Methods of making and isolating circular RNA and circular RNA compositions

Abstract

Provided herein are a method of preparing a circular RNA, a method of isolating a circular RNA, and a composition comprising a circular RNA.

Description

[0001] Cross Reference to Related Applications

[0002] 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 by reference herein in its entirety.

[0003] Reference to Sequence Listing

[0004] The.XML entitled “10063-079WO1_ST26.xml” created on January 25, 2024, and having a file size of 100,751 bytes, was submitted on January 26, 2024, is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND

[0005] Circular RNAs (circRNAs) are a promising platform in the field of RNA-based therapeutics. Like linear RNAs, circRNAs can also be translated into therapeutic proteins or vaccines. However, because circRNAs do not have ends, they are not susceptible to degradation by most RNAses. Thus, they are more stable than linear RNAs and persist for longer durations, and are able to produce more protein per unit of RNA. Prolonging the duration of protein expression is particularly important for diseases that require long-lasting therapeutic effects, as well as for improving the efficacy of RNA vaccines. CircRNAs have many other advantages, including: (a) unlike linear RNAs, they do not require the use of expensive modified nucleosides; (b) they evade detection by cells as foreign RNAs that are known to induce immune responses; (c) they are known to be more resistant to high temperatures than linear RNAs, which can make their storage more cost-effective.

[0006] Prior to this, circRNAs were created using a method called PIE, which stands for permutation of intron-exon. The PIE method involves chemically splicing under conditions that are harsh. These conditions give rise to two major limitations: (a) low splicing efficiency, and (b) “breaks” in the circRNA that allow for the accumulation of linear “cut-open” loop contaminant byproducts. Thus, there is a need for improved methods of creating circRNAs. SUMMARY

[0007] Included herein is a method of making a circular RNA, the method comprising: transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and excluding a complementary sequence: 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; and contacting the precursor RNA with a paired Group I or Group II mature enzyme polypeptide to allow formation of a circular RNA. In some embodiments, the vector further comprises an internal ribosome entry site (IRES) and a protein coding sequence and a second non-coding sequence.

[0008] Also included herein is a method of making a circular RNA, the method comprising: transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order: a 5' complementary sequence, a 3' Group I or 3' Group II intron sequence comprising a 3' splice site dinucleotide, a non-coding sequence, a corresponding 5' Group I or 5' Group II intron sequence comprising a 5' splice site dinucleotide, and a 3' complementary sequence; and contacting the precursor RNA with a paired Group I or Group II mature enzyme polypeptide to allow formation of a circular RNA. In some embodiments, the vector further comprises an internal ribosome entry site (IRES) and a protein coding sequence and a second non-coding sequence in the following order.

[0009] In certain aspects, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of 20 °C to 45 °C and a magnesium concentration of 100 micromolar to 25 millimolar.

[0010] 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 a 5' splice site dinucleotide.

[0011] 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 a 5' splice site dinucleotide.

[0012] In certain aspects, the Group I intron is an Aspergillus nidulans COB1 intron. The A. nidulans I-Anil mature enzyme can be paired with an A. nidulans COB1 intron. In some embodiments, the A. nidulans I-Anil mature enzyme has reduced DNA endonuclease activity and / or increased splicing activity. The A. nidulans I-Anil mature enzyme can also be N-terminally truncated.

[0013] In some embodiments, the Group II intron is selected from the Oryza sativa trnK gene, tRNA (V-UAC, I-GAU, A-UGC, and K-UUU), ribosomal proteins (rpl2 and rpsl2), and chloroplast ATPase (atpF). These Group II introns can pair with Oryza sativa mature ribozyme K.

[0014] In some embodiments, the Group II intron is the Lactococcus lactis LtrA intron. The Lactococcus lactis LtrA intron can pair with Lactococcus lactis LtrA mature ribozyme.

[0015] In some embodiments, the Group II intron is the Lactococcus lactis LtrB intron. The Lactococcus lactis LtrB intron can pair with Lactococcus lactis LtrB mature ribozyme.

[0016] In some embodiments, the mature ribozyme is cyt-19 from Neurospora crassa and the paired intron is selected from the aI5y and bI1 Group II introns. In some embodiments, the mature ribozyme is cyt-19 from Saccharomyces cerevisiae and the paired intron is Group II intron aI2. In some embodiments, the mature ribozyme is Mssl 16p from Saccharomyces cerevisiae and the paired intron is Group II intron aI2. In some embodiments, the mature ribozyme is MatR from Brassicaceae plant family and the paired intron is nadl i4. In some embodiments, the intron and the paired mature ribozyme are as described in Table 2.

[0017] In certain aspects, one or more of the non-coding sequences is about 10 to 50 nucleotides, or about 20 to 30 nucleotides. In certain aspects, one or more of the non-coding sequences comprises a poly(A) sequence. In other aspects, one or more of the non-coding sequences comprises only A and C nucleotides.

[0018] The IRES element of the vector can have a sequence selected from the 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 1 IRES, Canine distemper IRES, Classical swine fever virus IRES, Common faecal associated virus (Cosavirus), Coxsackievirus A (CVB1 / 2) IRES, Coxsackievirus B3 (CVB3) IRES, Crickets paralysis virus IRES, Crucifer tobamo virus IRES, Drosophila antennapedia IRES, Diresapivirus B1 IRES, Drosophila C virus IRES, Drosophila bald IRES, Drosophila reaper IRES, Drosophila Ubx IRES, Ectropis oblique picornavirus IRES, Encephalomyocarditis virus (EMCV) IRES, Equine rhinitis virus IRES, Foot-and-mouth disease virus IRES, Guanxi changeable lizard picornavirus 2 IRES, Hepatitis A virus IRES, Hepatitis C virus IRES, Hepatitis G virus IRES, Hippokrinis wiltis ring spot virus IRES, HimetobiP virus IRES, Homalodisca coagulata virus-1 IRES, Human AMLl / RUNXl gene IRES, Human AQP4 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 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 papilloma virus IRES, human PDGF2 / c-sis gene IRES, human Pim-1 gene IRES, human rhinovirus 2 IRES, human SFTPA1 gene IRES, human UNR gene IRES, human VEGF-A gene IRES, human XIAP gene IRES, human ELG1 IRES, human caspase 8-associated protein 2 (CASP8AP2), Kashmir bee virus IRES, 14 containing mouse eukaryotic translation initiation factor 1A domain (Eif1ad14) gene IRES, mouse Gtx gene IRES, mouse HIF1a gene IRES, mouse NDST4L gene IRES, mouse Rbm3 gene IRES, mouse UtrA gene IRES, mouse Line 1-ORF1 IRES, Parechovirus IRES, Patek duckling picornavirus IRES, Plautia stali intestine virus IRES, poliovirus 1 IRES, reticuloendotheliosis virus IRES, Rhopalosiphum padi virus IRES, Rous sarcoma virus IRES, S. Cerevisiae TFIID gene IRES, S. Cerevisiae YAP1 gene IRES, Salivavirus IRES, Simian picornavirus IRES, Simian virus 40 IRES, Solenopsis invicta virus 1 IRES, Taura syndrome virus IRES, Theiler's encephalomyelitis virus IRES, and Triatoma virus IRES, Aplysia californica ELH IRES, a synthetic IRES, or a novel synthetic IRES. In some embodiments, the synthetic IRES is generated by creating a chimera that adds a fragment to a known IRES. In some embodiments, the synthetic IRES is PPT19. In some embodiments, the synthetic IRES is KMI1.

[0019] 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 or 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.

[0020] Also included herein are circular RNAs prepared according to the methods described herein. In some embodiments, the circular RNA comprises 300 to 12000 nucleotides.

[0021] Further included herein are pharmaceutical compositions comprising a circular RNA described herein, and a pharmaceutically acceptable nanocarrier selected from the group consisting of a lipid nanoparticle, a lipid, a lipid polymer, a lipopolymeric hybrid, an exosome, and a leukosome.

[0022] Still further included herein are methods of isolating a circular RNA described herein. In some embodiments, the method comprises obtaining a mixture of linear RNAs 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 S. cerevisiae DXO 5' binding protein or a homolog thereof. In other embodiments, the method of isolating circular RNAs comprises obtaining a mixture of linear RNAs and circular RNAs produced by the methods described herein, and isolating the circular RNAs using size exclusion chromatography. In other embodiments, a polymerase A, RtcB RNA ligase, or a polynucleotide specific for the cyclization junction is used for isolation. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic showing the maturation enzyme assisted generation of circular RNA.

[0024] Figure 2 shows that expression of the reporter protein GFP (green fluorescent protein) from circular RNA is still increasing 48 hours after transfection, while protein from linear expression starts to decrease after 16 hours.

[0025] Figure 3 shows chemical splicing of A. n. Cobl linear RNA (full size (middle cob) and short size (small intron) over time.

[0026] Figure 4 (A-B) shows purification of I-aniI maturation enzyme (A) and expression of I-aniI maturation enzyme from total bacterial lysate (B).

[0027] Figure 5 shows test conditions for I-aniI maturation enzyme expression in E. coli using SDS-page gel, arrow indicates overexpressed recombinant maturation enzyme.

[0028] Figure 6SDS-page gel showing different steps for I-aniI mature enzyme purification to obtain pure recombinant I-aniI protein.

[0029] Figure 7 Schematic of the general mechanism of intron splicing in permutation intron exon (PIE) constructs used to generate circRNAs.

[0030] Figure 8 PIE (permutation intron exon) version of the A.n.Cob1 intron is shown to retain the same structure as originally, according to RNAfold web server prediction.

[0031] Figure 9 Design of PIE constructs (based on A.n.Cob1 intron) A to E and their corresponding predicted structures using the RNAfold web server are shown.

[0032] Figure 10 E-gel showing the initial splicing reactions of test constructs A, C and E. The 5' intron (225 bases) is undergoing some level of splicing.

[0033] Figure 11 E-gel showing the chemical splicing of construct D under different temperatures, incubation times and buffers. The splicing efficiency of construct D appears to be higher under condition 6.

[0034] Figure 12 E-gel showing the chemical splicing of construct F, with the generated circRNA and splicing intermediates increasing over time.

[0035] Figure 13 Schematic showing the method used to isolate circular RNAs using 5' and 3' RNA binding proteins.

[0036] Figure 14 (A-B) shows the successful expression of recombinant proteins for DXO, truncated I-aniI (NusA) (A) and I-aniI (OEC-1 and OEC-2) (B). Bands of the expected size with high protein expression were observed (kD shown under each protein).

[0037] Figure 15 I-aniI purification N-terminal truncation was optimized to prevent the protein from being cleaved during the bacterial lysis step.

[0038] Figure 16 (A-E) shows that in the presence of I-aniI, the designed constructs' mature enzyme assisted circularization shows that most produce circRNA, consistent with the structure prediction. An extended study was performed to characterize construct B, which was more efficient at producing circRNA than the other constructs. The new RNA entity's resistance to RNase R treatment confirmed the likelihood that it was a circular RNA. (A) Construct B RNA obtained by in vitro transcription was incubated with or without the mature enzyme I-aniI at a near 1 : 1 molar ratio (RNA to mature enzyme) and incubated at 37 °C for 1 hour. Only in the presence of the mature enzyme did a new band corresponding to a circRNA appear. (B) The use of a dose increase of I-aniI with construct B ribozyme's circRNA production shows an increase in the yield of circRNA (thicker circRNA band). The observation that the new band is resistant to RNase R when treated with RNase R that degrades linear RNA but not circRNA confirms that it is a circRNA entity. (C) All RNAs of constructs A, B, C, D, E were spliced with I-aniI at a 1 : 1 molar ratio for 1 hour:30 minutes at 37 °C. A, B, and C have circRNA bands. Construct E (negative control) did not produce circRNA and construct D is splicing but the size of the circRNA band is not visible on the gel. Constructs F, G, and H were spliced without a 1 : 1 molar ratio. While there is some splicing, the reaction was not optimized. (D) Construct B was incubated with or without I-aniI mature enzyme over the incubation time. The presence of the mature enzyme clearly produced circRNA and the reaction was well completed after 160 minutes. (E) Incubation of circRNA or precursor RNA from construct B with RNase R degrades most of the precursor (-I-aniI + R) and the circRNA resists degradation (+I-aniI + R). This indicates that the RNA entity produced is a circular RNA.

[0039] Figure 17 RT-PCR and Sanger sequencing were used to show that the circRNA was produced using mature enzyme assisted RNA circularization. Only when the RNA is circular is it possible to have the unique junction sequence that joins the 5' shortened exon and the 3' shortened exon.

[0040] Figure 18 (A-C) shows that the newly designed circRNA sequence (A) is able to form a circRNA in vitro (B). In this figure, "placeholder" refers to "coding sequence". (C) Predicted diagram of the structure of all the ribozymes tested. Each structure was plotted using the RNAfold web server with standard parameters. A, B and C show that the use of homology arms (grey) enables the splicing arrangement to be conserved, whereas the lack of homology arms in E distorts the ribozyme structure. The predicted structure consists of intron, exon 1, exon 2, homology arm, IRES, Nluc ORF and MCS.

[0041] Figure 19 (A-C) shows a series of optimization experiments performed to improve the circularization efficiency. Constructs A, B and C successfully produced circRNAs. (A) Constructs A, B and C were incubated with I-Anil mature enzyme for three hours at 37°C. (B) Construct B was incubated with I-Anil mature enzyme for 20 minutes and 45 minutes, at temperatures varying between room temperature and 65C, in 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(pH 50 mM Tris 7, 1 mM DTT), both in the presence of 1 mM GTP. (C) Construct B was incubated with I-Anil mature enzyme in TKM buffer: 4 hours at 37°C, using twice the amount of mature enzyme (molar ratio) of the mature enzyme of construct B (third and fourth lanes); 3 hours at 37°C, using three times the amount of mature enzyme (molar ratio) of the mature enzyme of construct B (fifth and sixth lanes); 3 hours at 42°C, using twice the amount of mature enzyme (molar ratio) of the mature enzyme of construct B (seventh and eighth lanes).

[0042] Figure 20 It is shown that circRNAs produced with the aid of a mature enzyme (I-Anil) can be translated into proteins. Nanoluciferase activity was detected by a circRNA encoding a nanoluciferase reporter protein.

[0043] Figure 21 It is shown that the mature enzyme K was cloned and expressed.

[0044] Figure 22 (A-B) shows recombinant LtrA mature enzyme expression in E. coli, as seen on a non-stained SDS-PAGE gel and protein detection with anti-V5 antibody (A). Recombinant LtrA mature enzyme was purified from E. coli cell lysate (B). The arrow indicates the expected LtrA fusion protein band.

[0045] Figure 23 (A-C) shows self-splicing and maturation enzyme assisted RNA circularization curves for P2.1 and P2.2 (A) as a working control. The P2.1 construct is P2 with a 15 nucleotide extension on the 5' exon (to create a long non-coding sequence) and 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. RNA circularization curves for P2.1 and P2.2 after adjusting incubation times and performing assays with a 1 :4 RNA to LtrA maturation enzyme ratio, as in 1.2% FlashGel TM visible on the RNA box (B). Double asterisks indicate circRNA. Detection of novel circRNA specific sequence junctions using reverse transcriptase PCR followed by Sanger sequencing, indicates that the circRNA is a complete circle (C).

[0046] Figure 24 (A-D) shows different sequence designs for circRNA using LtrA maturation enzyme, where "D" refers to the RNA domain of the LtrB intron (A). RNA structure predictions generated by RNAfold for both linear control RNA (Linl) and RNA circularization constructs during the initial design, validation, and optimization phase (B-C) of the project. The nomenclature Dl, D2, D3, D4, D5, and D6 in (A) correspond to the numbered I, II, III, IV, V, and VI RNA domains shown in the schematic provided in (D).

[0047] Figure 25 (A-B) shows RNA circularization curves for P2 and splicing curves for Linl after adjusting incubation times and performing assays with a 1 :2 RNA to LtrA maturation enzyme (A) ratio. Maturation enzyme assisted circRNA generation using optimized circularization sequences (P2.1 construct is P2 with extensions to facilitate RNA circularization) and splicing curves for the Linl linear RNA control (B). Double asterisks indicate circRNA.

[0048] Figure 26 NanoLuc reporter activity in U20S-K294A cells transfected with LtrA maturation enzyme assisted circRNA (circ5.1Nluc) and reference circRNA (circNluc) is shown. The LtrA maturation enzyme assisted circRNA (circ5.1Nluc) and reference circRNA (circNluc) were transfected into U20S-K294A cells and the NanoLuc reporter activity was measured.

[0049] Figure 27 (A-C) shows recombinant MatR mature enzyme expression in E. coli as seen on a non-stained SDS-PAGE gel and protein detection with anti-V5 antibody (A). Different conditions were evaluated to optimize MatR expression in E. coli (B). Recombinant MatR mature enzyme was purified from E. coli cell lysate as shown on a non-stained SDS-PAGE gel (C). The arrow indicates the expected MatR mature enzyme protein.

[0050] Figure 28 (A-B) shows that Cyt-19 dead box protein is highly expressed in bacteria (A) and successfully purified from E. coli lysate (B).

[0051] Figure 29 It was shown that increasing the amount of cyt19 protein in the circularization reaction with circRNA construct B Figure 17-1 9 described above had no positive effect on promoting circRNA formation.

[0052] Figure 30 It was shown that using reverse transcriptase PCR to detect the novel circRNA specific sequence junction followed by Sanger sequencing indicated that the circRNA was a complete circle. This particular circRNA design uses non-coding RNA sequences to improve the production efficiency of the circRNA.

[0053] Figure 31 (A-C) shows a schematic of the plasmid backbone used to generate an internal ribosome entry site (IRES) test library (up to 10 8 unique 150 nucleotide random sequences). Each random 150-nt sequence was placed in front of the mCherry reporter protein coding sequence (A). Linearized plasmid library was used as a template to generate circRNAs (B). Microscopy images show that a small number of cells can express the mCherry reporter protein from transfected circRNAs. Only circRNAs with functional IRES sequences can generate mCherry protein in cells (C).

[0054] Figure 32 The use of circRNA capture method to isolate pure circRNAs is shown. Biotinylated locked nucleic acid oligonucleotide (TT177) specific for the circularization junction of RNAs hybridizes to circRNAs and is used to purify intact circRNAs (marked by the ellipse) from linear RNA contaminants (bars). Incubation for 2 hours shows good recovery of circRNAs with streptavidin beads and that the circRNAs are intact.

[0055] Figure 33 (A-B) shows the isolation of pure circRNA using an RNA ligase and desulpho-biotin followed by recovery using streptavidin beads (Linear RNA capture method number 1) and used to purify intact circRNA (marked by the oval) from linear RNA contaminants (stripes). The experiment was performed using both a crude circRNA preparation (A) and a more pure circRNA preparation (B). Little enrichment of circRNA was observed.

[0056] Figure 34 Figure 34 shows the isolation of pure circRNA using RtcB protein binding and capture of linear RNA by-products (Linear RNA capture method number 2). The circRNA does pass through the procedure unmodified (sample 5 in different lanes of both gels), but in reduced amounts. The results do show a reduction in the major linear RNA species (fuzzy bands in sample 1, bottom gel), but the ability to capture linear RNA by-products was not quantitatively evaluated.

[0057] Figure 35 (A-B) shows the isolation of pure circRNA using DXO protein binding and capture of linear RNA by-products (Linear RNA capture method number 3.1 and number 3.2) first part. Expression (A) and purification (B) of wild-type and exonuclease mutant DXO proteins from E. coli cell lysates. Arrows indicate expected WT and D236A / E253A mutant DXO protein bands (expected 54 kDa).

[0058] Figure 36 (A-B) shows the isolation of pure circRNA using DXO protein binding and capture of linear RNA by-products (Linear RNA capture method number 3.1 and number 3.2) second part. Exonuclease assays using WT and D236A / E253A (no nuclease activity) mutant DXO on two different circRNAs. Purified WT DXO has nuclease activity as shown by RNA cleavage products (in brackets) in both of the two different RNA substrates treated with the two ways (circESAT6 IVT RNA (A) and circGFP-3XFL IVT RNA (B)). These cleavage products are not seen in the mutant DXO treatments, indicating that the nuclease activity in the generated D236A / E253A mutant DXO is abolished.

[0059] Figure 37 (A-B) shows the use of poly(A) polymerase to isolate pure circRNA using biotinylated adenosine extension of linear RNA byproducts that will be used to bind and capture linear RNA byproducts (Linear RNA capture method number 4). Products after poly(A) extension of a mixed pool of circRNA and linear RNA substrates (A, short arrows indicate linear byproducts and color-coded long arrows indicate polyadenylated linear RNA byproducts after the procedure). The resulting samples were assayed using TapeStation chromatograms (B). The intensity of the samples resolved in each lane of the chromatogram in (B) is shown in (C-H). DETAILED DESCRIPTION

[0060] Provided herein are methods of making circular RNA that greatly improve upon the methods of the prior art. In some embodiments, the methods are more efficient and more effective than the methods of the prior art. Also included herein are methods of isolating the circular RNAs described herein, circular RNA compositions, and pharmaceutical compositions comprising the circular RNA compositions. The methods provided herein utilize mature enzymes, and more particularly, mature enzyme intron pairs, which allow for the use of milder reaction conditions and improve circRNA yield.

[0061] The terms used throughout this application are to be understood in the ordinary and typical meaning for one of ordinary skill in the art. However, the Applicant wishes to make the following specific definitions for the following terms.

[0062] Definitions.

[0063] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes multiple cells, including mixtures thereof.

[0064] The terms "about" and "approximately" are defined as "close to" as understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms are defined as within 10%. In another non-limiting embodiment, the terms are defined as within 5%. In still another non-limiting embodiment, the terms are defined as within 1%.

[0065] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a particular target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each of the heavy chains has, at one end, a variable domain (VH) followed by various constant domains. Each of the light chains has, at one end, a variable domain (VL) and at its other end, a constant domain.

[0066] 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. A "functional fragment or analog" of an antibody refers to a compound that has the same qualitative biological activity as a full length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one that binds to IgE immunoglobulin in a manner that prevents or greatly reduces the ability of such molecule to bind with high affinity to the receptor FcεRI. As used herein, a "functional fragment" with respect to an antibody refers to Fv, F(ab), and F(ab')2 fragments. An "Fv" fragment is the minimum antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain (VH-VLdimer). It is in this configuration that the three CDRs from each variable domain interact to define an antigen binding site on the surface of the dimeric H -V L The dimeric VH-VLdimer. It is in this configuration that the three CDRs from each variable domain interact to define an antigen binding site on the surface of the dimeric H -V L The dimeric VH-VLdimer. It is in this configuration that the three CDRs from each variable domain interact to define an antigen binding site on the surface of the dimeric H and V L domains, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the sFv to form the desired structure for target binding.

[0067] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" shall mean excluding other elements of any importance to the composition and method. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification process, and pharmaceutically acceptable carriers, such as 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 this application. Embodiments defined by each of these transition terms are within the scope of the application.

[0068] A "control" is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0069] A "control sequence" is a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. The DNA for a pre-sequence or a secretory leader sequence, if present, is operably linked to DNA for a polypeptide involved in secretion of the polypeptide; the DNA for a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or the DNA for a ribosome binding site is operably linked to a coding sequence if it affects the translation of the sequence; or the DNA for a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. In general, "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.

[0070] "Decreasing" or "reducing" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. The decrease or reduction can be any individual, median, or average decrease in the condition, symptom, activity, composition by a statistically significant amount. Thus, a decrease 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% reduction, so long as the decrease is statistically significant.

[0071] "Homolog" is defined herein as two polynucleotides or two polypeptides having some identity. Homologs include allelic variants, orthologs, and paralogs that have the same general related function, such as the ability to act as a mature enzyme. 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% homology. In other embodiments, homologs have about 50% identity. In some embodiments, homologs have about 50%, 60%, 70%, or 80% identity.

[0072] The term "identity" should be construed as meaning the percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical with the corresponding sequence being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of identity over the whole sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions or insertions are to be construed as reducing 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, when aligned, the percentage of bases (or amino acids) that are the same in comparing the two sequences is that percentage. It should be understood that alignment over "its 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 percentage of sequence identity can be determined using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment, the BLAST program with default parameters is used. In one embodiment, the BLAST program BLASTN and BLASTP with the following default parameters are used: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR.

[0073] "An 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, 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.

[0074] The word "mature enzyme" is used herein to refer to any protein that can assist or facilitate splicing of an intron. In some embodiments, the mature enzyme is an RNA molecular chaperone, such as cyt-19.

[0075] "Non-coding sequence" refers to a polynucleotide sequence that does not encode an amino acid sequence.

[0076] "Pharmaceutical composition" is intended to encompass a combination of an active agent with a pharmaceutically acceptable inert or active carrier, which is suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0077] The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable nanocarrier" mean a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes a carrier or excipient that is acceptable for veterinary use and / or human pharmaceutical use. The term "pharmaceutically acceptable carrier" as used herein encompasses any standard pharmaceutical carrier, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, as well as various types of wetting agents. The term "carrier" as used herein encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and described further below. The pharmaceutical composition can also include a preservative. As used in the specification and claims, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable nanocarrier" includes 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 leukosome.

[0078] The term "protein coding sequence" as used herein refers to a polynucleotide sequence that encodes a polypeptide, including fragments of known functional polypeptides. Likewise, the word "protein" includes whole proteins and fragments of proteins.

[0079] The term "subject" refers to any individual who is the target of an administration or treatment. The subject can be a vertebrate, such as a mammal. In one aspect, the subject can be a human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or a mole. Thus, the subject can be a human or a veterinary patient. The term "patient" refers to a subject under treatment by a clinician, such as a physician.

[0080] The word "vector" refers to an agent that carries a polynucleotide into a cell for expression of the polynucleotide in the cell. Once transformed into a suitable host, a vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. In some embodiments, a vector is a DNA construct containing a DNA sequence operably linked to suitable control sequences capable of affecting expression of the DNA in a suitable host cell. In some embodiments, such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control transcription and translation termination.

[0081] Description

[0082] As described above, provided herein are methods of making circular RNA that greatly improve upon the methods of the prior art. Also included herein are methods of isolating the circular RNA described herein, circular RNA compositions, and pharmaceutical compositions comprising the circular RNA compositions. It was surprisingly found that the use of mature enzyme introns, among other things, improves splicing efficiency and reduces the formation of byproducts. Figure 1 A general schematic of a mature enzyme assisted process for generating circular RNA is provided.

[0083] In some embodiments, the method of making circular RNA comprises the steps of:

[0084] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and does not include complementary sequences:

[0085] i. a 3' Group I intron or 3' Group II intron sequence containing a 3' splice site di-nucleotide,

[0086] ii. a non-coding sequence, and

[0087] iii. a corresponding 5' Group I intron or 5' Group II intron sequence containing a 5' splice site di-nucleotide; and

[0088] b. contacting the precursor RNA with a paired Group I or Group II mature enzyme polypeptide to allow formation of the circular RNA.

[0089] It should be understood that for all embodiments described herein, the transcribing and contacting steps can be performed simultaneously or sequentially. In some embodiments, the contacting step begins before the transcribing step is complete.

[0090] In some embodiments, the method of making circular RNA comprises the steps of:

[0091] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and does not comprise a homologous sequence:

[0092] i. a 3' Group I intron sequence or a 3' Group II intron sequence comprising a 3' splice junction di-nucleotide,

[0093] ii. a first non-coding sequence,

[0094] iii. a protein coding sequence,

[0095] iv. a second non-coding sequence, and

[0096] v. a corresponding 5' Group I intron sequence or 5' Group II intron sequence comprising a 5' splice junction di-nucleotide; and

[0097] b. contacting the precursor RNA with a cognate Group I or Group II mature enzyme polypeptide to allow formation of the circular RNA.

[0098] In some methods, the vector further comprises an internal ribosome entry site (IRES) and a protein coding sequence and a second non-coding sequence in the following order between the elements of a. ii. and a. iii. Accordingly, included herein are methods of making a circular RNA, the method comprising the steps of:

[0099] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and does not comprise a homologous sequence:

[0100] i. a 3' Group I intron sequence or a 3' Group II intron sequence comprising a 3' splice junction di-nucleotide,

[0101] ii. a first non-coding sequence,

[0102] iii. an internal ribosome entry site (IRES),

[0103] iv. a protein coding sequence,

[0104] v. a second non-coding sequence,

[0105] vi. a corresponding 5' Group I intron sequence or 5' Group II intron sequence comprising a 5' splice junction di-nucleotide; and

[0106] b. contacting the precursor RNA with a cognate Group I mature enzyme or a cognate Group II mature enzyme polypeptide to allow formation of a circular RNA.

[0107] As used herein, a "group I intron" refers to a self-splicing ribozyme that can catalyze its own excision from a precursor RNA. Group I introns generally consist of nine paired regions, PI -P9, which generally fold into two domains, a P4-P6 domain formed by the stacking of P5, P4, P6, and P6a helices, and a P3-P9 domain formed by P8, P3, P7, and P9 helices. A "group II intron" also refers to a self-splicing ribozyme that can catalyze its own excision from a precursor RNA. However, group II introns differ from group I introns in that excision can occur in the absence of GTP. Group II introns generally have a secondary structure of six stem-loop domains, D1 through D6, which radiate from a central core such that the 5' and 3' splice junctions are in close proximity.

[0108] The term "3' group I intron sequence" refers to any DNA sequence that encodes 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 that encodes 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 that encodes 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 that encodes 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 group II sequence comprises a deletion. One example of such a deletion is the "minimal D2 and D3" element in construct P2. In some embodiments, the 5' or 3' group I or group II sequence is about 100 to 750 nucleotides in length. By "corresponding 5' group I intron sequence or 5' group II intron sequence" is meant the 5' group I or group II intron sequence, respectively, from the same group I or group II intron as the above 3' group I intron sequence or 3' group II intron sequence. In some embodiments, the 3' and 5' intron sequences are each about half of a group I or group II intron. In some embodiments, the 3' and 5' intron sequences are the result of differential splicing of a group I or group II intron. The 3' and 5' splice site dinucleotides can be any dinucleotides that are splice sites by a group I intron or group II intron. In some embodiments, the 5' splice site dinucleotide is GT.

[0109] In some aspects, the 3' I group 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' I group intron sequence comprises or is SEQ ID NO: 27. In some embodiments, the 3' I group intron sequence comprises or is SEQ ID NO: 30. In some embodiments, the 3' I group intron sequence comprises or is SEQ ID NO: 32. In some embodiments, the 3' I group intron sequence comprises or is SEQ ID NO: 34. In some aspects, the 5' I group 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' I group intron sequence comprises or is SEQ ID NO: 29. In some embodiments, the 5' I group intron sequence comprises or is SEQ ID NO: 31. In some embodiments, the 5' I group intron sequence comprises or is SEQ ID NO: 33. In some embodiments, the 5' I group intron sequence comprises or is SEQ ID NO: 35.

[0110] Thus, in some embodiments, the method of making a circular RNA comprises the steps of:

[0111] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and does not include a complementary sequence:

[0112] i. a 3' I group intron sequence comprising a 3' splice site dinucleotide,

[0113] ii. a non-coding sequence, and

[0114] iii. a corresponding 5' I group intron sequence comprising a 5' splice site dinucleotide; and

[0115] b. contacting the precursor RNA with a paired I group mature enzyme polypeptide to allow formation of a circular RNA.

[0116] In other embodiments, the method of making a circular RNA comprises the steps of:

[0117] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and does not include a complementary sequence:

[0118] i. a 3' II group intron sequence comprising a 3' splice site dinucleotide,

[0119] ii. a non-coding sequence, and

[0120] iii. a corresponding 5' Group II intron sequence containing a 5' splice site di-nucleotide; and

[0121] b. contacting the precursor RNA with a paired Group II maturation enzyme polypeptide to allow formation of a circular RNA.

[0122] In some methods, the vector further comprises an internal ribosome entry site (IRES) and a protein coding sequence and a second non-coding sequence between the elements of a. ii. and a. iii. in the following order.

[0123] Accordingly, included herein are methods of making a circular RNA, the method comprising the steps of:

[0124] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and excluding complementary sequences:

[0125] i. a 3' Group I intron sequence containing a 3' splice site di-nucleotide,

[0126] ii. a first non-coding sequence,

[0127] iii. an internal ribosome entry site (IRES),

[0128] iv. a protein coding sequence,

[0129] v. a second non-coding sequence,

[0130] vi. a corresponding 5' Group I intron sequence containing a 5' splice site di-nucleotide; and

[0131] b. contacting the precursor RNA with a paired Group I maturation enzyme polypeptide to allow formation of a circular RNA, wherein the 3' splice site di-nucleotide and the 5' splice site di-nucleotide are splice sites by a Group I intron.

[0132] In other embodiments, the method of making a circular RNA comprises the steps of:

[0133] a. transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order, and excluding complementary sequences:

[0134] i. a 3' Group II intron sequence containing a 3' splice site di-nucleotide,

[0135] ii. a first non-coding sequence,

[0136] iii. an internal ribosome entry site (IRES),

[0137] iv. a protein coding sequence,

[0138] v. Second non-coding sequence,

[0139] vi. the corresponding 5' group II intron sequence containing the 5' splice site dinucleotide; and

[0140] b. contacting the precursor RNA with a paired Group II maturase polypeptide to allow formation of a circular RNA, wherein the 3' splice site dinucleotide and the 5' splice site dinucleotide are splice sites from a Group II intron.

[0141] The non-coding sequence of a vector refers to any DNA sequence that does not encode an amino acid. In some embodiments, the length of the non-coding sequence is about 15 to 50 nucleotides or about 20 to 30 nucleotides. In some aspects, the non-coding sequence comprises a poly (A) sequence. In other or additional embodiments, the non-coding sequence comprises only A and C nucleotides. In some embodiments, each non-coding sequence is the same. In other embodiments, each non-coding sequence is different. "Non-coding" sequences are also referred to herein as "exons". For example, Figure 9 In some embodiments, each "exon" in the non-coding sequence is or comprises a non-coding sequence. In some embodiments, the length of the "short" non-coding sequence or exon is about 15-25 or 18-22 nucleotides. In other or additional embodiments, the length of the "long" non-coding sequence or exon is about 39 nucleotides. In some embodiments, the first non-coding sequence is or comprises 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.

[0142] The "protein coding sequence" of a vector refers to any DNA sequence that encodes one or more amino acids. Therefore, it should be understood that the term "protein coding sequence" includes DNA sequences that encode fragments or portions of complete proteins. In some embodiments, the protein coding sequence encodes viral proteins, eukaryotic proteins, prokaryotic proteins, fungal proteins, or protozoan proteins, 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 antibodies as defined herein, and in some aspects, the antibody is bispecific. In some embodiments, the protein coding sequence encodes more than one protein or protein fragment.

[0143] The present disclosure includes vectors comprising more than one protein coding sequence. In some embodiments, the vector comprises one, two, or three coding sequences. Accordingly, included herein are methods of making 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 containing a 5' splice site dinucleotide. In still further 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 I or Group II intron sequence containing a 5' splice site dinucleotide.

[0144] An internal ribosome entry site (IRES) is an RNA sequence that allows initiation of translation. In some embodiments, the IRES has the sequence of an aptamer for eIF4G, Homo sapiens cDNA FLJ43058, Acute bee paralysis virus IRES, Aphid deadly paralysis virus IRES, Avian encephalomyelitis virus IRES, Bovine viral diarrhea virus 1 IRES, Canine distemper IRES, Classical swine fever virus IRES, Common faecal associated virus, Coxsackievirus A (CVB1 / 2) IRES, Coxsackievirus B3 (CVB3) IRES, Crickets paralysis virus IRES, Crucifer tobamovirus IRES, Drosophila antennapedia IRES, Drosophila melanogaster Leucovirus B1 IRES, Drosophila melanogaster C virus IRES, Drosophila melanogaster bald IRES, Drosophila melanogaster harvester IRES, Drosophila melanogaster Ubx IRES, Ectropis oblique picornavirus-like virus IRES, Encephalomyocarditis virus (EMCV) IRES, Equine rhinitis virus IRES, Foot-and-mouth disease virus IRES, Gekko kikuchii variable lizard picornavirus 2 IRES, Hepatitis A virus IRES, Hepatitis C virus IRES, Hepatitis G virus IRES, Hibiscus chlorotic ring spot virus IRES, Himetobi P virus IRES, Icerya purchasi virus-1 IRES, Human AMLl / RUNXl gene IRES, Human AQP4 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 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 papilloma virus IRES, human PDGF2 / c-sis gene IRES, human Pim-1 gene IRES, human rhinovirus 2 IRES, human SFTPA1 gene IRES, human UNR gene IRES, human VEGF-A gene IRES, human XIAP gene IRES, Kashmir bee virus IRES, 14 containing the mouse eukaryotic translation initiation factor 1A domain (Eif1ad14) gene IRES, mouse Gtx gene IRES, mouse HIF1a gene IRES, mouse NDST4L gene IRES, mouse Rbm3 gene IRES, mouse UtrA gene IRES, entomopoxvirus IRES, pintail duck picobirnavirus IRES, spined soldier bug enterovirus IRES, poliovirus 1 IRES, reticuloendotheliosis virus IRES, Sitobion avenae virus IRES, Rous sarcoma virus IRES, Saccharomyces cerevisiae TFIID gene IRES, Saccharomyces cerevisiae YAP1 gene IRES, salivirus IRES, simian picornavirus IRES, simian virus 40 IRES, Solenopsis invicta virus 1 IRES, Thalia geniculata syndrome virus IRES, Theiler's encephalomyelitis virus IRES, or Triatoma 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 about 220 nucleotides in length.

[0145] As described above, the methods of preparing circular RNA described herein employ either a Group I or Group II maturase. The Group I or Group II maturase pairs with a Group I or Group II intron, respectively. In this context, "pairs" refers to a maturase that splices or assists in splicing a particular Group I or Group II intron. Table 2 provides exemplary Group I or Group II maturases and their Group I or Group II intron pairs, all of which are contemplated to be within the scope of the present disclosure. Homologs of the Group I or Group II maturases as described herein are also included.

[0146] In some embodiments, the Group I intron is an Aspergillus nidulans COBl intron and its paired mature enzyme is an Aspergillus nidulans I-Anil mature enzyme. The term “I-Anil mature enzyme” includes a polypeptide of SEQ ID NO: 3 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% identity to a polypeptide of SEQ ID NO: 3 or a polypeptide comprising a portion of a polypeptide of SEQ ID NO: 3. In some embodiments, the I-Anil mature enzyme is N-terminally truncated in a manner that preserves the function of the mature enzyme domain. In some aspects, about 5-10 amino acids preceding the mature enzyme domain are preserved in the N-terminally truncated I-Anil mature enzyme. In some embodiments, the I-Anil mature enzyme is a polypeptide of SEQ ID NO: 4 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% identity to a polypeptide of SEQ ID NO: 4 or a polypeptide comprising a portion of a polypeptide of SEQ ID NO: 4.

[0147] 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.

[0148] In some embodiments, the Group II intron is the rice lysine tRNA-K(UUU) intron, and its paired mature enzyme is the rice mature enzyme K. The term "mature enzyme K" includes a polypeptide encoded by SEQ ID NO: 1 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% 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 mature enzyme is cyt-19 from Neurospora Crassa, and the intron is the aI5y and bI1 Group II introns from yeast. The term "cyt-19" includes a polypeptide of SEQ ID NO: 2 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% 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 Lactococcus LI. LtrB intron, and its paired mature enzyme is the Lactococcus LtrA mature enzyme. The term "LtrA mature enzyme" includes a polypeptide of SEQ ID NO: 15 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% 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 LI. LtrB intron comprises or is SEQ ID NO: 45.

[0149] In some embodiments, the Group II intron is a Lactococcus lactis LtrB intron and its paired mature enzyme is a Lactococcus lactis LtrB mature enzyme. In some aspects, the intron is nadl i4 and its paired mature enzyme is a MatR mature enzyme. The term "MatR mature enzyme" includes a polypeptide of SEQ ID NO: 16 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% identity to a 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 Group IIA intron al2 and the paired mature enzyme is MSS116. The term "MSS116" includes a polypeptide of SEQ ID NO: 17 or a polypeptide sequence having or greater than about 50% identity, having or greater than about 75% identity, having or greater than about 80% identity, having or greater than about 85% identity, having or greater than about 90% identity, having or greater than about 95% identity, or having or greater than about 98% identity to a polypeptide of SEQ ID NO: 17 or a polypeptide comprising a portion of the polypeptide of SEQ ID NO: 17. In still other aspects, the Group I or Group II intron and paired mature enzyme are as described in Table 2 or homologs thereof.

[0150] It is understood that the Group I or Group II intron and its respective paired mature enzyme can be from different species. It is also understood that the mature enzyme can be mutated at one or more amino acids to reduce or eliminate DNA endonuclease activity and / or to increase splicing activity. The mature enzyme can also be N-terminally truncated. In some embodiments, the mature enzyme is mutated to increase its solubility.

[0151] Inclusion of the mature enzyme and the mature enzyme intron pair in the present disclosure allows for milder reaction conditions and / or increased circular RNA production. Accordingly, included herein are methods of making circular RNA, wherein the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of 20°C to 45°C. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 20°C. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 25°C. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 30°C. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 35°C. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 37°C. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a temperature of about 42°C.

[0152] In some aspects, the mature enzyme polypeptide and the precursor RNA are contacted for about 30 minutes, about one hour, about two hours, about three hours, or about four hours. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted for about two hours. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted for about three hours. In some embodiments, the mature enzyme polypeptide and the precursor RNA are contacted for about four hours.

[0153] In other or additional embodiments, the mature enzyme polypeptide and the precursor RNA are contacted under conditions comprising a magnesium concentration of 100 micromolar to 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.

[0154] Also included herein are methods of making circular RNA, wherein the vector further comprises two or more complementary sequences. Accordingly, the present disclosure includes a method of making circular RNA, the method comprising:

[0155] a. transcribing the vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order:

[0156] i. a 5' complementary sequence,

[0157] ii. a 3' I group intron or a 3' II group intron sequence comprising a 3' splice site dinucleotide,

[0158] iii. a first non-coding sequence,

[0159] iv. a corresponding 5' Group I or 5' Group II intron sequence containing a 5' splice site di-nucleotide, and

[0160] v. a 3' complement sequence; and

[0161] b. contacting the precursor RNA with a paired Group I or Group II mature enzyme polypeptide to allow formation of the circular RNA.

[0162] The present disclosure also includes a method of making a circular RNA, the method comprising:

[0163] a) transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order:

[0164] i. a 3' Group I or 3' Group II intron sequence containing a 3' splice site di-nucleotide,

[0165] ii. a first non-coding sequence,

[0166] iii. a 5' complement sequence,

[0167] iv. a 3' complement sequence;

[0168] v. a corresponding 5' Group I or 5' Group II intron sequence containing a 5' splice site di-nucleotide, and

[0169] b. contacting the precursor RNA with a paired Group I or Group II mature enzyme polypeptide to allow formation of the circular RNA.

[0170] In some methods, the vector further comprises an internal ribosome entry site (IRES) and a protein-coding sequence and a second non-coding sequence between the elements of a.iii. and a.iv. in the following order.

[0171] It will be understood that the 5' and 3' complement sequences are DNA sequences that are sufficiently complementary to one another to bind to one another, e.g., based on A-T complementarity and C-G complementarity. In these embodiments, each element of the vector or the transcribing or contacting steps can be any element described herein. In some embodiments, the complement sequences are 25 to 60 nucleotides in length. In some embodiments, the 5' complement sequence comprises or is SEQ ID NO: 37 and the 3' complement sequence comprises or is SEQ ID NO: 41.

[0172] In addition to methods of making circular RNA, provided herein are circular RNA made according to these methods and pharmaceutical compositions comprising the circular RNA. For example, the circular RNA described herein can be used as a virus-like protein vaccine for preventing infection, such as COVID-19. In some embodiments, the circular RNA comprises about 300 to 12,000 nucleotides, about 1,000 to 10,000 nucleotides, about 5,000 to 8,000 nucleotides, about 8,000 to 12,000 nucleotides, or about 10,000 to 12,000 nucleotides. The pharmaceutical compositions can 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 lipopolymer, a lipopolymeric hybrid, an exosome, or a leukosome.

[0173] Also provided herein are methods of isolating the circular RNA described herein. Any appropriate method can be used to perform such isolation, including the use of solid phase extraction (SPE) and size exclusion chromatography. In some embodiments, the method of isolating the circular RNA described herein comprises

[0174] a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein;

[0175] b. contacting the mixture with one or more immobilized 5' binding proteins and one or more immobilized 3' binding proteins; and

[0176] c. isolating the circular RNA.

[0177] Figure 13A general schematic of one such method is shown. The 5' and 3' binding proteins can be any protein that binds to the 5' and 3' RNA ends. In some embodiments, the 5' binding protein is a S. cerevisiae DXO 5' binding protein or homolog thereof. The term "DXO" includes a polypeptide encoded by SEQ ID NO: 5 or a polypeptide sequence having or being greater than about 50% identity, having or being greater than about 75% identity, having or being greater than about 80% identity, having or being greater than about 85% identity, having or being greater than about 90% identity, having or being greater than about 95% identity, or having or being greater than about 98% 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 changes its temperature or pH sensitivity. In some embodiments, the RNA binding protein is mutated, truncated, or fused to another protein domain to improve its solubility and / or RNA binding affinity.

[0178] In other or additional embodiments, the methods of isolating circular RNA described herein comprise

[0179] a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein;

[0180] b. adding biotinylated adenosine to the linear RNA using a poly(A) polymerase to generate biotinylated linear RNA; and

[0181] c. isolating the circular RNA by removing the biotinylated linear RNA using immobilized streptavidin.

[0182] In other or additional embodiments, the methods of isolating circular RNA described herein comprise

[0183] a. obtaining a mixture of linear RNA and circular RNA produced by any of the methods described herein;

[0184] b. using an RtcB RNA ligase to modify the linear RNA;

[0185] c. contacting the mixture and immobilized His-Tag streptavidin bound to the modified linear RNA; and

[0186] d. isolating the circular RNA from the flow-through.

[0187] In other or additional embodiments, the methods of isolating circular RNA described herein comprise

[0188] a. obtaining a mixture of linear RNAs and circular RNAs produced by any of the methods described herein;

[0189] b. contacting the mixture and a polynucleotide specific for the circularization junction of the circular RNA bound to biotin to generate a second mixture;

[0190] c. contacting the second mixture with immobilized streptavidin;

[0191] d. removing the linear RNAs; and

[0192] e. isolating the circular RNAs by releasing them from the one or more immobilized polynucleotides.

[0193] In some aspects, the polynucleotide specific for the circularization junction of the circular RNA is a locked nucleic acid (LNA), and the polynucleotide is complementary to a sequence in the circularization junction of the circular RNA.

[0194] It is to be understood that the above description is directed to preferred embodiments of the application and that numerous modifications can be made thereto without departing from the scope of the application. The present application is further illustrated by the following examples which should not be construed as imposing any limitations upon the scope thereof in any way. On the contrary, it is to be clearly understood that resort might be had to various other embodiments, modifications, and equivalents thereof which may

[0195] Example

[0196] Example 1: Performance of circRNAs is superior to linear RNAs.

[0197] The performance of circRNAs versus linears was compared using expression of green fluorescent protein (GFP). Briefly, circRNAs and linear RNAs were transfected into HEK293 and the fluorescence intensity was monitored using a live cell imaging system (Incucyte by Sartorius). It was observed that GFP expression from circRNAs lasted longer and was still increasing after 48 hours, while GFP expression from linear RNAs started to decrease after 24 hours. See Figure 2 .

[0198] Example 2: A. n. Cob 1 intron pre-RNAs are successfully spliced in vitro and can be visualized with agarose gel.

[0199] Methods: RNA was prepared using in vitro transcription with the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) starting with linearized plasmid. Purification was then performed using Monarch RNA Cleanup 500 ug (New England Biolabs). RNA was pre-incubated in 2X TN buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 300 mM MgCl2) at 37C for 20 minutes. 2x GTP solution (2 mM rGTP (Promega)) was then added to initiate splicing. Aliquots were collected at the indicated times and the reactions were column purified using Monarch RNA Cleanup columns. RNA was visualized using an e-gel system (denatured with 50% formamide, 3 minutes at 70C).

[0200] Results: Linear RNA can be self-spliced at 37C using these conditions in a solution of 50 mM Tris-HCL, pH 7.5; 100 mM NaCl, 150 mM MgCl2, and 1 mM GTP. Over time, the precursor RNA decreases and the ligated exons and free introns as well as intermediates of the splicing reaction are visualized. See Figure 3 .

[0201] After verifying that linear RNA can be self-spliced in vitro under high magnesium conditions, the focus shifted to mature enzyme assisted splicing. To successfully produce macRNA, the mature enzyme needs to be purified and generate the rearranged intron / exon (PIE) version from the RNA to be spliced.

[0202] Example 3: I-aniI purification

[0203] The I-aniI expression vector was designed using its codon optimization tool, cloned into its primary expression vector (optimized for expression in E. coli), and purchased as a complete plasmid from Geneart (Thermofisher). The plasmid was first transformed into a custom cell line of plasmid BL21-DE3-star (Thermofisher). A colony was used to inoculate 5 ml of culture in LB + ampicillin (Ambp). The next day, 1 ml of culture was used to inoculate 50 ml of LB + amp and the OD was followed until reaching 0.6-0.8 log phase. IPTG was added to the culture at a final concentration of 1 mM and incubated at low temperature (30C) overnight. Expression was tested by running SDS gels on lysed bacterial pellets. See Figure 4.

[0204] Preliminary attempts showed limited expression of I-aniI mature enzyme using this plasmid. Troubleshooting was done by changing the used medium (Premium Fermentation Broth (Athena ES) to give the bacteria additional nutrients and reach higher densities); lowering the incubation temperature to 18°C to allow proper protein folding; changing the used IPTG stock and performing a positive control. Possible problems could be codon optimization (fast translation could lead to protein misfolding), trying a different plasmid expression system, etc. See Fig. 4.

[0205] In a subsequent attempt to express I-aniI mature enzyme, I-aniI was subcloned into a new expression backbone from addgene by exchanging the ORF from GFP to the mature enzyme. Also a new full size open reading frame of I-aniI was obtained from geneart. All constructs were tested using the same method as described above (Premium Fermentation Broth, induction at log phase with 0.5 mM or 1 mM IPTG). Different incubation temperatures and different time points were tested. Figure 5 Expression of mature enzyme with new full size open reading frame can be seen in Fig. 5.

[0206] The mature enzyme I-aniI was purified as follows: the pellet of bacteria was resuspended in lysis buffer (lx PBS, 100 mM PMSF, 250 mM NaCl, 5 mM imidazole). After that, the mixture was sonicated for 15 minutes at 4°C (15 seconds on, 15 seconds off, high frequency of cycles). 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 (Fisher) was equilibrated with buffer (lx PBS, 10 mM imidazole). The equilibration buffer was added to the supernatant, as the volume obtained was equivalent to 3 His-Pur resin bed. The solution was incubated with the equilibrated resin and rotated on a rolling shaker for 90 minutes at 4°C. The flow-through was discarded and the resin was subjected to 4 rounds of washing (wash buffer: lx PBS, 25 mM imidazole, 100 mM NaCl). Finally, the protein was eluted stepwise by incubating the resin with elution buffer (lx PBS, 250 mM imidazole, 100 mM NaCl). The sample was then run in a SDS page gel. Below is shown the different steps of the purification, the eluate shows a band corresponding to I-aniI (marked with an arrow). See Fig. 6. Figure 6 .

[0207] Example 4: It is essential for the pie version to retain the splicing position and folding of the ribozyme.

[0208] Ribozyme function relies heavily on proper folding into a catalytically active form. Structure prediction methods were used to try to match the folding of the native RNA to the synthetic pie version. RNAfold was used to predict the structure. As mentioned previously, the native ribozyme consists of an intron sandwiched between 5' and 3' exons. To make the ribozyme form a loop, the positions of the intron and exons are twisted and the intron is split into two parts. See Figure 7 .

[0209] The structure of different intron splits were tested using RNAfold. It was determined that splitting the intron in two produced very similar structures and the splice sites were similarly arranged. It was also determined that having a shorter 5' intron part would still preserve the overall structure, as opposed to (longer 5' intron part) producing a scrambled structure. See Figure 8 . Five (5) constructs were generated from A to E using different layouts obtained through geneart. See Figure 9 . The selection of these constructs is explained in Table 1 below.

[0210] Table 1.

[0211]

[0212]

[0213] Example 5: Adjustment of splicing conditions

[0214] RNA for each construct was obtained using in vitro transcription (IVT) and tested for self-splicing using the same conditions as for the un-torqued RNA. It appears that some degree of splicing has occurred during or after IVT. Some degree of degradation also occurred, likely due to the high magnesium content. See Figure 10 . In the presence of the mature enzyme, the concentration of magnesium will be greatly reduced (from 150 mM down to 5 mM final concentration), which can reduce the degradation of the RNA.

[0215] Various splicing conditions were tested for construct D (no cargo) and differences in splicing were observed, especially when pre-incubating the RNA in buffer for 10 minutes instead of 20 minutes before adding the splicing trigger (GTP). These steps will allow for the adjustment of splicing conditions when using the mature enzyme. See Figure 11 .

[0216] Constructs F, G and H described in Table 1 were also developed. This was done to try to improve the splicing constructs A to E, which involved splitting the intron exactly in half by using shortened exons. Thus, for constructs F, G and H, the intron was split at 2 positions (creating long or new short 5’ ends). Constructs G and H were generated by choosing random positions for the intron to be split and the exons were of the same size as used on the small intron construct (SEQ ID NO: 14). Construct F was generated to modulate the percentage of GC content, which can influence splicing efficiency. As shown in Table 1, construct F seems to react to the splicing conditions (showing extra bands appearing over time) and can be the preferred construct. Figure 12

[0217] Between the two exons, that is where the cargo is going to arrive, a placeholder can be added (a small sequence that adds multiple cloning sites). Also a spacer sequence with many AAATTT that can base pair with the intron can be added. Downstream of the whole sequence, a sap1 restriction site was added for future IVT.

[0218] Example 6: Mature enzyme-intron pairs

[0219] Other mature enzyme / intron pairs can work with the I-aniI mature enzyme-A.n.Cob1 intron system. The following Table 2 provides exemplary pairs.

[0220] Table 2.

[0221]

[0222]

[0223] Table 3: List of IRES sequences tested for specificity of circRNA.

[0224] Label Full name IRES tested A14 circPPT19-mcherry PPt19 A15 circA1-mcherry IRES00103 (Rous SarV IRES) A16 circA3-mcherry IRES488 (AQP4 IRES) A17 circA4-mcherry IRES190 (HPV IRES) A18 circA5-mcherry IRES498 (Line ORF1) A19 circA7-mcherry IRES485 (bag1) A20 circA9-mcherry IRES487 (ELG1) A21 circA13-mcherry RBM3 IRES A22 circA14-mcherry At1R IRES A23 circA6-mcherry IRES494 (Line ORF1) A24 circA8-mcherry IRES476 (KMI1) A25 circA15-mcherry Powan duck IRES A26 circA16-mcherry Caspase 8 IRES A27 circ-mcherry CVB3 A28 circA2-mcherry IRES240 (ELH)

[0225] Selection sequence:

[0226] 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.

[0227]

[0228] SEQ ID NO: 7 Construct B; underlined: intron sequence; bold: nanoluciferase; bold underlined: CVB3 IRES; bold underlined: homology arms; italic: short exon. ​

[0229]

[0230] SEQ ID NO: 8 Construct C; underlined: intron sequence; bold: nanoluciferase; bold underlined: CVB3 IRES; bold underlined: homology arm; italic: short exon; italic underlined: extended exon;

[0231]

[0232]

[0233] SEQ ID NO: 9 Construct D; underlined: intron sequence.

[0234]

[0235]

[0236] SEQ ID NO: 10 Construct E; underlined: intron sequence; bold: nanoluciferase; bold underlined: CVB3 IRES; italic: short exon; italic underlined: extended exon.

[0237]

[0238]

[0239] SEQ ID NO: 11 Construct F; underlined: intron sequence.

[0240]

[0241] SEQ ID NO: 12 Construct G; underlined: intron sequence.

[0242]

[0243] SEQ ID NO: 13 Construct H; underlined: intron sequence.

[0244]

[0245]

[0246] SEQ ID NO: 14 Intron; underlined: intron sequence.

[0247]

Claims

1. A method for preparing circular RNA, comprising: a) transcribing the vector to form a precursor RNA, wherein the vector comprises the operably linked elements in the following order, and does not include complementary sequences: i. a 3' group I intron or a 3' group II intron sequence containing a 3' splice site dinucleotide, ii. the first non-coding sequence, and iii. the corresponding 5' group I intron or 5' group II intron sequence containing the 5' splice site dinucleotide; and b) contacting the precursor RNA with a paired Group I or Group II maturase polypeptide to allow formation of the circular RNA.

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

3. A method for preparing circular RNA, the method comprising: a) transcribing a vector to form a precursor RNA, wherein the vector comprises operably linked elements in the following order: i. a 3' group I intron or a 3' group II intron sequence containing a 3' splice site dinucleotide, ii. the first non-coding sequence, iii.5' complementary sequence, iv.3' complementary sequence; v. the corresponding 5' group I intron or 5' group II intron sequence containing the 5' splice site dinucleotide, and vi. and b) contacting the precursor RNA with a paired Group I or Group II maturase polypeptide to allow formation of the circular RNA.

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

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

6. The method according to 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 containing the 5' splice site dinucleotide. 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 containing 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. The method of claim 8, wherein the paired Group I maturase is the Aspergillus nidulans I-AniI maturase.

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

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

12. The method according to any one of claims 1 to 7, wherein the group II intron is selected from the group consisting of rice (Oryza sativa) trnK gene, tRNA (V-UAC, I-GAU, A-UGC and K-UUU), ribosomal proteins (rpl2 and rps12) and chloroplast ATPase (atpF).

13. The method of claim 12, wherein the paired Group II maturase is rice maturase K.

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

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

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

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

18. 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. The method of any one of claims 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 nadli4 and the group II paired intron is MatR.

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

22. The method according to any one of claims 1 to 20, wherein one or more of the non-coding sequences are 15 to 50 nucleotides.

23. The method according to any one of claims 1 to 20, wherein one or more of the non-coding sequences are 18 to 22 nucleotides.

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

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 method of any one of claims 1 to 25, wherein the IRES has a sequence that is 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 1 IRES, Canine Stridor IRES, Classical Swine Fever Virus IRES, Common Feces-Associated Virus (Cosavirus), Coxsackievirus A (CVB1 / 2) IRES, Cricket Paralysis Virus IRES, Crucifer Tobamo Virus IRES, Drosophila Antennapedia IRES, Diresapivirus B1 IRES, Drosophila C virus IRES, Drosophila hairless IRES, Drosophila harvester IRES, Drosophila Ubx IRES, tea geometrid picornavirus-like virus IRES, encephalomyocarditis virus (EMCV) IRES, equine rhinitis virus IRES, foot-and-mouth disease virus IRES, Guanxi changeable lizard picornavirus 2 IRES, hepatitis A virus IRES, hepatitis C virus IRES, hepatitis G virus IRES, hibiscus chlorosis ringspot virus IRES, HimetobiP virus IRES, Homalodisca glabra coagulata) virus-1 IRES, human AML1 / RUNX1 gene IRES, human AQP4 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 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, Kashmir beevirus IRES, mouse eukaryotic translation initiation factor 1A domain-containing 14 (Eif1ad14) gene IRES, mouse Gtx gene IRES, mouse HIF1α gene IRES, mouse NDST4L gene IRES, mouse Rbm3 gene IRES, mouse UtrA gene IRES, Parechovirus IRES, pink-eared duck picornavirus IRES, Plautia staliintestine virus IRES, poliovirus 1 IRES, reticuloendotheliosis virus IRES, Rhopalosiphum padivirus IRES, Rous sarcoma virus IRES, Saccharomyces cerevisiae TFIID gene IRES, Saccharomyces cerevisiae YAP1 gene IRES, salivirus IRES, simian picornavirus IRES, simian virus 40 IRES, fire ant virus 1 IRES, Taura syndrome virus IRES, Theileria encephalomyelitis virus IRES, or Triatoma 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. 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. 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 antigen or a bacterial antigen.

32. A circular RNA prepared according to the method of any one of claims 1 to 31.

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

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

35. A method for isolating circular RNA, the method comprising: a) obtaining a mixture of linear RNA and circular RNA produced 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; and c) isolating the circular RNA.

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

37. The method of claim 35 or claim 36, wherein the 3' binding protein is nuclease inactivated RNase R.

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

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