVB3) IRES, cricket paralysis virus IRES, crucifer tobamovirus IRES, Drosophila antennapedia IRES, Diresapivirus B1 IRES, Drosophila C virus IRES, Drosophila hairless IRES, Drosophila reaper IRES, Drosophila Ubx IRES, tea tortoise picorna-like virus IRES, encephalomyocarditis virus (EMCV) IRES, equine rhinitis virus IRES, foot-and-mouth disease virus IRES, Guangxi grass lizard picornavirus type 2 IRES, hepatitis A virus IRES, hepatitis C virus IRES, hepatitis GB virus IRES, hibiscus yellow ringspot virus IRES, small brown planthopper P virus IRES, homalogicis coagulata virus type 1 IRES, human AML1 / RUNX1 gene IRES, human A QP4 gene IRES, human AT1R gene IRES, human BAG-1 gene IRES, human BCL2 gene IRES, human BiP gene IRES, human c-IAPl gene IRES, human c-myc gene IRES, human c-src gene IRES, human eIF4G gene IRES, human enterovirus type 71 IRES, human FGF-1 gene IRES, human FGF2 gene IRES, human immunodeficiency virus type 1 IRES, human LEF1 gene IRES, human n.myc gene IRES, human p27kip1 gene IRES, human p53 gene IRES, human papillomavirus IRES, human PDGF2 / c-sis gene IRES, human Pim-1 gene IRES, human rhinovirus type 2 IRES, human SFTPAl gene IRES, human UNR gene IRES, human VEGF-A gene IRES, human XIAP gene IRES, Kashmir bee virus IRES, mouse eukaryotic translation initiation factor 1A domain-containing 14 (Eif1ad14) gene IRES, mouse Gtx gene IRES, mouse HIF1 alpha gene IRES, mouse NDST4L gene IRES, mouse Rbm3 gene IRES , mouse UtrA gene IRES, parechovirus IRES, pink-eared duck picornavirus IRES, German-winged stink bug enteric virus IRES, poliovirus type 1 IRES, reticuloendotheliosis virus IRES, Rhopalosiphum oryzae virus IRES, Rous sarcoma virus IRES, S. cerevisiae TFIID gene IRES, S. cerevisiae YAP1 gene IRES, salivirus IRES, simian picomavirus IRES, simian virus type 40 IRES, red fire ant virus type 1 IRES, taura syndrome virus IRES, Theileria encephalomyelitis virus IRES, or Triatomine bug virus IRES. In some embodiments, the IRES is a Coxsackievirus B3 (CVB3) IRES. In certain aspects, the CVB3 IRES comprises SEQ ID NO: 36. In some embodiments, the IRES is approximately 220 nucleotides in length.

[0058] As noted above, the methods for generating circular RNA described herein employ group I or group II maturases. Group I or group II maturases are paired with group I or group II introns, respectively. In this context, "paired" refers to a maturase that splices or assists in the splicing of a particular group I or group II intron. Table 2 provides exemplary group I or group II maturases with group I or group II intron pairs, all of which are contemplated within the scope of the present disclosure. Also included are homologs of the group I or group II maturases described herein.

[0059] In some embodiments, the Group I intron is an Aspergillus nidulans COB1 intron and its paired maturase is an Aspergillus nidulans I-AniI maturase. The term "I-Anil maturase" includes the polypeptide of SEQ ID NO: 3, or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to the polypeptide of SEQ ID NO: 3, or a polypeptide comprising a portion of the polypeptide of SEQ ID NO: 3. In some embodiments, the I-Anil maturase is N-terminally truncated in a manner that retains maturase domain function. In some aspects, about 5-10 amino acids preceding the maturase domain are retained in the N-terminally truncated I-Anil maturase. In some embodiments, the I-Anil maturase is a polypeptide of SEQ ID NO: 4, or a polypeptide sequence having at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity to the polypeptide of SEQ ID NO: 4, or a polypeptide comprising a portion of the polypeptide of SEQ ID NO: 4.

[0060] In some embodiments, the 3' Group I intron sequence is or comprises SEQ ID NO:27, and the 5' Group I intron sequence is or comprises SEQ ID NO:28. In some embodiments, the 3' Group I intron sequence is or comprises SEQ ID NO:27, and the 5' Group I intron sequence is or comprises SEQ ID NO:29. In some embodiments, the 3' Group I intron sequence is or comprises SEQ ID NO:30, and the 5' Group I intron sequence is or comprises SEQ ID NO:31. In some embodiments, the 3' Group I intron sequence is or comprises SEQ ID NO:32, and the 5' Group I intron sequence is or comprises SEQ ID NO:33. In some embodiments, the 3' Group I intron sequence is or comprises SEQ ID NO:34, and the 5' Group I intron sequence is or comprises SEQ ID NO:35.

[0061] In some embodiments, the group II intron is an Oryza sativa din tRNA-K(UUU) intron and the maturase paired therewith is Oryza sativa maturase K. The term "maturase K" includes a polypeptide encoded by SEQ ID NO: 1 or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to the polypeptide encoded by SEQ ID NO: 1, or a polypeptide comprising a portion of the polypeptide encoded by SEQ ID NO: 1. In some embodiments, the maturase is cyt-19 from Neurospora crassa and the introns are aI5γ and bI1 group II introns from yeast. The term "cyt-19" includes the polypeptide of SEQ ID NO:2, or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to the polypeptide of SEQ ID NO:2, or a polypeptide comprising a portion of the polypeptide of SEQ ID NO:2. In some embodiments, the group II intron is the L1.LtrB intron of Lactococcus lactis and the maturase paired therewith is the LtrA maturase of Lactococcus lactis. The term "LtrA maturase" includes the polypeptide of SEQ ID NO:15, or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to the polypeptide of SEQ ID NO:15, or a polypeptide comprising a portion of the polypeptide of SEQ ID NO:15. In some embodiments, the L1.LtrB intron comprises or is SEQ ID NO:45.

[0062] In some embodiments, the group II intron is the LtrB intron of Lactococcus lactis and its paired maturase is the LtrB maturase of Lactococcus lactis. In some aspects, the intron is nad1 i4 and its paired maturase is the MatR maturase. The term "MatR maturase" includes the polypeptide of SEQ ID NO: 16, or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to the polypeptide of SEQ ID NO: 16, or a polypeptide comprising a portion of the polypeptide of SEQ ID NO: 16. In other aspects, the intron is IIA intron aI2 and its paired maturase is MSS116. The term "MSS116" includes the polypeptide of SEQ ID NO: 17, or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to the polypeptide of SEQ ID NO: 17, or a polypeptide comprising a portion of the polypeptide of SEQ ID NO: 17. In yet other embodiments, the intron is a Group I or Group II intron and the paired maturase is as set forth in Table 2 or a homolog thereof.

[0063] It should be understood that the group I or group II intron and its corresponding maturase can be from different species. It should also be understood that the maturase can be mutated with one or more amino acids to reduce or eliminate DNA endonuclease activity and / or increase splicing activity. The maturase can also be N-terminally truncated. In some embodiments, the maturase is mutated to increase its solubility.

[0064] The inclusion of a maturase and a maturase-intron pair in the present invention allows for milder reaction conditions and / or improved circular RNA yield. Accordingly, included herein is a method for producing circular RNA by contacting a maturase polypeptide with a precursor RNA under conditions comprising a temperature between 20°C and 45°C. In some embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 20°C. In some embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 25°C. In some embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 30°C. In some embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 35°C. In some embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 37°C. In some embodiments, the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 42°C.

[0065] In some aspects, the maturase polypeptide and precursor RNA are contacted for approximately 30 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, or approximately 4 hours. In some embodiments, the maturase polypeptide and precursor RNA are contacted for approximately 2 hours. In some embodiments, the maturase polypeptide and precursor RNA are contacted for approximately 3 hours. In some embodiments, the maturase polypeptide and precursor RNA are contacted for approximately 4 hours.

[0066] In other or further embodiments, the maturase polypeptide and precursor RNA are contacted under conditions comprising a magnesium concentration of between 100 micromolar and 25 millimolar. In some embodiments, the magnesium concentration is about 100 micromolar. In some embodiments, the magnesium concentration is about 75 micromolar. In some embodiments, the magnesium concentration is about 50 micromolar. In some embodiments, the magnesium concentration is about 25 micromolar.

[0067] Also included herein is a method of making a circular RNA, wherein the vector further comprises two or more complementary sequences. a. Transcribing a vector to form a precursor RNA, wherein the vector contains elements operably linked in the following order: i. a 5' complementary sequence; and ii. a 3' Group I intron or a 3' Group II intron sequence including a 3' splice site dinucleotide; iii. a first non-coding sequence; and iv. the corresponding 5' Group I intron or 5' Group II intron sequence containing the 5' splice site dinucleotide; v. a 3' complementary sequence; and forming a precursor RNA comprising: b. contacting the precursor RNA with a paired Group I or Group II maturase polypeptide to allow for the formation of a circular RNA; The present invention also includes a method for producing circular RNA, comprising:

[0068] The present invention also provides a) transcribing a vector to form a precursor RNA, the vector comprising elements operably linked in the following order: i. a 3' Group I intron or a 3' Group II intron sequence including a 3' splice site dinucleotide; ii. a first non-coding sequence; and iii. a 5' complementary sequence; and iv. a 3' complementary sequence; and v. the corresponding 5' Group I intron or 5' Group II intron sequence including the 5' splice site dinucleotide; forming a precursor RNA comprising: b) contacting the precursor RNA with a pair of Group I or Group II maturase polypeptides to allow for the formation of a circular RNA; Also included is a method for producing circular RNA comprising:

[0069] In some methods, the vector further comprises, between elements a.iii. and a.iv., an internal ribosome entry site (IRES), a protein coding sequence, and a second non-coding sequence, in that order.

[0070] It should be understood that 5' and 3' complementary sequences are DNA sequences that are sufficiently complementary to each other to bind to each other, for example, based on AT complementarity and CG complementarity. Each element of the vector, or the transcription or contacting step, in these embodiments can be any of those described herein. In some embodiments, the complementary sequences are 25-60 nucleotides in length. In some embodiments, the 5' complementary sequence comprises or is SEQ ID NO:37, and the 3' complementary sequence comprises or is SEQ ID NO:41.

[0071] In addition to methods for producing circular RNAs, provided herein are circular RNAs produced according to these methods and pharmaceutical compositions containing circular RNAs. By way of example, the circular RNAs described herein can function as viral protein vaccines for preventing infectious diseases such as COVID-19. In some embodiments, the circular RNA contains between about 300 and 12,000 nucleotides, between about 1,000 and 10,000 nucleotides, between about 5,000 and 8,000 nucleotides, between about 8,000 and 12,000 nucleotides, or between about 10,000 and 12,000 nucleotides. The pharmaceutical composition may include a pharmaceutically acceptable carrier or nanocarrier for administering the pharmaceutical composition to a subject. Examples of pharmaceutically acceptable carriers and nanocarriers are provided herein. In some embodiments, the pharmaceutically acceptable nanocarrier is a lipid nanoparticle, a lipid, a lipid polymer, a lipopolymer hybrid, an exosome, or a leucosome.

[0072] Also provided herein is a method for isolating the circular RNA described herein.Such isolation can be performed by suitable methods, including solid phase extraction (SPE) and size exclusion chromatography.In some embodiments, the method for isolating the circular RNA described herein comprises: a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein; b. contacting the mixture with one or more immobilized 5' binding proteins and one or more immobilized 3' binding proteins; c. isolating the circular RNA; Includes.

[0073] Figure 13 shows a general schematic of one such method. The 5' and 3' binding proteins can be any that bind to 5' and 3' RNA ends. In some embodiments, the 5' binding protein is the DXO 5' binding protein from Saccharomyces cerevisiae or a homolog thereof. The term "DXO" includes a polypeptide encoded by SEQ ID NO:5, or a polypeptide sequence having about 50% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more identity to a polypeptide encoded by SEQ ID NO:5, or a polypeptide comprising a portion of a polypeptide encoded by SEQ ID NO:5. In some embodiments, the DXO 5' binding protein is inactivated. In some embodiments, the 5' binding protein is inactivated RNAse R or a homolog thereof. In some embodiments, the 5' or 3' binding protein has a mutation that increases or alters its temperature or pH sensitivity. In some embodiments, the RNA binding protein is mutated, truncated, or fused to other protein domains to increase its solubility and / or RNA binding affinity.

[0074] In other or further embodiments, the methods for isolating circular RNA described herein include: a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein; b. adding biotinylated adenosines to the linear RNA using poly(A) polymerase to produce biotinylated linear RNA; c. isolating the circular RNA by removing the biotinylated linear RNA using immobilized streptavidin.

[0075] In other or further embodiments, the methods for isolating circular RNA described herein include: a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein; b. Modifying linear RNA using RtcB RNA ligase; c. contacting the mixture with immobilized His-Tag streptavidin that binds the modified linear RNA; d. isolating the circular RNA from the effluent; Includes.

[0076] In other or further embodiments, the methods for isolating circular RNA described herein include: a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein; b. contacting the mixture with a polynucleotide specific for the circularization junction of the circular RNA bound to biotin to produce a second mixture; c. contacting the second mixture with immobilized streptavidin; d. Removing linear RNA; e. isolating the circular RNA by releasing the circular RNA from the one or more immobilized polynucleotides; Includes.

[0077] In some embodiments, the polynucleotide specific for the circularization junction of the circular RNA is a locked nucleic acid (LNA), wherein the polynucleotide is complementary to a sequence within the circularization junction of the circular RNA.

[0078] It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous modifications may be made thereto without departing from the scope of the present invention. The present invention is further illustrated by the following examples, which should not be construed as imposing any limitations on the scope of the present invention. On the contrary, after reading the description herein, it will be clearly understood that resort may be made to various other embodiments, modifications, and equivalents thereof, which may be suggested to those skilled in the art, without departing from the spirit of the invention and / or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes. [Example]

[0079] Example 1. CircRNAs outperform linear RNAs. We compared the performance of circRNAs and linear RNAs in green fluorescent protein (GFP) expression. Briefly, circRNAs and linear RNAs were transfected into HEK293 cells, and fluorescence intensity was monitored using a live cell imaging system (Incucyte, Sartorius). We observed that GFP expression from circRNAs persisted longer and was still increasing after 48 hours, whereas GFP expression from linear RNAs began to decrease after 24 hours. See Figure 2.

[0080] Example 2. Precursor RNA of the AnCob1 intron is successfully spliced ​​in vitro and can be visualized using agarose gels. Methods: Starting from linearized plasmid, RNA was generated by in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). It was then purified using 500 μg of Monarch RNA cleanup (New England Biolabs). The RNA was preincubated in 2x TN buffer (100 mM Tris-HCl pH 7.5, 200 mM NaCl, 300 mM MgCl2) at 37°C for 20 minutes. A 2x GTP solution (2 mM rGTP (Promega)) was then added to initiate splicing. Aliquots were withdrawn at the indicated times, and the reaction mixture was column-purified using a Monarch RNA cleanup column. RNA (denatured with 50% formamide at 70°C for 3 minutes) was visualized using the e-gel system.

[0081] Results: Using these conditions, linear RNA can self-splice at 37°C in a solution of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 150 mM MgCl, and 1 mM GTP. Precursor RNA decreased over time, allowing visualization of ligated exons and released introns, as well as intermediates in the splicing reaction. See Figure 3.

[0082] After verifying that linear RNAs could self-splice in vitro under high-magnesium conditions, we shifted our focus to maturase-assisted splicing. Successful production of macRNAs required the purification of maturase and the generation of permuted intron / exon (PIE) versions of splicing RNAs.

[0083] Example 3. Purification of I-aniI The I-aniI expression vector was designed using Geneart's (Thermofisher) codon optimization tool, cloned into their Premier Expression Vector (optimized for E. coli expression), and purchased as a complete plasmid from the company. The plasmid was first transformed into a custom cell line for the plasmid, BL21-DE3-star (Thermofisher). A colony was used to inoculate a 5 ml culture into LB + ampicillin (amp). The next day, 1 ml of the culture was used to inoculate 50 ml of LB + amp, and the OD was followed until the culture reached 0.6-0.8 during the logarithmic phase. IPTG was added to a final concentration of 1 mM, and the culture was grown overnight at a reduced temperature (30°C). Expression was tested by running an SDS gel of the lysed bacterial pellet. See Figure 4.

[0084] Initial attempts show limited expression of the I-aniI maturase using this plasmid. We are troubleshooting by changing the medium used (we used Superior broth (AthenaES), which provides additional nutrients to the bacteria and allows them to reach higher densities), lowering the incubation temperature to 18°C ​​to allow the protein to fold correctly, changing the IPTG stock used, and having a positive control. Possible issues include codon optimization (fast translation can lead to misfolding of the protein) and trying different plasmid expression systems. See Figure 4.

[0085] In subsequent attempts to express the I-aniI maturase, I-aniI was subcloned into a new expression backbone from addgene by swapping the ORF from GFP to the maturase. A new full-size open reading frame for I-aniI was also obtained from geneart. All constructs were tested using the same method described above (superior broth, induction with 0.5 mM or 1 mM IPTG upon reaching logarithmic phase). Different culture temperatures and different time points were tested. Expression of the maturase using the new full-size open reading frame is shown in Figure 5.

[0086] Maturase I-aniI was purified by resuspending the bacterial pellet in lysis buffer (1x PBS, 100 μM PMSF, 250 mM NaCl, 5 mM imidazole). The mixture was then sonicated for 15 minutes at 4°C (15 seconds on, 15 seconds off, high frequency cycle). The solution was centrifuged at 4500 rpm for 40 minutes at 4°C, and the supernatant was transferred to a new tube. His-Pur resin (Thermofisher) was equilibrated with buffer (1x PBS, 10 mM imidazole). Equilibration buffer was added to the supernatant until the resulting volume was equivalent to three His-Pur resin beds. This solution was incubated with the equilibrated resin and rotated on an end-over-end shaker at 4°C for 90 minutes. The flow-through was discarded, and the resin was washed four times (wash buffer: 1× PBS, 25 mM imidazole, 100 mM NaCl). Finally, the protein was slowly eluted by incubating the resin with elution buffer (1× PBS, 250 mM imidazole, 100 mM NaCl). The sample was then run on an SDS-PAGE gel. The different steps of the purification are shown below, and the band corresponding to I-aniI (indicated by an arrow) can be seen in the eluate. See Figure 6.

[0087] Example 4. Maintaining the splicing position and folding of the ribozyme is crucial for the pie version. Ribozyme function is highly dependent on proper folding into a catalytically active form. A structure prediction approach was used to attempt to match the folding of natural RNA with that of the synthetic pie RNA. To predict the structure, RNAfold was used. As previously mentioned, natural ribozymes consist of an intron sandwiched between a 5' exon and a 3' exon. To cleave the ribozyme and generate a circle, the positions of the intron and exon are swapped, splitting the intron into two parts. See Figure 7.

[0088] Different intron split structures were tested using RNAfold. It was found that splitting the intron in half resulted in very similar structures, with similarly positioned splice sites. It was also found that having a short 5' intron portion could still preserve the overall structure, while the opposite (a long 5' intron portion) resulted in a disrupted structure. See Figure 8. Five constructs, A through E, were generated using different layouts obtained through geneart. See Figure 9. The selection of these components is explained in Table 1 below. [Table 1]

[0089] Example 5. Adjustment of splicing conditions RNA for each construct was obtained using in vitro transcription (IVT) and tested for self-splicing using the same conditions used for the unshuffled RNA. Some splicing appears to occur during or even after IVT. Some degradation is also observed, likely due to the high magnesium content (see Figure 10). The magnesium concentration can be significantly reduced (from a final concentration of 150 mM to 5 mM) in the presence of maturase, which likely reduces RNA degradation.

[0090] We tested multiple splicing conditions for construct D (no cargo) and observed differences in splicing, particularly when the RNA was preincubated in buffer for 10 minutes rather than 20 minutes before adding the splicing primer (GTP). These steps may allow us to tailor the splicing conditions when using maturase. See Figure 11.

[0091] Constructs F, G, and H, listed in Table 1, were also developed. This was an attempt to improve on splicing constructs A through E and involved splitting the intron exactly in half by using a shortened exon. Thus, for constructs F, G, and H, the intron was split at two locations (generating either a longer or a new, shorter 5' end). Constructs G and H were generated by randomly selecting the location to split the intron, and the exon was the same size as that used in the small intron construct (SEQ ID NO: 14). Construct F was engineered to adjust the percentage of GC content, which may affect splicing efficiency. As shown in Figure 12, construct F appeared to respond to splicing conditions (appearance of additional bands over time) and was potentially a preferred construct.

[0092] Between the two exons, where the cargo would go, a placeholder (a small sequence that adds a multiple cloning site) can be added. A spacer sequence with many AAATTTs that can base pair with the intron can also be added. Downstream of the entire sequence, a sap1 restriction site was added for future IVT.

[0093] Example 6. Maturase-intron pair In addition to the I-aniI maturase-AnCob1 intron system, other maturase / intron pairs may also be effective. Exemplary pairs are shown in Table 2. [Table 2] [Table 3]

[0094] Selection Array: SEQ ID NO: 6 Construct A. Underlined: intron sequence, bold: nanoluciferase, bold underlined: CVB3 IRES, double underlined: homology arms, italic: short exon, italic underlined: extended exon. [ka]

[0095] SEQ ID NO: 7 Construct B. Underlined: intron sequence, bold: nanoluciferase, bold underlined: CVB3 IRES, double underlined: homology arms, italicized: short exon. [ka]

[0096] SEQ ID NO: 8 Construct C. Underlined: intron sequence, bold: nanoluciferase, bold underlined: CVB3 IRES, double underlined: homology arms, italic: short exon, italic underlined: extended exon. [ka]

[0097] SEQ ID NO: 9 Construct D. Underlined: intron sequence. [ka]

[0098] SEQ ID NO: 10 Construct E. Underlined: intron sequence, bold: nanoluciferase, bold underlined: CVB3 IRES, italic: short exon, italic underlined: extended exon. [ka]

[0099] SEQ ID NO: 11 Construct F. Underlined: intron sequence. [ka]

[0100] SEQ ID NO: 12 Construct G. Underlined: intron sequence. [ka]

[0101] SEQ ID NO: 13 Construct H. Underlined: intron sequence. [ka]

[0102] SEQ ID NO: 14 Small intron. Underline: intron sequence. [ka]

Claims

1. 1. A method for making circular RNA, comprising: a) transcribing a vector to form a precursor RNA, said vector comprising elements operably linked in the following order: i. a 3' Group I intron or a 3' Group II intron sequence comprising a 3' splice site dinucleotide; ii. a first non-coding sequence; and iii. the corresponding 5′ Group I intron or 5′ Group II intron sequence, including the 5′ splice site dinucleotide; and excluding the complementary sequence; b) contacting the precursor RNA with a pair of Group I or Group II maturase polypeptides to allow for the formation of a circular RNA; A method comprising:

2. 2. The method of claim 1, wherein the vector further comprises, between the elements of a) ii. and a) iii., an internal ribosome entry site (IRES), a protein-coding sequence, and a second non-coding sequence, in that order.

3. 1. A method for making circular RNA, comprising: a) transcribing a vector to form a precursor RNA, said vector comprising elements operably linked in the following order: i. a 3' Group I intron or a 3' Group II intron sequence comprising a 3' splice site dinucleotide; ii. a first non-coding sequence; and iii. a 5' complementary sequence; and iv. a 3' complementary sequence; and v. the corresponding 5′ Group I intron or 5′ Group II intron sequence, including a 5′ splice site dinucleotide; and forming a precursor RNA comprising: b) contacting the precursor RNA with a pair of Group I or Group II maturase polypeptides to allow for the formation of a circular RNA; A method comprising:

4. 4. The method of claim 3, wherein the vector further comprises, between the elements of a) iii. and a) iv., an internal ribosome entry site (IRES), a protein-coding sequence, and a second non-coding sequence, in that order.

5. 5. The method of any one of claims 1 to 4, wherein the maturase polypeptide and the precursor RNA are contacted under conditions comprising a temperature between 20°C and 45°C and a magnesium concentration between 100 micromolar and 25 millimolar.

6. 5. The method of claim 2 or claim 4, wherein the vector further comprises a second IRES, a second protein-coding sequence, and a third non-coding sequence between the second non-coding sequence and the 5′ Group II intron sequence comprising a 5′ splice site dinucleotide.

7. 7. The method of claim 6, wherein the vector further comprises a third IRES, a third protein-coding sequence, and a fourth non-coding sequence between the third non-coding sequence and the 5′ Group II intron sequence comprising a 5′ splice site dinucleotide.

8. The method of any one of claims 1 to 7, wherein the Group I intron is the Aspergillus nidulans COB1 intron.

9. 9. The method of claim 8, wherein the paired group I maturase is an Aspergillus nidulans I-AniI maturase.

10. 10. The method of claim 9, wherein the Aspergillus nidulans I-AniI maturase has reduced DNA endonuclease activity and / or increased splicing activity.

11. The method of claim 9 or claim 10, wherein the Aspergillus nidulans I-AniI maturase is N-terminally truncated.

12. 8. The method of claim 1, wherein the Group II intron is selected from the Oryza sativa trnK gene, tRNAs (V-UAC, I-GAU, A-UGC, and K-UUU), ribosomal proteins (rpl2 and rps12), and chloroplast ATPase (atpF).

13. 13. The method of claim 12, wherein the paired group II maturase is Oryza sativa maturase K.

14. The method of any one of claims 1 to 7, wherein the group II intron is a Lactococcus lactis LtrA intron.

15. 15. The method of claim 14, wherein the paired group II maturase is Lactococcus lactis LtrA maturase.

16. The method of any one of claims 1 to 7, wherein the group II intron is the LtrB intron of Lactococcus lactis.

17. 17. The method of claim 16, wherein the paired group II maturase is the LtrB maturase of Lactococcus lactis.

18. 8. The method of any one of claims 1 to 7, wherein the group II intron is IIA intron a12 and the paired group II maturase is cyt-19 or Mss116p.

19. 8. The method of any one of 1 to 7, wherein the group II introns are aI5γ and bI1, and the paired group II maturase is cyt-19.

20. The method of any one of claims 1 to 7, wherein the group II intron is nadl i4 and the paired group II intron is MatR.

21. 21. The method of any one of claims 1 to 20, wherein one or more of the non-coding sequences is between 10 and 40 nucleotides.

22. 21. The method of any one of claims 1 to 20, wherein one or more of the non-coding sequences is between 15 and 50 nucleotides.

23. 21. The method of any one of claims 1 to 20, wherein one or more of the non-coding sequences is between 18 and 22 nucleotides.

24. 24. The method of any one of 1 to 23, wherein one or more of the non-coding sequences comprises a poly(A) sequence.

25. 25. The method of any one of claims 1 to 24, wherein one or more of the non-coding sequences comprises only A and C nucleotides.

26. The IRES may be an aptamer for eIF4G, Homo sapiens cDNA FLJ43058, acute wasp paralysis virus IRES, aphid lethal paralysis virus IRES, avian encephalomyelitis virus IRES, bovine viral diarrhea virus type 1 IRES, dog camper IRES, classical swine fever virus IRES, cosavirus, coxsackievirus A (CVB1 / 2) IRES, cricket paralysis virus IRES, cruciferous tobamovirus IRES, Drosophila antennapedia IRES, Diresapivirus B1 IRES, Drosophila C virus IRES, Drosophila hairless IRES, Drosophila reaper IRES, Drosophila UbxIRES, smaller tea tortrix picorna-like virus IRES, encephalomyocarditis virus (EMCV) IRES, equine rhinitis virus IRES, foot-and-mouth disease virus IRES, Guangxi discolored lizard picornavirus type 2 IRES, hepatitis A virus IRES, hepatitis C virus IRES, hepatitis B virus IRES, hibiscus yellow ringspot virus IRES, Himetobi P virus IRES, Homalodisca coagulata virus type 1 IRES, human AML1 / RUNX1 gene IRE S, human AQP4 gene IRES, human AT1R gene IRES, human BAG-1 gene IRES, human BCL2 gene IRES, human BiP gene IRES, human c-IAP1 gene IRES, human c-myc gene IRES, human c-src gene IRES, human eIF4G gene IRES, human enterovirus type 71 IRES, human FGF-1 gene IRES, human FGF2 gene IRES, human immunodeficiency virus type 1 IRES, human LEF1 gene IRES, human n. myc gene IRES, human p27kipl gene IRES, human p53 gene IRES, human papillomavirus IRES, human PDGF2 / c-sis gene IRES, human Pim-1 gene IRES, human rhinovirus type 2 IRES, human SFTPAl gene IRES, human UNR gene IRES, human VEGF-A gene IRES, human XIAP gene IRES, Kashmir bee virus IRES, mouse eukaryotic translation initiation factor 1A domain-containing 14 (Eif1ad14) gene IRES, mouse Gtx gene IRES, mouse HI F1 alpha gene IRES, mouse NDST4L gene IRES, mouse Rbm3 gene IRES, mouse UtrA gene IRES, parechovirus IRES, pink-eared duck picornavirus IRES, brown marmorated stink bug enteric virus IRES, poliovirus type 1 IRES, reticuloendotheliosis virus IRES, wheat aphid virus IRES, Rhodospirillum sarcoma virus IRES, budding yeast TFIID gene IRES, budding yeast YAP1 gene IRES, salivirus IRES, monkey picornavirus IRES, simian virus 40The method of any one of claims 1 to 25, wherein the IRES has a sequence of a red fire ant virus type 1 IRES, a taura syndrome virus IRES, a Theiler's encephalomyelitis virus IRES, or a Triatomine bug virus IRES.

27. The method of any one of claims 4 to 26, wherein the protein coding sequence encodes a viral protein, a eukaryotic protein, or a prokaryotic protein.

28. 27. The method of any one of claims 4 to 26, wherein the protein coding sequence encodes a human protein.

29. The method of any one of claims 4 to 26, wherein the protein coding sequence encodes an antibody.

30. 30. The method of claim 29, wherein the antibody is bispecific.

31. The method of any one of claims 4 to 26, wherein the protein coding sequence encodes a viral or bacterial antigen.

32. Circular RNA produced according to the method of any one of claims 1 to 31.

33. 33. The circular RNA of claim 32, wherein the RNA comprises between 300 and 12,000 nucleotides.

34. A pharmaceutical composition comprising the circular RNA of claim 29 or 30 and a pharmaceutically acceptable nanocarrier selected from the group consisting of lipid nanoparticles, lipids, lipid polymers, lipopolymer hybrids, exosomes and leucosomes.

35. 1. A method for isolating circular RNA, comprising: a) obtaining a mixture of linear RNA and circular RNA prepared by the method according to any one of claims 1 to 31; b) contacting the mixture with one or more immobilized 5' binding proteins and one or more immobilized 3' binding proteins; c) isolating the circular RNA; and A method comprising:

36. 36. The method of claim 35, wherein the 5'-binding protein is Saccharomyces cerevisiae DXO 5'-binding protein, or a homolog thereof.

37. 37. The method of claim 35 or claim 36, wherein the 3'-binding protein is a nuclease-inactivating RNAse R.

38. 1. A method for isolating circular RNA, comprising: a) obtaining a mixture of linear RNA and circular RNA prepared by the method according to any one of claims 1 to 31; b) isolating said circular RNA using size exclusion chromatography; A method comprising:

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