Compositions and methods for preparing capped circular RNA molecules

Abstract

The present disclosure provides compositions, reagents and methods for producing capped circular RNA molecules, cyclized RNA molecules, and in particular cyclized mRNA molecules encoding polypeptides such as therapeutic proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 355,456 filed on June 24, 2022 and U.S. Provisional Application No. 63 / 480,291 filed on January 17, 2023, the disclosures of which are expressly incorporated herein by reference. Incorporation by Reference into this Electronically Available Sequence Listing

[0003] This application contains a sequence listing submitted in the form of an electronic text file, named "21-1401-WO_SequenceListing.xml", with a size of 24,183 bytes, created on June 26, 2023. The information contained in this electronic file is hereby incorporated by reference in its entirety. Background Art

[0004] RNA therapeutics as a field has recently developed rapidly, as evidenced by the recent clinical performance of successful mRNA vaccines against SARS-CoV-2. See Polack et al., 2020, Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 383, 2603–2615; Lombardi et al., 2021, Mini Review Immunological Consequences of Immunization With COVID-19 mRNA Vaccines: Preliminary Results. Front. Immunol. http: / / paperpile.com / b / RTDeyI / 5uWI12, 657711. The inherent programmability and relative ease of production of mRNA underlie its potential to replace traditional protein-based therapies. See Sahin et al., 2014, mRNA-based therapeutics—developing a new class of drugs," Nature reviews Drug discovery 13:759-780. In addition to proven clinical applications exemplified by COVID vaccines, mRNA has been used experimentally to express angiogenic factors and generate vaccines against influenza and Zika viruses (Zangi et al., 2013, Nature biotechnology 31:898; Bahl et al., 2017, Molecular Therapy 25:1316-1327; Richner et al., 2017, Cell 168:1114-1125).

[0005] mRNA is also an emerging therapeutic modality due to its ability to rapidly produce a protein of interest (POI) in vivo. Some of the main advantages of mRNA as a platform compared to DNA-based therapeutics are its programmability, transient capabilities, ease of production, and lack of risk of genomic integration. In eukaryotic cells, typical mRNAs are linear and contain a 5' 7-methylguanosine cap (m 7 G) and 3' poly(A) tails, both of which are indispensable for efficient translation in cells. Recent studies have shown that circular mRNAs (circRNAs) have a longer half-life in cells compared to linear mRNAs due to their reduced sensitivity to exonucleases. However, cap-dependent translation of circRNAs is not as efficient as that of linear RNAs.

[0006] Therefore, there remains a need in the art for reagents and methods for producing and using compositions comprising circularized RNA molecules, particularly circularized mRNA molecules, encoding polypeptides such as therapeutic proteins, to produce useful, particularly therapeutically useful, phenotypic effects on recipient cells. Summary of the invention

[0007] The present invention provides compositions, reagents and methods comprising RNA molecules, preferably encoding polypeptides, wherein the RNA molecules are circularized RNA molecules and particularly circularized mRNA molecules.

[0008] In certain embodiments, a type 2 capped circular RNA molecule comprises an mRNA region encoding a polypeptide, a 5' end containing a cap structure, a derivatized nucleotide located between the cap structure and the mRNA region; and a 3' end covalently linked to the derivatized nucleotide.

[0009] In certain embodiments, a Type 1 capped circular RNA molecule comprises an RNA oligonucleotide comprising a 5' end and a 3' end portion comprising a cap structure; a circular RNA molecule comprising an mRNA encoding a polypeptide; and a derivatized nucleotide located within the circular RNA molecule, wherein the 3' end portion of the oligonucleotide is covalently linked to the derivatized nucleotide on the circular RNA molecule.

[0010] In certain alternative embodiments, a Type 3 capped circular RNA molecule comprises an RNA oligonucleotide comprising a 5' end and a 3' end portion containing a cap structure; a circular RNA molecule comprising a twister ribozyme, an mRNA encoding a polypeptide, an oligonucleotide portion forming a hairpin, and a derivatized nucleotide located within the hairpin, wherein the 3' end portion of the oligonucleotide is covalently linked to the derivatized nucleotide within the hairpin of the circular RNA molecule.

[0011] In certain embodiments, the derivatized nucleotide includes a moiety that can react with the 3' end moiety via bioconjugation chemistry, such as click chemistry. Additionally, the cap structure includes 7-methylguanosine (m 7 G), 7-benzylguanosine (Bn 7 G), 7-chlorobenzylguanosine (ClBn 7 G), chlorobenzyl-O-ethoxyguanosine (ClBnOEt 7 G), or any derivative thereof. In certain embodiments, the 7-methylguanosine cap structure further comprises one or more locked nucleic acids (LNA), or one or more 2'-methoxy groups (2OMe), or any derivative thereof.

[0012] In certain embodiments, type 2 and type 1 capped circular RNA molecules include one or more modified nucleotides, such as pseudouridine, N 1 -methyl pseudouridine (m 1 Ψ), 6-methyladenosine (m6 A), 5-methylcytidine, inosine, or any derivative thereof. In some embodiments, the modified nucleotide comprises a locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethyl ether (2-MOE) sugar backbone, or any derivative thereof.

[0013] In certain embodiments, the type 3 capped circular RNA molecule comprises one or more modified nucleotides, such as 6-methyladenosine (m 6 A), 5-methylcytidine, inosine, or any derivative thereof. In certain embodiments, the modified nucleotide comprises a locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethyl ether (2-MOE) sugar backbone, or any derivative thereof.

[0014] In certain embodiments, type 1 and type 3 capped circular RNA molecules include circular RNAs that include multiple mRNA regions encoding multiple polypeptides. In these embodiments, type 1 and type 3 capped circular RNA molecules may also include multiple RNA oligonucleotides, including a 5' end and a 3' end portion containing a cap structure, and multiple derivatized nucleotides, which are located at the 5' position of each of the mRNA regions encoding a peptide or polypeptide in the circular RNA, wherein each 3' end of each of the multiple RNA oligonucleotides is covalently linked to each of the multiple derivatized nucleotides. In some embodiments, each mRNA region encoding a peptide or polypeptide includes a 3' poly A sequence, wherein the polypeptide encodes Cas9, a base editor, or a derivative thereof, or a therapeutic protein.

[0015] Further provided is a pharmaceutical composition of the capped circularized RNA molecule provided by the present invention, which comprises a specific embodiment of the capped circularized RNA molecule provided by the present invention and a pharmaceutically acceptable adjuvant, excipient, carrier or diluent.

[0016] The present invention also provides a method for producing a type 2 capped circular RNA molecule of this aspect of the present invention, the method comprising synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure, an mRNA encoding a peptide or polypeptide, a derivatized nucleotide located between the cap structure and the mRNA region encoding the polypeptide, and a 3' end containing a portion; and reacting the derivatized nucleotide with the 3' end portion to form a covalently linked capped circular RNA molecule. In certain embodiments, the synthesis of the RNA oligonucleotide comprises the following steps: synthesizing a first RNA oligonucleotide comprising a 5' end containing a cap structure, an mRNA encoding a peptide or polypeptide, and a hairpin structure between the capped 5' end and the mRNA encoding the peptide or polypeptide; derivatizing the nucleotides within the hairpin structure of the first RNA; synthesizing a second RNA oligonucleotide comprising a 3' end portion reactive with the derivatized nucleotide; and connecting the 3' end of the first RNA molecule to the 5' end of the second RNA molecule. In certain embodiments, the synthesis of RNA oligonucleotides comprises the following steps: synthesizing a first RNA oligonucleotide primer comprising a 5' end comprising a cap structure, a derivatized nucleotide, and a complementary sequence to a DNA template encoding a peptide or polypeptide; transcribing the first RNA oligonucleotide from the primer along the DNA template to produce an mRNA encoding the peptide or polypeptide; synthesizing a second RNA oligonucleotide comprising a 3' end comprising a portion; and connecting the 3' end of the first RNA oligonucleotide encoding the peptide or polypeptide sequence to the 5' end of the second RNA molecule.

[0017] The present invention also provides a method for producing a type 1 capped circularized RNA molecule of this aspect of the present invention, the method comprising the following steps: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end containing a portion reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent connection between the RNA oligonucleotide and the circular RNA. In certain embodiments, the derivatized nucleotide comprises a portion capable of reacting with the 3' end portion by bioconjugation chemistry, wherein the bioconjugation chemistry is click chemistry. In addition, the circularized RNA is produced by ribozyme-mediated splicing, enzymatic ligation, or click chemistry-mediated circularization.

[0018] In certain embodiments, the synthesis of a circular RNA oligonucleotide of a type 1 capped circularized RNA molecule comprises the following steps: synthesizing an RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide and complementary sequences on the 5' and 3' ends to facilitate circularization, wherein the derivatized nucleotide is located within the complementary sequence; and circularizing the RNA oligonucleotide. In certain embodiments, the complementary sequence comprises a single cytidine nucleotide, wherein the single cytidine is a derivatized nucleotide. In certain embodiments, the synthesis of a circular RNA oligonucleotide comprises the following steps: synthesizing an RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide and a hairpin structure containing an enzyme recognition site for introducing the derivatized oligonucleotide into the RNA oligonucleotide; reacting the RNA oligonucleotide with an enzyme to produce a derivatized nucleotide within the hairpin structure; and circularizing the RNA oligonucleotide. In certain embodiments, the synthesis of a circular RNA oligonucleotide comprises the following steps: synthesizing a first RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide, and hydroxyl groups on both the 5' and 3' ends; synthesizing a second RNA oligonucleotide comprising a derivatized nucleotide, and phosphates on both the 5' and 3' ends; connecting the 5' phosphate end and the 3' hydroxyl end; and connecting the 5' hydroxyl end and the 3' phosphate end of the first oligonucleotide and the second oligonucleotide, respectively, to produce a circularized RNA oligonucleotide. In certain embodiments, the synthesis of circular RNA oligonucleotides comprises the following steps: synthesizing a first RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide, a 5' end containing a triphosphate, and a 3' end containing a hydroxyl group; synthesizing a second RNA oligonucleotide comprising a derivatized nucleotide and phosphates on both the 5' and 3' ends; connecting the 3' end of the first oligonucleotide to the 5' end of the second oligonucleotide to produce a third oligonucleotide; hydrolyzing the triphosphate on the 5' end of the third oligonucleotide; and connecting the 5' end of the third oligonucleotide to the 3' end to produce a circularized RNA oligonucleotide. In certain embodiments, the synthesis of circular RNA oligonucleotides comprises the following steps: synthesizing an RNA oligonucleotide primer comprising a derivatized nucleotide and a complementary sequence of a DNA template encoding a peptide or polypeptide; transcribing the RNA oligonucleotide to further include an mRNA encoding a peptide or polypeptide; and circularizing the RNA oligonucleotide.

[0019] The present invention also provides a method for producing the type 3 capped circularized RNA molecule of this aspect of the present invention, the method comprising the following steps: producing a circularized RNA molecule, the circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide, the RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end containing a portion reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the circular RNA,

[0020] The synthesis of circular RNA oligonucleotide further comprises the following steps: synthesizing RNA oligonucleotide, which includes an mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme recognition site, and twister ribozyme sequences on both the 5' and 3' ends; reacting the RNA oligonucleotide with an enzyme to produce a derivatized nucleotide within the hairpin structure; and circularizing the RNA oligonucleotide using the twister ribozyme sequence.

[0021] The capped circularized RNA molecules provided by the present invention advantageously improve the stability and translation efficiency of peptides and polypeptides encoded therefrom. They can be used in particular to promote therapeutic substitution of polypeptide variants encoded by genetic polymorphisms, particularly such polymorphisms associated with genetic diseases. The capped circularized RNA molecules provided by the present invention can advantageously provide transient expression of encoded peptides or polypeptides, thereby providing therapeutic flexibility that traditional gene replacement therapy cannot achieve. The capped circularized RNA molecules provided by the present invention have advantages that other circular RNA processing does not have, including resistance to nuclease exonucleases and higher ribosome loading. The capped circularized RNA molecules provided by the present invention further advantageously provide translation initiation that is not limited to internal ribosome entry sites (IRES) or translation enhancing elements (TEEs), thereby providing more powerful cap-dependent translation initiation. Another advantage of the capped, circularized RNA molecules provided by the present invention is to replace protein-based therapeutics (i.e., where the protein is delivered and must be specifically introduced into the target cell in a functional manner and targeted to the appropriate intracellular niche; see, Lagasse et al., 2017, F1000 Research 6:113; doi:10.12688 / f1000research.9970.1) with the delivery of RNA encoding the necessary peptide or polypeptide, the form of which (capped, circular) is resistant to nuclease degradation and provides robust expression due to the presence of the eukaryotic cap.

[0022] These and other features, objects and advantages of the present invention will become better understood from the following description. In the description, reference is made to the accompanying drawings, which form a part of this article, in which embodiments of the present invention are shown by way of illustration and not limitation. The description of the preferred embodiments is not intended to limit the present invention to cover all modifications, equivalents and alternatives. Therefore, the scope of the present invention should be interpreted with reference to the claims described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A to 1H The conceptualization of capped circular mRNA (QRNA) is shown. Figure 1A Schematic diagram showing the regular circular RNA structure and IRES-mediated translation initiation. Figure 1B Schematic diagram showing the regular linear RNA structure and m7G-cap-dependent translation initiation. Figure 1C We demonstrate the design of a capped circular mRNA (qRNA): a circular RNA with a click-reaction handle in its 5' untranslated region (UTR) is chemically conjugated to the 3' end of a chemically capped linear oligomer. The qRNA hijacks the cap-dependent translation initiation machinery to increase ribosomal loading on the circular transcript. Figure 1D and Figure 1E The general structures of type 1 and type 2 QRNAs are shown. Figure 1F The synthesis scheme of QRNA encoding type 3 Flag is shown, where oligo 3.1 is a sequence-designed RNA containing twister ribozyme (red), tRNA-like hairpin (green), 5'UTR (blue), and 3xFlag-peptide coding sequence (orange); oligo 3.2 is a circularized RNA; oligo 3.3 is a circular RNA with a 5-methyl-tetrazine click chemistry handle; oligo 3.4 is a capped oligo with a 3'-TCO click chemistry handle; and oligo 3.5 is the desired 3xFlag encoding type 2 QRNA product with a click chemistry linkage shown in pink. Figure 1G The structure of 5-methyl-tetrazine containing preQ1 cofactor analog is shown. Figure 1H The structure of the 3'-TCO click chemistry handle is shown.

[0024] Figures 2A to 2H The general synthesis workflow of type 1 qRNA is shown. Figure 2A Representative chemical structures of the various components in a capped circular mRNA are shown: the cap, the alkyne handle, the phosphate group, and the azide handle, each color-coded and shown in the lower panel. Figure 2B Chemically synthesized oligomers with 3'-terminal click chemistry handles were shown to be chemically capped and purified by high performance liquid chromatography (HPLC). Figure 2C It is shown that alternatively chemically synthesized oligomers with 3'-terminal click chemistry handles can be capped enzymatically. Figure 2DWe show that chemically / enzymatically synthesized mRNA transcripts bearing click chemistry handles on their 5'-UTR and 5'-phosphate can be circularized using T4 RNA ligase; complementary sequences in both the 5' and 3' UTRs facilitate circularization. CircRNAs bearing click chemistry handles were chemically conjugated to capping oligos. Figure 2E It is shown that chemically / enzymatically synthesized mRNA transcripts with click chemistry handles on their 5'-UTR and 5'-phosphate can be circularized using T4 RNA ligase II with DNA splint probes complementary to the 5' / 3'-ends. The circRNA with click chemistry handles is chemically conjugated to a capping oligomer. Figure 2F It is shown that mRNA constructs with RNA stem-loops in their 5'-UTR can be synthesized by IVT and cyclized using the methods in 2C / 2D, or cyclized using ribozyme-mediated backsplicing. The stem-loops can be modified using RNA modifying enzymes (e.g., tRNA transferases) to introduce click reaction handles on the circular RNA. The circRNA with the click chemistry handle is chemically conjugated to a capping oligomer. Figure 2G It was demonstrated that in vitro transcribed mRNAs with 3' / 5'-hydroxyl groups can be enzymatically ligated to chemically synthesized oligomers with click chemistry handles and phosphates at both the 5' and 3' ends. Ligation of the 5'-phosphate and 3'-OH was achieved using T4 RNA ligase II, and ligation of the 5'-OH and 3'-phosphate was achieved using RNA ligase RtcB. DNA splints facilitated both ligations. CircRNAs with click chemistry handles were chemically conjugated to capping oligomers. Figure 2H It was demonstrated that in vitro transcribed mRNA 5' triphosphate / 3' hydroxyl groups can be enzymatically ligated to chemically synthesized oligomers with click chemistry handles and phosphates on both the 5' and 3' ends. Ligation of the 5'-phosphate and 3'-OH was achieved using T4 RNA ligase II. The 5'-triphosphate was hydrolyzed to the 5'-hydroxyl using calf intestinal alkaline phosphatase (CIAP). Ligation of the 5'-OH and 3'-phosphate was achieved using RNA ligase RtcB. DNA splints facilitated both ligations. CircRNAs with click chemistry handles were chemically conjugated to capping oligomers. Fig.2I It is shown that alternatively chemically synthesized oligomers with click chemistry handles can be used as primers annealed to a DNA template and in vitro transcribed using an RNA polymerase engineered from a DNA polymerase. The IVT synthesized mRNA can then be used for qRNA synthesis, such as Figure 2D / Figure 2E shown.

[0025] Figures 3A to 3B A general synthesis workflow for type 2 qRNA is shown. Figure 3AShown is the synthesis of a capped RNA with a stem-loop in its 5'-UTR by IVT and its subsequent ligation to a chemically synthesized oligonucleotide with a 5'-phosphate and a 3'-click chemistry handle using T4 RNA ligase. The product undergoes intramolecular circularization to generate a type 2 QRNA. Figure 3B It is shown that alternatively chemically synthesized oligomers with click chemistry handles are capped and used as primers to anneal to DNA templates and in vitro transcribed using RNA polymerases engineered from DNA polymerases. The IVT synthesized mRNA can then be used for type 2 qRNA synthesis.

[0026] Figures 4A to 4F A proof-of-concept experiment using type 1 qRNA encoding a HiBit tag is shown. Figure 4A Shown is the synthesis of HiBit QRNA type 1. HiBit encoding RNA was codon optimized to contain only a single C in the 5'-UTR, and an azide handle was incorporated during IVT by complete replacement of CTP with azido-CTP. Figure 4B Representative HPLC traces and gel electrophoresis of azide-circRNA purification are shown. Figure 4C is a representative HPLC trace of a chemically capped EU-containing oligomer. Figure 4D Precursors 1 to 6 produced during the synthesis reaction are shown. Figure 4E This is the gel electrophoresis characterization of Hibit QRNA. Figure 4F Bar graph showing luminescence of HiBit QRNA 8 hours after transfection in HeLa cells. Mean ± sem. P values ​​were calculated by unpaired t-test with Welch correction. ****P < 0.0001; **P < 0.0021; *P < 0.0332, no significant difference P > 0.1234.

[0027] FIG. 5A to FIG. 5B A summary of the oligonucleotide chemical conjugation methods is shown. Screening was performed using a 15-nt dA model substrate at micromolar concentrations. Modification handles were incorporated by solid phase synthesis followed by amine-NHS labeling and HPLC purification as required. Figure 5C Gel electrophoresis of crude thiol-ene / yne oligonucleotide conjugates of a 15-nt model substrate containing only one conjugation handle. Figure 5D Gel electrophoresis of crude CuAAC and IEDDA 30-nt oligonucleotides with three EU / TCO handles reacted with 30-nt N3 / Tz modified oligos.

[0028] Figure 6The chemical structure of the engineered branched poly(A) tail is shown. The branched oligonucleotides are conjugated via triazole bonds and flanked by 15A in the middle. Nuclease resistance modifications are incorporated into the last 6 nucleotides of each branch. A chain terminating nucleotide is introduced at the 3' end to prevent self-ligation.

[0029] 7A to 7F The synthesis of 100% capped, chemically and topologically enhanced oligonucleotides is shown. Fig. 7A Shown is a schematic representation of chemical capping and HPLC purification of solid phase synthesized oligonucleotides and a diagram of capped oligonucleotides produced using the methods disclosed herein. Figure 7B The scalability of oligomer capping in the 4 to 12 nanomolar range is shown. Figure 7C PAGE characterization of uncapped / fully capped oligonucleotides (15% TBU) is shown. Fig.7D is a graph of HPLC purification of capped oligonucleotides with various sugar backbones. Fig. 7E is a graph showing HPLC purification of a branched oligomer with two caps. Figure 7F PAGE representation of a double-capped oligomer (15% TBU) with the illustrated structure is shown. M, marker.

[0030] FIG. 8A to FIG. 8H Multidimensional chemical optimization of the mRNA cap and 5'-UTR is shown. Fig. 8A is a schematic illustration showing the use of an integrated chemoenzymatic approach to access the 5'-mRNA chemical modification landscape. FIG. 8B to FIG. 8E Screening for first base identity, phosphodiester bond, sugar backbone and m 7 Bar graph of a time course dual luciferase assay of chemical modifications on the G cap, as indicated by the bar graph corresponding to each of the bars in the figure. Luciferase luminescence normalization The luminescence of Renilla luciferase (transfection control) was then normalized to wild-type cap (m 7 Protein expression was measured using the GG) constructs (red line). Mean ± sem. P values ​​were calculated by ordinary one-way ANOVA (α = 0.05) at 24 h compared with m 7 GG (Figure b) / m 7 GA( FIG. 8C to FIG. 8E ) for multiple comparisons. *P<0.0332, **P<0.0021, ***P<0.0002, ****P<0.0001, no significant difference P>0.05. Fig.8F Combinatorial optimization of cap and 5'-UTR sugar backbone modifications is shown. Figure 8G is a graph showing the effect of chemical modification on oligonucleotide affinity for eIF4E by EMSA. Figure 8H is a graph showing the effect of chemical modification on oligonucleotide resistance against hDcp2.

[0031] FIG. 9A to FIG. 9B Chemical topological engineering of multi-capped branched mRNA is shown. Fig.9A is a comparison of branched caps versus regular caps by bioluminescence assay (8 hours after transfection). Fig. 9B is a bar graph showing the bioluminescence decay of branched caps relative to regular caps from 8 to 24 hours after transfection. P values ​​were calculated by unpaired t-test. ***P<0.001; ****P<0.0001; no significant difference P>0.05.

[0032] FIG. 10A to FIG. 10G The synthesis of capped circular mRNAs by a branching topology is shown. Fig. 10A IRES-based circRNA translation initiation by the eIF4G-4F complex is shown. Fig. 10B The branched cap is shown to induce translation while lacking protection of the mRNA from exonuclease degradation. Fig. 10C A conceptualization of capped circular mRNAs (qRNAs) to capture the eIF4E-dependent pathway for circRNA translation initiation is shown. Fig. 10D Figure 2 is a schematic diagram of the qRNA synthesis workflow combining enzymatic labeling and click chemistry. Fig. 10E is a graphic representation of the dual RNase H assay characterization of qRNA and its PAGE analysis. Fig.10F The results show that QRNA efficiently induces translation on circRNA in the absence of IRES. 7 G-capped linear mRNA was used as a positive control. CircRNAs bearing iHRV IRES were also compared. Translation activity was measured 6 h after transfection. Figure 10G It shows that uridine globally reduces mRNA translation. 7 G cap, m 1 Ψ modification) co-transfection with / without m 1 Ψm 7 G-capped linear mRNA and QRNA encoding Nluc (with uridine). When Nluc mRNA containing uridine was transfected, the translation of the regulated Fluc mRNA was also reduced. DETAILED DESCRIPTION

[0033] A more detailed description of the compositions, reagents and methods comprising the present invention is provided herein, which is provided to illustrate and enhance but not to supersede or replace the claims set forth below.

[0034] All publications cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference as if set forth in their entirety in this application.

[0035] definition

[0036] As used in the specification, the articles "a" and "an" herein refer to one or more than one (ie, at least one) grammatical object of the article. For example, "an element" means at least one element and may include more than one element.

[0037] "About" is used to provide flexibility to numerical range endpoints by specifying that a given value can be "slightly above" or "slightly below" the endpoint without affecting the desired result. The term "about" in relation to a numerical value means that the numerical value can vary within a range of ±5% or less of the numerical value.

[0038] Throughout this specification, the words "comprise" and "including" shall apply unless the context requires otherwise. (include) and variations such as “comprises”, “comprising” and “includes”, “including” will be understood to imply the inclusion of stated components, features, elements or steps or groups of components, features, elements or steps but not the exclusion of any other integers or steps or groups of multiple integers or steps.

[0039] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative form ("or").

[0040] Unless otherwise expressly indicated herein, the recitation of numerical ranges herein is intended only as a concise method of individually referring to each individual value falling within the range. Furthermore, each individual value is otherwise incorporated into the specification as if it were individually cited herein. For example, if a range is stated as 1 to 50, it is intended that values ​​such as 2 to 4, 10 to 30, or 1 to 3, etc., are expressly recited in the present disclosure. These are merely examples of specific intent, and all possible combinations of values ​​between and including the lowest and highest values ​​recited are deemed to be expressly stated in the present disclosure.

[0041] As used in accordance with the present disclosure, unless otherwise defined, all technical and scientific terms should be understood to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0042] The present application discloses and claims compositions, reagents and methods including capped circular RNA molecules, particularly circularized mRNA molecules that preferably encode peptides or polypeptides. As provided herein, the cap used in the capped circular RNA molecules of the present invention may include 7-methylguanine (m 7G), but in addition may also include cap analogs, especially as described below: U.S. Patent Application No. 2020 / 0055891 to Walczak et al.; Holstein et al., 2016, Agnew Chem. Int. Ed. Engl. 55: 10899-10903; Walczak et al., 2017, Chem. Sci. 8: 260-267; Muttach et al., 2017, J. Org. Chem. 13: 2819-2832) can be incorporated into the circularized RNA molecule precursor to produce the capped circular RNA molecule provided herein.

[0043] Capped circular mRNA (QRNA)

[0044] Despite advances in circular RNA (circRNA) engineering, current constructs rely on IRES (internal ribosome entry site) or TEE (translation enhancing element)-mediated translation, which are examples of approaches to achieve cap-independent translation ( Figure 1A Linear mRNAs are capable of cap-dependent translation through interaction with eIF4E and other eukaryotic translation initiation factors ( Figure 1B ), which is the major form of translation in cells (Sonenberg and Hinnebusch, 2009, Cell 136:731-745) and is generally more efficient than cap-independent translation (Koch et al., 2020, Nat. Struct. Mol. Biol. 27:1095-1104).

[0045] As described herein, "capped circular mRNA" is a circular mRNA characterized by one or more covalent attachments to one or more cap structures (or derivatives thereof). A circular mRNA may contain all the canonical elements of a linear mRNA: (1) a cap, (2) a 5'UTR (untranslated region), (3) a protein coding region (CDS), (4) a 3'UTR, and (5) a poly(A) tail. By circularizing these features into a capped circular RNA, it is intended to extend the half-life of a typical circular mRNA (increased nuclease resistance) while retaining the benefits of efficient cap-dependent translation, such as in a linear mRNA.

[0046] The RNA embodiments and methods disclosed herein take advantage of the exonuclease resistance characteristics of circRNAs while taking advantage of a strong m7G-cap-dependent translation initiation mechanism. Such characteristics can be achieved by chemical conjugation of capping oligonucleotides to circRNAs through click chemistry reactions, such as copper-catalyzed azide-alkyne cycloaddition (CuAAC) or tetrazine-trans-cyclooctene reverse electron demand Diels-Alder reaction (IEDDA) ( Figure 1C ).like Figure 1D and Figure 1E As shown, the present invention contemplates two general structures of capped circular messenger RNA (QRNA): type 1 QRNA and type 2 QRNA. In type 1 QRNA, there is a cyclic polyphosphodiester backbone, and capping is achieved by chemically connecting a short capping oligonucleotide to the internal handle of the circular mRNA by click chemistry. The 5' cap may include 7-methylguanosine or an alternative common mRNA cap structure that can effectively translate mRNA, such as shown in Mccaffreyanton, 2019, Genetic Engineering & Biotechnology News. 39. In type 2 QRNA, there is a continuous mRNA polyphosphodiester backbone; cyclization is achieved by chemical conjugation between the 3'-end of the mRNA and the 5'-UTR by click chemistry.

[0047] Various components of circRNA Figure 2A 5' capping and 3' poly(A) tailing steps can be used to generate active synthetic mRNA; these modifications protect mRNA from degradation and promote translation initiation in eukaryotic cells. As used herein, "capping" refers to the addition of a "cap" molecule, such as 7-methylguanosine (m 7 G) Cap, modified at the 5' end of the mRNA. Other cap structures and modifications of the cap as described below can be used to optimize translation efficiency.

[0048] Enzymes capable of catalyzing the reaction of attaching a cap molecule to mRNA include, but are not limited to, the vaccinia capping system ( Figure 2C ), tRNA guanine transglycosylase (TGT), Faustovirus capping enzyme and T4-RNA ligase. Capping can also occur during mRNA synthesis, which is called co-transcriptional capping.

[0049] As used herein, the term "molecular handle" or "handle" refers to a chemical group attached to a nucleotide on an mRNA, which can form a covalent bond with another molecule separated from the mRNA, thereby connecting the other molecule to the mRNA. Covalent bonds can be formed by various appropriate functional cross-linking reactions. In some embodiments described herein, the cross-linking reaction is click chemistry. As used herein, the term "click handle" refers to a molecule on an mRNA, which can be covalently bound to another molecule by a click chemistry reaction. The example of the handle includes but is not limited to alkynes or azides (when CuAAC is used in click chemistry), or trans-cycloolefins or tetrazines (when IEDDA is used in click chemistry), or hydrazones or oximes, or any equivalent structure thereof. Other crosslinking chemistries include thiol and thiol-yne reactions (Escorihuela et al., 2014, Bioconjug. Chem. 25:618-627), phosphoamine-based reactions (El-Sagheer and Brown, 2017, Chem. Commun. 53:10700-10702; Kalinowski et al., 2016, Chembiochem. 17:1150-1155) (respectively). Figure 5A and Figure 5B ), thiol-alkyne, amino-alkyne, and hydroxyl-alkyne reactions (Worch et al., 2021, Chem Rev. 121(12): 6744-6776), and other bioconjugation reactions (Gassensmith, https: / / chem.libretexts.org / Bookshelves / Organic_Chemistry / Supplemental_Modules_(Organic_Chemistry) / Reactions / Introduction_to_Bioconjugation, accessed on June 23, 2023) have also been considered.

[0050] As used herein, the term "hairpin" or "hairpin oligonucleotide" refers to a single-stranded oligonucleotide having complementary base pair sequences at both ends that are capable of forming a "stem-loop" structure.

[0051] As used herein and understood in the art, the term "click chemistry" is intended to encompass chemical methods for joining chemical components together, including but not limited to joining nucleotides into polynucleotides and amino acids into peptides and polypeptides, which are "simple to operate, high yield, require no or minimal purification, and are versatile in joining different structures without the need for protection steps" (see, e.g., Hein et al., 2006, Pharm. Res. 10: 2216-2230). In current chemical synthesis practice, four major reactions are employed: 1) cycloaddition reactions (including, for example, the most widely used copper-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes); 2) nucleophilic ring opening (including ring systems containing strained heterocyclic electrophiles); 3) non-aldol carbonyl chemistry (including, for example, hydrazone / oxime ether formation); and 4) carbon multiple bond additions (including, for example, certain Michael additions and the formation of various three-membered rings, especially by epoxidation). Click chemistry has been found to be particularly useful for polymerizing substances such as proteins and nucleic acids, as shown herein.

[0052] As used herein, the term "equivalent structure" refers to any molecule that is sufficiently similar in structure to perform the same function in a chemical reaction.

[0053] As used herein, the term "derivatization" or "functionalization" refers to a modification of a nucleotide that results in some functional outcome in terms of its chemical properties or reactivity or both. These two terms should be understood as equivalent to the extent to which a particular embodiment of a capped circular RNA molecule is functional by virtue of its derivatization, particularly with respect to the cross-link-dependent cyclization embodiments provided herein. In some embodiments, a derivatized nucleotide is a nucleotide that is modified to include a chemical group / handle that can participate in a cross-linking reaction.

[0054] As used herein, the term "QRNA" is intended as a general term to refer to a capped circular messenger RNA. The term specifically encompasses various types of circularized RNA molecules, and in particular the circularized mRNA molecules disclosed herein, but these examples are not intended to be limiting.

[0055] In some embodiments, the synthetic pathways of type 1 and type 3 QRNAs enable multiple oligonucleotides containing 5' caps to be bound to circular RNAs. For example, circular RNAs may include multiple derivatized nucleotides that can covalently bind multiple oligonucleotides containing 5' caps. Alternatively, a single circular RNA backbone can encode multiple TGT sites to enable multiple oligonucleotides containing 5' caps to be bound to circular RNAs simultaneously.

[0056] In some embodiments, the capped circular RNA molecule comprises an mRNA region encoding one or more peptides or polypeptides.

[0057] Cap modification

[0058] Several changes in the cap structure were considered here to optimize the translation efficiency of qRNA. These changes include: including multiple cap structures (caps 0, 1, and 2; Shanmugasundaram et al., 2022, Chem Rec. 22(8): e202200005); including N 6 , 2'-O-dimethyladenosine (m 6 Am) as a terminal modification adjacent to the mRNA cap (Sun et al., 2021, Nat Commun. 12(1):4778); using a cap structure with a modified triphosphate bridge (Sun et al., 2021, Nat Commun. 12(1):4778; Wojtczak et al., 2018, J Am Chem Soc. 140(18):5987-5999); incorporating locked nucleic acid (LNA) modified cap analogs (Kore et al., 2009, J Am Chem Soc. 131(18):6364-5); introducing cap analogs with alternative functions such as photoreactivity and click groups (Klocker et al., 2022, Nat Chem. 14(8):905-913; Nowakowska et al., 2014, Org. biomol. Chem. 12:4841-4847); hydrophobic cap analogs (WO 2017066782A1); and others (Wojcik et al., 2021, Pharmaceutics 13(11):1941; Grudzien et al., RNA 10(9):1479-1487; Grzela et al., 2023, RNA 29(2):200-216).

[0059] In some embodiments, 7-methylguanosine (m 7 G) The methyl group in the cap structure can be modified to produce 7-benzylguanosine (Bn 7 G), 7-chlorobenzylguanosine (ClBn 7 G) and chlorobenzyl-O-ethoxyguanosine (ClBnOEt 7 G). One or more locked nucleic acids (LNA), 2'-methoxy groups (2OMe) and 2-methoxyethoxy groups (2MOE) are introduced into m 7 The G structure can significantly increase mRNA translation. In some embodiments, the cap structure includes but is not limited to m 7 G-LNA、LNAm 7 G-LNA、LNAm 7 G-LNAx6, LNAm 7 G-2OMex6. In some embodiments, the cap structure is m 7 G imidazole diphosphate (m 7GDP-Im).

[0060] Nucleotide modification

[0061] In some embodiments, as disclosed and recognized herein, it is beneficial to change the type of nucleotide / nucleotide identity, particularly in mRNA, preferably incorporating adenosine (A), guanosine (G), 6-methyladenosine (m-1), or guanosine (G) at the +1 position. 6 A) or atypical inosine (I), increases translation efficiency. In some embodiments, some or all uridine residues in the mRNA are replaced with N 1 -methyl pseudouridine (m 1 Ψ) also promotes translation. Nucleotides are numbered according to their position immediately downstream of the cap structure. For example, the cap structure found at the 5' end of eukaryotic mRNA consists of 7-methylguanosine (m 7 G) and is linked to the first nucleotide (+1 position) of the transcript via a 5'-5' triphosphate bridge.

[0062] Other modified nucleotides include, but are not limited to, pseudouridine, 5-methylcytidine, 2-thiouridine, 5-methoxyuridine, 4-acetylcytidine, xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, digoxigenated adenine, digoxigenated cytosine, digoxigenated guanine, digoxigenated uracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil pyrimidine, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethyl ester uracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, arabinoadenine, cytosine arabinoside, arabinoguanine, arabinouracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallylcytosine, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-amino Allylcytosine, cyanine 7-aminoallyluracil, dabcyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thiocytosine, thioguanine, thiouracil, xanthine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-da-aminoguanine, 5-formamido-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methylthio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine is also considered. (m6t6A), N6-hydroxypentanoylaminoformyl adenine (hn6A), 2-methylthio-N6-hydroxy N-pentanoylaminoformyl adenine adenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A) at +1 and other positions.

[0063] In some embodiments, the modified phosphate backbone can be phosphorothioate (PS), phosphorothioate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5-hydroxyphosphonate, hydroxyphosphate, phosphoselenate, selenophosphate, phosphoamide, carbon phosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinyl ring, triazole ring, borophosphate (BP), methylphosphonate, or guanidinylpropylphosphonamide.

[0064] In some embodiments, the introduction of locked nucleic acids (LNA), 2'-methoxyribose (2-OMe), and 2-methoxyethoxy (2-MOE) into the ribose backbone increases mRNA translation. Adding multiple bases modified with 2-OMe and 2-MOE further increases translation. LNA specifically increases expression at the +1 position.

[0065] In some embodiments, the modified sugar can be 2-thioribose, 2,3-dideoxyribose, 2-amino-2-deoxyribose, 2'deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2,3-dideoxyribose , 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methyl ribose, 5'-amino ribose, 5'-thio ribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, or 2'-O,4'-C-thio-linked ribose.

[0066] Among these backbone modifications, stereoisomeric structures are also considered as they have been shown to affect the nuclease resistance of RNA (Iwamoto et al., 2017, Nat. Biotech. 35:845-851; Jahns et al., 2022, Nucleic Acids Res. 50(3):1221-1240).

[0067] Nucleotide modifications on traditional circRNAs are limited because not all nucleotides are compatible with internal ribosome entry sites (IRES). QRNA translation does not require an IRES; therefore, more modified nucleotides can be tolerated over a wide range of percentages. These modifications can be incorporated into the circular backbone at different percentages (m6A is typically incorporated at 5%). "Stem" oligomers containing caps or 5' / 3'UTRs and tails may tolerate higher percentages of modifications. Alternatively, these modifications may be present at different percentages along different regions of the circular RNA backbone (e.g., in the 5'UTR, or 3'UTR, or CDS, or near the cap structure, or a combination thereof). In addition, the "stem" oligomers (capped oligonucleotides) of type 1 QRNAs are chemically synthesized and may tolerate more complex structures that are difficult to enzymatically incorporate, such as locked nucleic acids (LNAs), 2'O-methyl nucleotides, peptide nucleic acids (PNAs), morpholinos, and various internal chemical linkers provided herein.

[0068] QRNA-encoded peptides and polypeptides

[0069] The polypeptides encoded by the capped circular RNA molecules provided by the present invention include any therapeutically useful polypeptides for treating or intervening any disease process associated with or dependent on polymorphic or mutant polypeptide species, which are heritable or obtained due to environmental damage or injury. QRNA can encode a variety of polypeptides, such as self-amplifying mRNA boxes, or a variety of therapeutic peptides or polypeptides. In some embodiments, the capped circular RNA molecule includes an mRNA region encoding one or more peptides or polypeptides. Multiple polypeptides include multiple copies of the same polypeptide or multiple copies of different polypeptides.

[0070] IRES or self-cleaving peptides such as T2A sequences can be present between multiple polypeptide coding sequences on the QRNA. Alternatively, an RNA oligonucleotide containing a cap residue site is located before each polypeptide coding sequence, which will ultimately produce a QRNA with multiple RNA oligonucleotides containing cap residues and ensure that all coding sequences are effectively translated.

[0071] The peptides encoded by the capped circularized RNA molecules of the present invention may include, but are not limited to, therapeutic peptides or antigenic peptides, particularly antigenic peptides suitable for presentation to humoral (B cells) or cellular (T cells) immune system cells by antigen presenting cells. In certain embodiments, these antigenic peptides are suitable and effective for use as vaccines. In other embodiments, the antigenic peptides are suitable for or effectively inhibit immune responses, such as in autoimmune diseases or transplant patients. In further embodiments, the antigenic peptides are suitable and effective for eliciting specific anti-tumor immune responses or attracting cytotoxic natural (natural killer cells) or engineered (e.g., CAR-T) cells in tumor cells. The therapeutic peptides encoded by the capped circularized RNA molecules of the present invention may include, but are not limited to, human parathyroid hormone, filgrastim, oxytocin, somatostatin, calcitonin, glucagon, insulin, liraglutide, vasopressin, etc. (see, Fosgerau and Hoffman, 2015, Drug Discovery Today 20:122-128; al Musaimi et al., 2021, Pharmaceuticals (Basil) 14:145; Wang et al., 2022, Signal Transduct. and Targeted Therap. 7:1-27).

[0072] In some embodiments, peptides encoded by the capped circular RNA molecules of the present invention may include, but are not limited to, Cas9 or derivatives (Rothgangl et al., 2021, Nat. Biotechnol. 39:949-957) and adenine base editors or other base editors (Gaudelli et al., 2017, Nature 551:464-471), or RNA base editors for delivering genome or epigenome editing therapies.

[0073] In some embodiments, the peptides encoded by the capped circular RNA molecules of the present invention can be selected from any of several target categories, including but not limited to biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane-bound proteins, nuclear proteins, proteins associated with human diseases, or targeting moieties.

[0074] Synthesis of qRNA

[0075] Type 2: The present invention also provides a method for producing Type 2 capped circular RNA molecules of this aspect of the present invention, the method comprising: synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure, an mRNA encoding a peptide or polypeptide, a derivatized nucleotide located between the cap structure and the mRNA region encoding the polypeptide, and a 3' end containing a portion; and reacting the derivatized nucleotide with the 3' end portion to form a covalently linked capped circular RNA molecule.

[0076] Type 1: The present invention also provides a method for producing a type 1 capped circularized RNA molecule of this aspect of the present invention, the method comprising the following steps: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5' end comprising a cap structure and a 3' end comprising a portion reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent connection between the RNA oligonucleotide and the circular RNA. In certain embodiments, the derivatized nucleotide comprises a portion capable of reacting with the 3' end portion by bioconjugation chemistry, wherein the bioconjugation chemistry is click chemistry. In addition, the circularized RNA is produced by ribozyme-mediated splicing, enzymatic ligation, or click chemistry-mediated circularization.

[0077] Type 3: The present invention also provides a method for producing Type 3 capped circularized RNA molecules according to this aspect of the present invention, the method comprising the following steps: producing a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region; synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end containing a portion reactive with the derivatized nucleotide; and reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent connection between the RNA oligonucleotide and the circular RNA, wherein the synthesis of the circular RNA oligonucleotide further comprises the following steps: synthesizing an RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme recognition site, and a twister ribozyme sequence on both the 5' and 3' ends; reacting the RNA oligonucleotide with an enzyme to produce a derivatized nucleotide within the hairpin structure; and circularizing the RNA oligonucleotide using the twister ribozyme sequence.

[0078] like FIG. 2D to FIG. 2I , Figure 4A and Fig. 10D (Type 1), Figure 3A and Figure 3B (Type 2) and Figure 1FAs shown in (Type 3), the derivatized nucleotides in these three QRNAs can be generated using different strategies. That is, by having a hairpin structure containing a specific enzyme recognition site, the derivatized nucleotides can be specifically targeted. The enzyme is described in some examples of tRNA guanine transferase (TGT). In other examples, the derivatized nucleotides are generated by replacing a single cytidine with azide-cytidine.

[0079] Circularization of type 1 and type 3 RNA molecules can be achieved by T4 ligase, RtcB ligase, or ribozyme-mediated splicing. In these embodiments, the 5' and 3' ends of the linear oligonucleotide include appropriate moieties to participate in the enzymatic reaction to form the circular RNA. Alternatively, click chemistry moieties are also contemplated for circularization. In some embodiments, additional splint probes containing complementary sequences to the 5' and 3' ends of the linear oligonucleotide can be used, such as Figure 2E or Figure 2G as shown, to bring the two ends into proximity and promote circularization.

[0080] Pharmaceutical compositions and methods for delivery

[0081] The present invention provides pharmaceutical compositions comprising the capped circular RNA molecules of the present invention, in particular circularized mRNA molecules. In certain embodiments, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable excipient, and in certain other embodiments comprise one or more additional therapeutic agents.

[0082] In some embodiments, the composition is suitable for administration to a human subject in need thereof.In the context of the present disclosure, "active ingredient" generally refers to the capped circular RNA molecules described herein, particularly circularized mRNA molecules, and any additional therapeutic agent provided therewith.

[0083] It is generally understood by those of ordinary skill in the art that the compositions described herein are also suitable for application to any non-human subject. It will be understood by those of ordinary skill in the veterinary field that the pharmaceutical compositions described herein may be suitable for application to mammals, including but not limited to primates, cattle, pigs, horses, sheep, goats, cats, dogs, mice, rats, whales, and other mammals. It will also be understood by those of ordinary skill in the veterinary field that the pharmaceutical compositions described herein may be suitable for application to birds, including but not limited to chickens, ducks, geese, turkeys and other domesticated birds, as well as wild birds, particularly endangered species in such birds. Additionally, it will be understood by those of ordinary skill in the veterinary field that the pharmaceutical compositions described herein may be suitable for application to a variety of fish, including commercial or wild salmon, tuna, cod, sardines, zebrafish, sharks, etc.

[0084] The pharmacological compositions described herein may be prepared by any method known or developed in the fields of pharmacology, immunology, virology, or general biotechnology.

[0085] In some embodiments, in addition to at least one other pharmaceutically acceptable excipient, the formulation of the pharmacological composition described herein may include a unit dose of at least one derivatized RNA, particularly a circularized mRNA molecule. Such excipients may include, but are not limited to, solvents, dispersants, buffers, diluents, surfactants, emulsifiers, isotonic agents, preservatives, thickeners, lubricants, oils, etc.

[0086] In some embodiments, the pharmacological composition may include a delivery mechanism that further includes lipid nanoparticles. The size of the lipid nanoparticles may be varied to counteract an immunogenic response in a subject, or to allow for increased potency and pharmacological activity.

[0087] In other embodiments, the pharmacological composition may include a delivery mechanism that further includes lipids previously described in the art. See Akinc et al., 2008, Nat Biotechnol. 26: 561-596; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105: 11915-11920; Akinc et al., 2009, Mol Ther. 17: 872-879; Love et al., 2010. Proc Natl Acad Sci USA 107: 1864-1869; Leuschner et al., 2011, Nat Biotechnol. 29: 1005-1010, all of which are hereby incorporated herein in their entirety. Lipids generally refer to lipid nanoparticles, liposomes, lipid emulsions, lipid micelles, etc. The lipid containing pharmacological composition comprising the derivatized RNA can be administered parenterally by means including, but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, via dialysate, intrathecal injection, or intracranial injection.

[0088] Those of ordinary skill in the art will also recognize that there are other nucleotide delivery mechanisms, such as the use of virus-like particles or virus-derived particles. See Rohovie et al., 2016, Bioengineering & Translational Med. 2 (1): 43-57. Virus-like particles may include viral coat proteins or viral capsids. Such particles may be pegylated or further annealed with compounds that avoid phagocytic clearance. Additionally, the surface of the virus-like particles may be further functionalized to provide cell-specific targeting, promote extravasation, promote radiolabeling, improve permeability across cell boundaries, or pass through the blood-brain barrier. Virus-like particles may be derived from animal viruses, bacteriophages, or plant viruses. Examples of suitable viruses for deriving virus-like particle delivery mechanisms include, but are not limited to, cowpea chlorosis mottle virus, cowpea mosaic virus, hepatitis B virus (core), enterobacteriaceae phage MS2, Salmonella typhimurium P22, enterobacteriaceae phage Qβ, and other suitable viruses. The derivatized RNA payload may be loaded into the virus-like particles by electrostatic adsorption or any other suitable method known to those of ordinary skill in the art.

[0089] Various exemplary embodiments of the compositions and methods according to the present invention are now described in the following non-limiting examples. These embodiments are provided for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. In fact, except those shown and described herein, various modifications of the present invention will become apparent to those skilled in the art according to the preceding description and the following examples, and fall within the scope of the appended claims.

[0090] Examples

[0091] Example 1: Synthesis of Type 1 Capped Circular RNA

[0092] The synthesis of type 1 QRNA can be divided into two main steps: the synthesis of a 5'-capping oligonucleotide with a 3'-click chemistry handle; and the synthesis of a circular RNA containing the corresponding click chemistry handle in its untranslated region ("UTR", i.e., outside the part of the RNA encoding, in particular, a protein or peptide). The 5'-capping oligomer can be synthesized from an oligonucleotide generated by solid phase synthesis (with a click handle and a 5'-phosphate incorporated therein), followed by chemical capping of the RNA using N7-methylated GDP imidazole ( Figure 2B)(Abe et al., 2022, ACS Chem. Biol. 17: 1308-1314) or enzymatic capping using a capping enzyme such as a vaccine capping enzyme with guanine methyltransferase (FIG.2C) (Shuman in Progress in Nucleic Acid Research and Molecular Biology, 50: 101–129 (Cohn and Moldave, eds. Academic Press, 1995; Kyrieleis et al., 2014, Structure 22: 452-465). circRNAs containing click chemistry handles are synthesized by a variety of means. In a non-limiting example, a linear mRNA with a click handle in its 5' / 3'-UTR is generated by chemical or enzymatic synthesis, and the transcript is designed to contain complementary sequences in the 5' / 3'-UTR to allow circularization using a single-stranded RNA ligase (T4 RNA ligase) ( Figure 2D )(Wang and Ruffner, 2014, Nucleic Acids Res. 26:2502-2504). Figure 2D Examples of qRNAs constructed using the strategy described in Figure 4A and as described in Example 4 below. Alternatively, transcripts containing click handles can be circularized using a splint probe complementary to sequences on the 5' / 3'-ends, allowing circularization using a double-stranded RNA ligase such as T4 RNA ligase II ( Figure 2E )(Chen et al., 2020, Nucleic Acids Res. 48:e54). Circular RNAs with click handles can also be produced by a tandem ligation process: RNA transcripts containing 5' / 3'-hydroxyl groups can be synthesized by in vitro transcription (IVT) and ligated to chemically synthesized oligonucleotides that have click chemistry handles and phosphate groups at both the 5'- and 3'-ends. The 5'-phosphate / 3'-hydroxyl ends can be ligated by T4 RNA ligase, and the 5'-hydroxyl / 3'-phosphate ends can be ligated using RNA ligase RctB to produce the desired circRNA. The ligation reaction can also be guided by a splint to increase yield ( Figure 2G ). Alternatively, RNA transcripts can be produced by in vitro transcription and carry a 5'-triphosphate and a 3'-hydroxyl group. Chemically synthesized oligonucleotides with 5'-phosphate, 3'-phosphate, and derivatized nucleotides are first ligated to the mRNA using T4 RNA ligase (linking the mRNA 3'-hydroxyl group to the oligonucleotide 5'-phosphate). Calf intestinal alkaline phosphatase (CIAP) is then used to hydrolyze the mRNA 5'-triphosphate to the 5'-hydroxyl group. The mRNA is then cyclized by ligating the 5'-hydroxyl group and the 3'-phosphate using RtcB ligase ( Figure 2H ). Instead of incorporating a click handle before / during RNA cyclization, RNA with a stem-loop motif can be synthesized by IVT and cyclized by ribozyme-mediated backsplicing (Wesselhoeft et al., 2018, Nat Commun. 9:2629), and the click handle can introduce a hairpin modification enzyme, such as tRNA guanyl transferase (TGT) (Alexander et al., 2015, J. Am. Chem. Soc. 137:12756-12759) ( Figure 2F ).

[0093] Another approach to combine co-transcriptional circularization and click handle incorporation is to use a synthetic oligonucleotide containing a click handle as a primer to anneal on a DNA template and perform in vitro transcription using an engineered DNA polymerase that synthesizes RNA from the primer (Cozens et al., 2012, Proc. Natl. Acad. Sci. USA 109, 8067–8072), followed by ligation / backsplicing-based circularization ( Fig.2I ). After successful synthesis, click chemistry is used to cross-link the circRNA and the capping oligomer to generate type 1 circRNA. As an alternative to the above strategy, circular mRNA scaffolds (e.g., RNAs cyclized by non-natural / non-phosphodiester chemical bonds) can be constructed by chemical cyclization, for example by incorporating click handles on the 5' and 3' ends of the linear RNA. After successful chemical cyclization, the circular scaffold is further modified by covalent addition of a 7-methylguanosine cap or a cap-containing oligonucleotide.

[0094] Example 2: Synthesis of Type 2 Capped Circular RNA

[0095] For type 2 QRNA, a 5'-capped mRNA transcript containing an RNA hairpin in the 5'-UTR can be synthesized by IVT and co-transcriptional capping, and a hairpin tagging enzyme such as TGT can be used to introduce a first click chemistry handle, such as an alkyne. Subsequently, a second click chemistry is introduced by attaching a chemically synthesized oligomer with a 3'-terminal azide handle to the 3'-end of the transcript, and click chemistry can be used to intramolecularly circularize the mRNA to produce type 2 QRNA ( Figure 3A ). Alternatively, synthetic oligonucleotides containing click handles can be used as primers, annealed to DNA templates, and in vitro transcribed using engineered DNA polymerases that synthesize RNA from the primers (Cozens et al., 2012, Proc. Natl. Acad. Sci. USA 109:8067-8272; Freund et al., 2023, Nat. Chem. 15:91-100). The IVT product is then ligated to a 3'-azide / 5'-phosphate synthetic oligomer, allowing intramolecular chemical cyclization ( Figure 3B )

[0096] Example 3: Synthesis of Type 3 Capped Circular RNA

[0097] Description of the synthetic strategy: The synthesis scheme of type 3 qRNA is in Figure 1F to Figure 1H A short QRNA encoding a FLAG peptide (DYKDDDDK-SEQ ID NO: 1, wherein D = aspartic acid, Y = tyrosine, and K = lysine) was prepared and tested. The 3xFLAG peptide (DYKDDDDKDYKDDDDKDYKDDDDK-SEQ ID NO: 2) having a sequence described herein can be substituted or recoded into other reporter peptides (e.g., NanoBiT 11-amino acid peptides, as shown at www.promega.com / products / protein-interactions / live-cell-protein-interactions / nanobit-ppi-starter-systems / ?catNum=N2014) or therapeutic peptides (e.g., for peptide-based vaccines).

[0099] Oligomer 3.1 was generated by in vitro transcription and co-transcriptionally cyclized by twister ribozyme and RNA ligase RtcB (see, Litke et al., 2019, Nature Biotechnol. 37: 667-675) to produce circular RNA 3.2. tRNA guanine transglycosylase (TGT) and synthetic preQ1 cofactor analog ( Figure 1G ) Circular RNA 3.2 was post-transcriptionally and site-specifically labeled to incorporate a 5-methyl-tetrazine handle in the tRNA-like hairpin structure (see, Alexander et al., 2015, J. Amer. Chem. Soc. 137: 12756-12759 2015), generating circular RNA 3.3. Oligonucleotide 3.4 ( ) was prepared similarly to that described in Example 1 by replacing 3-azide-2,3-ddUTP with 5-TCO-PEG4-dUTP to generate a capped oligomer with a 3'-TCO handle. Figure 1H ). Oligo 3.4 is then linked to circular RNA 3.3 by tetrazine-TCO cyclization to produce the desired type 1 QRNA 3.5 encoding 3xFlag. The sequences of oligonucleotides 3.1 and 3.4 are listed in Table 1.

[0100] Table 1. Synthetic oligonucleotide sequences used for QRNA type 3 design

[0101]

[0102] Note: Unless otherwise stated, the bases listed are ribonucleotides.

[0103] AG = m7G(5')ppp(5')(2'OMeA)pG (e.g., "CleanCapAG" 5' cap analog from TriLink Biotechnologies); U =5-TCO-PEG4-2-deoxyuridine; U=N 1 -Methyl pseudouridine or uridine

[0104] Example 4: Type 1 capped circular mRNA has higher translation efficiency than uncapped circular mRNA

[0105] To facilitate synthesis, purification, and characterization, small RNA qRNA constructs were used. RNA templates ( Figure 4A ) contains 5' and 3' complementary regions (shown by dashed lines) to facilitate annealing and subsequent circularization (e.g., enzymatic ligation of the 5' and 3' ends). This RNA scaffold contains a short coding sequence encoding a 12 amino acid HiBit tag (shown in blue; MVSGWRLFKKIS - SEQ ID NO:5), and a short poly(A) region following the coding sequence (CDS) to mimic the structure of a linear mRNA. Either the 5'UTR or the 3'UTR contains a single C site for the installation of cytidine triphosphate (CTP)-azide during transcription. This single CTP-azide incorporation allowed us to click a 5' 7-methylguanosine cap oligo onto the circularized RNA scaffold.

[0106] The RNA scaffold containing a 5' C-azide site has the following sequence (5' to 3'):

[0107]

[0108] The RNA scaffold containing a 3' C-azide site has the following sequence (5' to 3'):

[0109]

[0110] The above sequence is used as a scaffold for the "circular" coding portion of the test mRNA. The underlined 5' and 3' regions are complementary and promote enzymatic cyclization of the RNA by enzymatic ligation. The bold text shows the protein coding sequence encoding the HiBit tag (MVSGWRLFKKIS*-SEQ ID NO:5), where * is a stop codon. Additionally, a single C site (shown in bold uppercase text) is encoded by this template and is located in the 5' or 3' complementary region. Site-specific azide functionalization in the circular RNA scaffold is achieved by encoding only a single cytidine in these templates ( Figure 4A ).

[0111] The above RNAs were synthesized from the corresponding DNA templates by in vitro transcription (IVT) using the following nucleotide triphosphates as precursors: GTP, ATP, 5-azido-PEG4-CTP (Jena Bioscience, catalog number: CLK-0523) instead of normalized CTP and N1-methyl pseudo-UTP (Jena Bioscience, catalog number: NU-890) instead of UTP. Except for a single site (shown in uppercase bold text), all internal instances of cytidine were removed to prevent nonspecific azide incorporation in the coding region or other sites within these templates.

[0112] The RNA produced by IVT contains a 5' triphosphate and a 3' hydroxyl group. After IVT synthesis, the RNA is treated with RNA 5' pyrophosphohydrolase (RppH) (New England BioLabs, catalog number: M0356S) to generate a 5' phosphate. Subsequently, these RNAs are annealed and the 5' and 3' ends are ligated together using T4 RNA ligase 1 (New England BioLabs, catalog number: M0437M) to generate a circular "scaffold" that will serve as the basis for the QRNA structure ( Figure 4A ). Circularized RNA was separated from contaminants using high performance liquid chromatography (HPLC) purification ( Figure 4B ), and served as a subsequent scaffold for click functionalization.

[0113] An oligomer containing a 5'7-methylguanosine cap and a 3'alkynyl group was synthesized ( Figure 4A and Figure 4C ). Synthetic RNA oligonucleotides bearing a 5'-phosphate and a 3'-alkyne were generated by solid phase synthesis (ordered by IDT, sequence order code: / 5Phos / rArGrArArUrArA / 35OCTdU / ). The synthetic oligomers with ammonia as counterions were dissolved in DMSO, treated with 100 equivalents of m7GDP-imidazole in 4% (v / v) 1-methyl-imidazole in DMSO at 55°C for 3 hours, and HPLC purified. The 5' cap oligomers were covalently linked to the circular RNA scaffold using copper-catalyzed azide-alkyne click chemistry ( Figure 4D ).

[0114] For expression testing, qRNA expression was compared with various precursors, including a cap-oligomer covalently linked to a linear template, or a circular template without a cap ( Figure 4D ). Lipofectamine (Thermo Fisher Scientific) was used (ThermoFisher Scientific, catalog number: LMRNA001) qRNA and related synthetic precursors (in Figure 4D 1 to 6) were transfected into HeLa cells, and bioluminescence was measured using the Hibit lysis assay (Promega, catalog number: N3030) 8 hours after transfection. A significant enhancement of translation was observed when the linear precursor was conjugated to a capping oligomer on the 5' end instead of the 3' end, indicating that the triazole-linked cap induces an enhancement in translation efficiency (TE) ( Figure 4F 1, 3, 4). Similarly, when the circular RNA was conjugated to a capping oligomer, the amount of luminescence increased, in which case the click handle further upstream in the original 3'-UTR outperformed the click handle in the more proximal 5'-UTR (as shown in Figure 4E 2, 5, and 6), confirming that QRNA is a feasible strategy to improve circRNA translation.

[0115] Example 5: Considering cross-linking reactions for synthesizing capped circular RNA

[0116] The experiments described in this disclosure demonstrate the synthesis of branched poly(A) oligonucleotide mRNAs, such as Figure 6 Similar conditions described herein can be applied to chemical conjugation and intramolecular conjugation in type 1 and type 2 QRNA synthesis, respectively. Chemically modified poly (A) oligonucleotides were obtained from Integrated DNA Technologies and suspended in RNase-free water at a final concentration of 100 uM.

[0117] For thiol-ene / alkyne conjugation (thiol (R-SH) and alkene (R2C=CR2) or alkyne to form Figure 5A For organic reactions of the thiol (RS-R') with the chemical structure shown, the disulfide protected thiol-oligomer was deprotected with 100 molar excess of TCEP (tris(2-carboxyethyl)phosphine) and immediately mixed with an equimolar amount of the alkene / alkyne modified oligomer and incubated at 37°C for 30 minutes to 1 hour. In the case of using free radical conditions, a substoichiometric amount of 2,2-dimethoxy-2-phenylacetophenone was added and incubated at room temperature under 370 nm irradiation (Kessil, catalog number: KSPR160L370). The sizes of the products and precursors of the crude thiol-ene / alkyne oligonucleotide conjugation of the 15-nt model substrate containing only one conjugated handle are shown in Figure 2. Figure 5C shown.

[0118] For amine-phosphate conjugation (upper panel, Figure 5B ), the oligonucleotide was mixed with an excess of imidazole and 1-2 equivalents of EDC as an additive. The reaction mixture was incubated at 37°C for 30 minutes to 1 hour.

[0119] For IEDDA (Inverse Demand Diels-Alder Reaction) conjugation, methyl tetrazine (Me-Tz) and trans-cyclooctene (TCO) labeled oligonucleotides were obtained from the corresponding amine-modified oligonucleotides after labeling with tetrazine-PEG5-NHS ester (Click Chemistry Tools, Catalog No.: 1143) or TCO-PEG4-TFP ester (Click Chemistry Tools, Catalog No.: 1198) at a molar ratio of 500:1 in 100mM NaHCO3 at 4°C overnight. The NHS labeled products were purified using ethanol precipitation. Me-Tz / TCO labeled oligonucleotides were suspended in RNase-free water and incubated at 55°C for 30 minutes.

[0120] For CuAAC (copper-catalyzed azide-alkyne conjugation) conjugation, the azide / alkyne-containing oligonucleotides were mixed at the indicated molar ratios (1:1 for single-branched oligos, 2:1 for double-branched oligos, and 3:1 for triple-branched oligos). The sizes of the products and precursors of the reactions of CuAAC and IEDDA 30-nt oligonucleotides with three EU / TCO handles and 30-nt N3 / Tz-modified oligos are shown in Figure 5D The branched oligomer structure is shown in Figure 6 As shown. The oligonucleotide mixture was diluted in a modified 1.5x click chemistry buffer (Lumiprobe, catalog number: 61150, with 5% super enzyme inhibitor, 5% DMSO and 5% 10mM dNTP mixture [Thermo Fisher Scientific, catalog number: 18427089]) and briefly degassed by purging with argon for 20 minutes before the reaction. For a typical 100 μL reaction, 33 μL of oligonucleotide solution was mixed with 66 μL of click chemistry buffer, and 2 μL of 100mM L-ascorbic acid solution (Sigma Aldrich, catalog number: A5960) was added immediately before the reaction. The mixture was Incubate at 37°C for 1 h and remove the precipitate by adding 1 μL of 500 mM The reaction was terminated with EDTA (pH 8.0). The reaction was purified using an h RNA purification kit (NEB, catalog number: T2040), and the crude product was repurified by RNase-free HPLC on an Agilent 1260 Infinity II HPLC using acetonitrile [Sigma-Aldrich, 34851] and 100 mM hexylamine / acetic acid (pH 7.0, containing 10% urea w / v) as the mobile phase. HPLC fractions were analyzed by Novex TBE urea gel analysis, stained with 1× SYBR Gold (Thermo Fisher Scientific, catalog number: S11494), and visualized using the BioRad ChemiDoc MP imaging system (catalog number: 12003154). The desired fractions were then pooled, desalted, and concentrated using the Monarch RNA purification kit for small-scale preparations or ethanol precipitation for large-scale preparations.

[0121] Example 6: Covalent internal caps drive robust translation of linear and circular RNAs

[0122] Systematic interrogation of 5'-modifications of mRNA translation

[0123] Traditional methods for preparing capped therapeutic mRNAs include enzymatic capping or co-transcriptional capping. For enzymatic capping, mRNA transcripts are treated with a capping enzyme and a methyltransferase after in vitro transcription (IVT) (Ramanthan et al., 2016, Nucleic Acids Res. 44:7511-7526). Co-transcriptional capping is achieved by incorporating synthetic cap analogs into the IVT reaction. The first generation of dinucleotide cap analogs m 7 G(5')ppp(5')G will lead to m 7 The problem of unintended "reverse" orientation of the G cap was solved by using anti-reverse cap analogs (ARCAs) (Grudzien-Nogalska et al., in Methods in Enzymology (Academic Press, 2007), 431:203-227; Stepinski et al., 2001, RNA 7:1486-1495). Recent developments of tri / tetranucleotide cap analogs for direct incorporation into cap-1 / cap-2 structures and most cap modifications use tri / tetranucleotides for screening (Ishikawa et al., 2009, Nucleic Acids Symp Ser. 53: 129-130; Sikorski et al., 2020, Nucleic Acids Res. 48: 1607-1626; Jurga et al., Messenger RNA Therapeutics (Springer Nature, 2022)). However, due to the different incorporation efficiencies of various cap structures, both methods are unable to obtain modifications beyond the first two bases and lead to potential biases in screening. Additionally, given that the two species have similar physicochemical properties, it is not easy to purify capped mRNA from uncapped mRNA.

[0124] To address these challenges, the capping step was separated from mRNA synthesis. Oligonucleotides with well-defined chemical modifications were easily synthesized on solid phase and subsequently cleaved using mRNA. 7 G imidazole diphosphate (m 7GDP-Im) derivatives were chemically capped (Abe et al., 2022, ACS Chem. Biol. 17: 1308-1314). Changing the oligonucleotide counterion to ammonium ions enabled stable capping without any divalent ion additives, and fine-tuning the reversed-phase high-performance liquid chromatography (RP-HPLC) gradient using more hydrophobic hexyl ammonium ions enabled the separation of 100% capped products on a large scale ( FIG. 7A to FIG. 7D These capping oligonucleotides were then ligated to the N-containing 1 -methyl pseudouridine (m 1 Ψ) were then treated with bacterial RNA 5' pyrophosphohydrolase (RppH).

[0125] The modular nature of this workflow allows for the differential construction and evaluation of mRNAs with a range of cap and 5'UTR modifications. Fig. 8A ). mRNA structure is divided into four dimensions: (1) first base identity, (2) phosphodiester bond, (3) sugar backbone, and (4) cap modification. First, the effect of nucleotide identity at the "+1 position" on translation was evaluated, where protein production changes in the order of A>G~C~U according to literature reports (Sikorski et al., 2020, Nucleic Acids Res. 48:1607-1626). As expected, changing A to m 6 A further enhanced total protein production, and interestingly, incorporation of the atypical inosine (I) showed a similar effect ( Figure 8B ). Then use m 7 GG, the “wild-type” base identity in ARCA-capped synthetic mRNA was used as a benchmark for subsequent screening and m 7 GA served as a control construct for other aspects of mRNA modification.

[0126] In terms of phosphodiester bonds, although it has been previously reported that the introduction of phosphorothioate (PS) to the cap triphosphate bridge can increase protein yield (Kawaguchi et al., 2020, Angew. Chem. Int. Ed Engl. 59: 17403-17407), the introduction of PS between the +1 and +2 positions reduced translation. Further introduction of PS to the +1 to +7 positions rescued translation to normalized levels, but still had no benefit ( Figure 8C ).

[0127] Regarding modifications of the ribose backbone, replacement of the adenosine 2'-hydroxyl with 2'-deoxyfluor (2FA) impeded translation. Switching to chirality-inverted L-adenosine (LA) or 2'-deoxyadenosine (dA) resulted in no significant changes in expression. Introduction of locked nucleic acids (LNA), 2'-methoxy (2OMe), and 2-methoxyethoxy (2MOE) significantly increased mRNA translation, with introduction of a single LNA base resulting in a 4.8-fold increase in mRNA translation. Extending the dA backbone to positions +1 to +6 did not significantly alter expression, while increasing the number of bases modified with 2OMe and 2MOE resulted in a 6.9-fold and 5.4-fold increase at 24 h, respectively. However, LNA increased expression only at the +1 position, while modification of the +1 position to the +6 position resulted in decreased activity. ( Fig.8D )

[0128] To evaluate cap modifications, we synthesized m-cap structures reported previously. 7 GDP-Im analogs to enhance translation. 7 G) and chlorobenzyl (ClBn 7 G) Replace m 7 G methyl group or with LNA sugar backbone (m 7 G-LNA) to modify the cap structure (Kore et al., 2009, J. Am. Chem. Soc. 131: 6364-6365; Wojcik et al., 2021, Pharmaceutics 13(11): 1941). We also incorporated chlorobenzyl-O-ethoxy (ClBnOEt 7 G), a previously developed structure that does not function as an mRNA cap but as a high-affinity eIF4E inhibitor (Chen et al., 2012, J. Med. Chem. 55:3837-3851). In contrast to previous reports, none of the aromatic substitutions showed significant increases in mRNA binding. 7 G had better performance, although all aromatic substitutions successfully triggered translation compared to uncapped mRNA. This difference may be due to the fact that these hydrophobic modifications lead to better separation of capped mRNA during purification after co-transcriptional capping, and in accordance with a recent report, we also observed a larger retention time shift on HPLC (Inagaki et al., 2023, Nat. Commun. 14:2657). At 24 hours, m 7 G-LNA indeed succeeded in enhancing translation by 4.5-fold ( Fig. 8E We then demonstrated that these modifications from different dimensions can be combined to further amplify the effect, where the m at the +1 base is simultaneously introduced. 7 G-LNA+LNA or 2OMe at +1 to +6 bases further enhanced translation by 8.6-fold and 7.5-fold, respectively ( Fig.8F ). In addition, chemical modifications of the 5' cap and downstream nucleotides can be optimized in combination to maximize affinity for eIF4E or increase resistance to hDcp2 decapping, potentially altering mRNA translation ( Figure 8G to H).

[0129] Internal capping drives robust translation on circRNAs

[0130] Because the addition of an internal cap structure enhances translation of linear transcripts, we sought to apply this approach to drive translation of circRNAs. Traditionally, circRNAs lack a cap and a poly(A) tail and require an IRES for translation initiation ( Fig. 10A However, the initial rate of IRES is known to be slower than that of typical cap-dependent mechanisms (Koch et al., 2020, Nat. Struct. Mol. Biol. 27:1095-1104). Although branch caps do not prevent exonuclease degradation of the uncapped mRNA "stem" ( Fig. 9B ), but circRNAs have previously been reported to have enhanced exonuclease resistance and stability in vivo (Wesselhoeft et al., 2018, Nat. Commun. 9-2629; Chen et al., 2022, Nat. Biotechnol. 41(2): 262-272). Therefore, the internal capping strategy simultaneously maintains the high stability of circRNAs while hijacking the cap-dependent translation initiation mechanism to enhance their translatability ( Fig. 10B and Fig. 10C ). We named this type of capped circular mRNA QRNA because this structure resembles the letter "Q".

[0131] In terms of qRNA synthesis, achieving RNA circularization and incorporation of site-specific click chemistry handles simultaneously is a major challenge. As an initial proof of concept, we synthesized a minimal RNA encoding HiBiT and engineered its sequence to contain only a single cytosine in its UTR, allowing us to introduce an azide handle during IVT by replacing CTP with an azide-labeled CTP (5-azido-PEG4-CTP). This minimal mRNA was then circularized by T4 RNA ligase with the help of homology regions in the 5' and 3' UTRs to generate an azide-labeled circRNA that was purified by HPLC and confirmed to be RNase R-resistant. The m 7 G-capped, OU-labeled oligos were conjugated to azide-circRNAs to generate minimal QRNAs ( Figure 4A To C). Encouragingly, enhanced translation was observed in qRNAs compared to circRNA precursors ( Figures 4D to 4F ).

[0132] To generalize qRNA synthesis to longer transcripts, alternative workflows for nucleotide removal are needed. To this end, we synthesized nanoluciferase (Nluc) encoding mRNA and cyclized it by intron backsplicing, a standard method for preparing circRNA. To introduce the click chemistry handle, a minimal hairpin sequence was incorporated upstream of the CDS, which can be specifically recognized and labeled by tRNA guanine transglycosylase (TGT) using the prequinone 1 (preQ1) site (Ehret et al., 2018, Mol. Pharm. 15: 737-742). Using TGT and synthetic preQ1-azide, we introduced a single azide handle on the circRNA and conjugated it to a 5' capping oligomer containing a single 5-octadiynyl dU site (OU) ( Fig. 10D ). In order to enrich the abundance of QRNA, hydrophobic Bn 7 G-cap analogs to enhance their interaction on RP-HPLC, and the enriched products were characterized by dual RNase H assay ( Fig. 10E ). Although still not as good as conventional m 7 G-capped linear mRNAs are efficiently translated, but qRNAs are more efficiently translated than their circRNA counterparts (with or without IRES) ( Fig.10F On the other hand, since the synthesis of circRNA precursors still depends on ribozyme splicing, this is different from m 1 Ψ is incompatible, so QRNA is similarly compatible only with uridine, while m 1 Linear mRNA expression was significantly enhanced after Ψ substitution ( Figure 10G This difference was not due to the intrinsic translatability of the QRNA construct but rather to the toxicity of uridine, as expression of the co-transfected control Fluc mRNA was also significantly reduced when the uridine-containing linear / QRNA was transfected ( Figure 10G ). Therefore, only polycapped linear mRNA is used for animal experiments because it is closely related to m 1 Ψ compatible, with higher therapeutic value.

[0133] Materials and methods

[0134] Plasmid cloning, characterization, and purification (linearization + circularization): mRNA expression vectors were generated as described previously. Briefly, the target protein coding sequence (CDS) was inserted into an optimized backbone that included (in order) a T7 promoter sequence, a 5' human α-globin UTR, a CDS, a 3' human α-globin UTR, a 100×A template-encoded poly(A) tail, and an Esp3I linearization site. Plasmids / gene blocks containing the CDS were PCR amplified, gel purified, and assembled into an optimized backbone using NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621S), transformed into stable cells, and sequence verified by whole plasmid sequencing.

[0135] The firefly luciferase construct was obtained from the pmirGLO dual luciferase miRNA target expression vector (Promega, E1330). The Renilla luciferase construct was obtained from pmirGLO without cloning into an optimized vector. The nanoluciferase construct was obtained by gene synthesis from Genewiz.

[0136] Linear mRNA synthesis and characterization: DNA plasmid was obtained as described above and linearized by Esp3I (NEB, R0734S). Linearization was purified using the DNAClean & Concentrator-25 kit from Zymo Research (D4033). Quality The particles were characterized by agarose gel electrophoresis. -1] 100% replacement of UTP and addition of 1:50 Superase RNase inhibitor [Thermo Fisher Scientific, AM2694], mRNA constructs were synthesized by in vitro transcription (IVT) using the HiScribe T7 High Yield RNA Synthesis Kit [NEB, E2040S] (for T7 promoter constructs) according to the manufacturer's protocol. After the IVT reaction, the DNA template was digested with TURBO DNase and purified with the Monarch RNA Purification Kit [NEB, T2040L]. The mRNA concentration was quantified using the Qubit RNA HS assay [Thermo Fisher Scientific, Q32852] or the Qubit RNA BR assay [Thermo Fisher Scientific, Q10210]. Unless otherwise stated, the mRNA product was suspended in 1:50 (v / v) RNase-free water containing RNase inhibitor (hereinafter referred to as RNase-free water) and stored at -80°C.

[0137] General conditions for RP-HPLC purification: All purifications were performed on an Agilent 1260 Infinity II HPLC. Acetonitrile (solvent A) [Sigma-Aldrich, 34851], 100 mM hexylamine / acetic acid aqueous solution (pH 7.0, containing 20% ​​acetonitrile w / v) (solvent B), 50 mM diethylamine / acetic acid + 50 mM ammonium acetate aqueous solution (pH 7.0) (solvent C) were used as mobile phases, and a PLRP-S column was used as the stationary phase.

[0138] Method 1: using 100A pore size, 0% A+100% B (0-5 minutes, hold); 10% A+90% B (5-10 minutes, linear increase); 25% A+75% B (10-55 minutes, linear increase).

[0139] Method 2: using 4000A pore size, 0% A+100% B (0 min); 20% A+80% B (0-2 min, linear increase); 70% A+30% B (2-30 min, linear increase).

[0140] Method 3: using 4000A pore size, 0% A + 100% C (0 min); 25% A + 75% B (0-25 min, linear increase).

[0141] Capped oligonucleotide synthesis: 12nmol of solid phase synthesized oligonucleotide (with ammonium as counter ion) was dissolved in 40mMm7GDP-Im (or corresponding cap analog) in 42μL of anhydrous DMSO, and 8μL of 1-methyl-imidazole solution was added. The reactants were mixed thoroughly and heated at 55°C for 3 hours. 50μL of water was then added to quench the reaction, and HPLC purification was performed directly using method 1. The fractions containing the capped product were pooled, lyophilized, and resuspended in RNase-free water and stored at -80°C until use. The concentration of the capped oligonucleotide was quantified using the Qubit microRNA assay kit [Invitrogen, Q32880] and nanodrop.

[0142] Enzymatic ligation of modified oligonucleotides to mRNA: 5'-triphosphorylated mRNA was first treated with RppH [NEB, M0356S] according to the manufacturer's protocol to produce 5P-mRNA and purified using the Monarch RNA purification kit. The synthesized oligomer and 5P-mRNA were mixed at a molar ratio of 25:1 and diluted in 2×50% PEG-8000, 10×T4 RNA ligase buffer, 10×T4 RNA ligase [Promega, M1051] and RNase-free water. The reaction was incubated at 37°C for 30 minutes and inactivated by adding 50×500mM EDTA (pH 8.0). The product was first purified by the Monarch RNA purification kit and then by RNase-free HPLC (Method 2). The purified fractions were extracted and desalted using the Monarch RNA purification kit, and the ligation efficiency was characterized using the RNase H assay as described above. (17) In the case of incomplete ligation, a second round of reaction was performed.

[0143] Modification screening using a time-course dual-luciferase assay: HeLa cells [ATCC, CCL-2] were maintained in DMEM medium [Thermo Fisher Scientific, 119951] containing 10% FBS and 1% penicillin-streptomycin [Thermo Fisher Scientific, 15070063] in a 37°C incubator with 5% CO2 and passaged every 3 days at a ratio of 1:10. One day before mRNA transfection, HeLa cells were seeded in individual wells of a 24-well plate to a confluency of 90%. The following day, 50 ng of Renilla luciferase (internal control) mRNA and 50 ng of modified firefly luciferase mRNA were transfected using Lipofectamine MessengerMAX transfection reagent [Thermo Fisher Scientific, LMRNA003] according to the manufacturer's protocol. Additional controls containing either Renilla luciferase mRNA alone or lipofectamine transfection reagent alone were included. Three separate transfections were performed for each condition. After 6 hours of transfection, the transfection medium was removed, and the cells were trypsinized and re-seeded in phenol red-free medium in three white transparent bottom 96-well plates [Corning (Corning), 3610]. 8 / 24 / 48 hours after transfection, the cell culture medium was removed and the cells were rinsed with DPBS. After cell lysis, luciferase activity was measured using the Promega Dual Glo luciferase assay system [Promega, E2920]. In short, 50 μL PBS and 50 μL firefly luciferase working solution (prepared according to the manufacturer's protocol) were added to each well using a multichannel pipette and mixed by pipetting. After gently shaking at room temperature and incubating in the dark for 10 minutes, the firefly luciferase luminescence was measured using a microplate reader. Then 50 μL of freshly prepared Renilla luciferase Stop & Glow working solution (prepared according to the manufacturer's protocol) was added. After incubation for 10 minutes, the Renilla luciferase luminescence was measured in a similar manner. For both firefly and Renilla luminescence, background was measured and subtracted by cells treated with lipofectamine reagent alone. Firefly luminescence / Renilla luminescence in each well was used as mRNA activity readout. In the case of using Nluc and Fluc, a similar operation was performed using the Nano-Glo Dual Luciferase Reporter Gene Assay System [Promega, N1610].

[0144] Circular mRNA synthesis and characterization: DNA templates were cloned as described in the previous section, PCR amplified and gel purified for use as IVT templates. CircRNAs were synthesized as described using the HiScribe T7 High Yield RNA Synthesis Kit [NEB, E2040S]. After IVT, the DNA templates were digested with Turbo DNase [Thermo Fisher Scientific, AM2238]. The reaction mixture was heated to 70°C for 5 min and then immediately cooled on ice for 3 min, after which GTP was added to a final concentration of 2 mM and the reaction mixture was incubated at 55°C for 15 min. CircRNAs were enriched by treatment with RNase R [Lucigen Introduces, RNR07250] for 1.5 h, and the products were column purified. CircRNA products were characterized by gel electrophoresis.

[0145] QRNA synthesis and characterization: circRNAs with TGT hairpins were synthesized as described in the previous section. TGT enzyme was expressed in Escherichia coli as described in the literature. (18) To label circRNAs with preQ1-azide, 1 μM circRNA, 100 μM preQ1-azide, 10 μM TGT, 10 μL SUPERase-In RNase inhibitor were incubated in a total of 100 μL reaction at 37 °C for 2 h in 1× TGT reaction buffer (100 mM HEPES, pH 7.3, 5 mM DTT, and 20 mM MgCl2). The labeled circRNAs were purified and cleaved with Bn using the general conditions of the click reaction. 7 The G-capped alkyne-labeled oligomers were subjected to click reactions for 30 minutes. The circRNAs were then purified by RP-HPLC to remove the linearized portion (Method 2), pooled and desalted, and subjected to another round of RP-HPLC purification to isolate the qRNA products. The qRNA products were characterized by RNase H assay using 2 primers upstream / downstream of the TGT site.

[0146] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0147] Table 2. Sequences described in this application.

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

Claims

1. A type 2 capped circular RNA molecule comprising: mRNA encoding a peptide or polypeptide; The 5' end contains the cap structure; derivatized nucleotides located between the cap molecule and the region of the mRNA encoding the polypeptide; and The 3' end to which the derivatized nucleotide is covalently linked.

2. A type 1 capped circular RNA molecule comprising: An RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end portion; A circular RNA molecule comprising an mRNA encoding a peptide or polypeptide, and a derivatized nucleotide located within the circular RNA molecule, The 3' end portion of the oligonucleotide is covalently linked to the derivatized nucleotide on the circular RNA molecule.

3. A type 3 capped circular RNA molecule comprising: An RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end portion; as well as A circular RNA molecule comprising a twister ribozyme, an mRNA encoding a peptide or polypeptide, an oligonucleotide portion forming a hairpin, and a derivatized nucleotide located within the hairpin, wherein the 3' end portion of the oligonucleotide is covalently linked to the derivatized nucleotide within the hairpin of the circular RNA molecule. 4 . The capped circular RNA molecule according to claim 1 , wherein the derivatized nucleotide comprises a moiety capable of reacting with the 3′ end portion via bioconjugation chemistry.

5. The capped circular RNA molecule of claim 4, wherein the bioconjugation chemistry is click chemistry.

6. The capped circular RNA molecule according to any one of claims 1 to 3, wherein the cap structure comprises 7-methylguanosine (m 7 G), 7-benzylguanosine (Bn 7 G), 7-chlorobenzylguanosine (ClBn 7 G), chlorobenzyl-O-ethoxyguanosine (ClBnOEt 7 G), or any derivative thereof.

7. The capped circular RNA molecule according to any one of claims 1 to 3, wherein the cap structure comprises: 7-methylguanosine cap, which further comprises one or more locked nucleic acids (LNA), or one or more 2'-methoxy groups (2OMe), or any derivatives thereof.

8. The capped circular RNA molecule according to any one of claims 1 and 2, further comprising one or more modified nucleotides.

9. The capped circular RNA molecule of claim 8, wherein the modified nucleotides include pseudouridine, N 1 -methyl pseudouridine (m 1 Ψ), 6-methyladenosine (m 6 A), 5-methylcytidine, inosine, or any derivative thereof.

10. The capped circular RNA molecule of claim 8, wherein the modified nucleotide comprises a locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethyl ether (2-MOE) sugar backbone, or any derivative thereof. The capped circular RNA molecule according to claim 3 , further comprising one or more modified nucleotides.

12. The capped circular RNA molecule according to claim 11, wherein the modified nucleotides include 6-methyladenosine (m 6 A), 5-methylcytidine, inosine, or any derivative thereof.

13. The capped circular RNA molecule of claim 8, wherein the modified nucleotide comprises a locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethyl ether (2-MOE) sugar backbone, or any derivative thereof.

14. The capped circular RNA molecule according to any one of claims 2 to 3, wherein the circular RNA comprises a plurality of mRNA regions encoding one or more polypeptides.

15. The capped circular RNA molecule according to claim 14, further comprising: a plurality of RNA oligonucleotides comprising a 5' end and a 3' end portion containing the cap structure; as well as a plurality of derivatized nucleotides located at the 5' position of each of the mRNA regions encoding a peptide or polypeptide in the circular RNA, wherein each 3' end of each of the plurality of RNA oligonucleotides is covalently linked to each of the plurality of derivatized nucleotides. 16 . The capped circular RNA molecule according to claim 15 , wherein each mRNA region encoding the peptide or the polypeptide comprises a 3′ poly A sequence.

17. The capped circular RNA molecule of claim 16, wherein the one or more polypeptides encode Cas9, a base editor, or a derivative.

18. The capped circular RNA molecule of claim 16, wherein the one or more polypeptides comprise a therapeutic protein.

19. A pharmaceutical composition comprising the capped circular RNA according to any one of claims 17 and 18 and a pharmaceutically acceptable carrier.

20. A method for producing a capped circular RNA molecule according to claim 1, comprising the following steps: a) synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure, an mRNA encoding a peptide or polypeptide, a derivatized nucleotide located between the cap structure and the mRNA region encoding the polypeptide, and a 3' end containing a moiety; as well as b) reacting the derivatized nucleotide with the 3' end moiety to form a covalently linked capped circular RNA molecule.

21. The method of claim 20, wherein the synthesis of the RNA oligonucleotide comprises the following steps: a) synthesizing a first RNA oligonucleotide, which comprises the 5' end containing a cap structure, the mRNA encoding a peptide or polypeptide, and a hairpin structure between the capped 5' end and the mRNA encoding the peptide or polypeptide; b) derivatizing nucleotides within the hairpin structure of the first RNA; c) synthesizing a second RNA oligonucleotide, the second RNA oligonucleotide comprising a 3' terminal portion reactive with the derivatized nucleotide; d) ligating the 3' end of the first RNA molecule to the 5' end of the second RNA molecule.

22. A method for producing a capped circular RNA molecule according to claim 3, wherein the synthesis of the RNA oligonucleotide comprises the following steps: a) synthesizing a first RNA oligonucleotide primer, wherein the first RNA oligonucleotide primer comprises the 5' end containing a cap structure, the derivatized nucleotides and a complementary sequence of a DNA template encoding a peptide or polypeptide; b) transcribing the first RNA oligonucleotide from the primer along the DNA template to produce mRNA encoding the peptide or polypeptide; c) synthesizing a second RNA oligonucleotide, wherein the second RNA oligonucleotide comprises a 3' end containing a moiety; d) connecting the 3' end of the first RNA oligonucleotide encoding the peptide or polypeptide sequence to the 5' end of the second RNA molecule.

23. A method for producing a capped circular RNA molecule according to claim 2, comprising the following steps: a) generating a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and derivatized nucleotides outside the mRNA region; b) synthesizing an RNA oligonucleotide comprising a 5' end comprising a cap structure and a 3' end comprising a portion reactive with the derivatized nucleotide; as well as c) reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the circular RNA.

24. The method of claim 23, wherein the synthesis of the circular RNA oligonucleotide comprises the following steps: a) synthesizing an RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide and complementary sequences on the 5' and 3' ends to facilitate cyclization, wherein the derivatized nucleotides are located within the complementary sequences; b) circularizing the RNA oligonucleotide.

25. The method of claim 24, wherein the complementary sequence comprises a single cytidine nucleotide, wherein the single cytidine is the derivatized nucleotide.

26. The method of claim 23, wherein the synthesis of circular RNA oligonucleotides comprises the following steps: a) synthesizing an RNA oligonucleotide, the RNA oligonucleotide comprising the mRNA region encoding the peptide or polypeptide, and a hairpin structure containing an enzyme recognition site for introducing a derivatized oligonucleotide into the RNA oligonucleotide; b) reacting the RNA oligonucleotide with an enzyme to produce the derivatized nucleotides within the hairpin structure; c) circularizing the RNA oligonucleotide.

27. The method of claim 23, wherein the synthesis of circular RNA oligonucleotides comprises the following steps: a) synthesizing a first RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide and hydroxyl groups on both the 5' and 3' ends; b) synthesizing a second RNA oligonucleotide comprising the derivatized nucleotide and phosphates on both the 5' and 3' ends; c) ligating the 5' phosphate end and the 3' hydroxyl end; and ligating the 5' hydroxyl end and the 3' phosphate end of the first oligonucleotide and the second oligonucleotide, respectively, to generate a circularized RNA oligonucleotide.

28. The method of claim 23, wherein the synthesis of circular RNA oligonucleotides comprises the following steps: a) synthesizing a first RNA oligonucleotide, wherein the first RNA oligonucleotide comprises the mRNA region encoding a peptide or polypeptide, a 5' end containing a triphosphate and a 3' end containing a hydroxyl group; b) synthesizing a second RNA oligonucleotide comprising the derivatized nucleotide and phosphates on both the 5' and 3' ends; c) ligating the 3' end of the first oligonucleotide to the 5' end of the second oligonucleotide to produce a third oligonucleotide; d) hydrolyzing the triphosphate on the 5' end of the third oligonucleotide; as well as e) ligating the 5' end of the third oligonucleotide to the 3' end to generate a circularized RNA oligonucleotide.

29. The method of claim 23, wherein the synthesis of circular RNA oligonucleotides comprises the following steps: a) synthesizing an RNA oligonucleotide primer, the RNA oligonucleotide primer comprising the derivatized nucleotide and a complementary sequence of a DNA template encoding a peptide or polypeptide; b) transcribing the RNA oligonucleotide to further include an mRNA encoding a peptide or polypeptide; c) circularizing the RNA oligonucleotide.

30. A method for producing a capped circular RNA molecule according to claim 3, comprising the following steps: a) generating a circularized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and derivatized nucleotides outside the mRNA region; b) synthesizing an RNA oligonucleotide comprising a 5' end comprising a cap structure and a 3' end comprising a portion reactive with the derivatized nucleotide; as well as c) reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the circular RNA, The synthesis of the circular RNA oligonucleotide further comprises the following steps: i. Synthetic RNA oligonucleotides comprising the mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme recognition site, and twister ribozyme sequences on both the 5' and 3' ends; ii. reacting the RNA oligonucleotide with an enzyme to produce the derivatized nucleotide within the hairpin structure; iii. Circularizing the RNA oligonucleotide using the twister ribozyme sequence.

31. The method of claim 23, wherein the derivatized nucleotide comprises a moiety capable of reacting with the 3' end moiety via bioconjugation chemistry.

32. The method of claim 31 , wherein the bioconjugation chemistry is click chemistry.

33. The method of claim 23, wherein the circularized RNA is produced by ribozyme-mediated splicing, enzymatic ligation, or click chemistry-mediated circularization.

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