EP4642914A1

EP4642914A1 - Compositions and methods for generating circular rna

Info

Publication number: EP4642914A1
Application number: EP23913587.4A
Authority: EP
Inventors: Hyun-Bae JIE
Original assignee: ST Pharm Co Ltd
Current assignee: ST Pharm Co Ltd
Priority date: 2022-12-28
Filing date: 2023-12-22
Publication date: 2025-11-05
Also published as: CN120813695A; WO2024145248A1; JP2026504812A; KR20250153190A

Abstract

Provided are compositions and methods for generating circular RNA (circRNA). Provided are nucleic acid molecules, such as RNA molecules, comprising ribozymes and insert sequences. In some aspects, the ribozymes in the RNA molecule is cleaved. In some embodiments, the cleaved RNA molecule is circularized by a ligase. Also provided are methods and systems for generating circRNA, and methods of using circRNA, including therapeutic and prophylactic methods.

Description

COMPOSITIONS AND METHODS FOR GENERATING CIRCULAR RNA

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/435,767, filed December 28, 2022 and U.S. Provisional Patent Application No. 63/501,318, filed May 10, 2023. The contents of each of these provisional applications are incorporated by reference in their entirety.

Sequence Listing

[0002] This application contains a ST.26 compliant Sequence Listing, which was submitted in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on December 20, 2023, is named 153908_8001_WO00_SL.xml and is 216,644 bytes in size.

Field

[0003] The present disclosure relates to compositions and methods for generating circular RNA (circRNA). Provided are nucleic acid molecules, such as RNA molecules, comprising ribozymes and insert sequences. In some aspects, the ribozymes in the RNA molecule is cleaved. In some embodiments, the cleaved RNA molecule is circularized by a ligase. Also provided are methods and systems for generating circRNA, and methods of using circRNA, including therapeutic and prophylactic methods.

Background

[0004] Circular RNA (circRNA) is a single-stranded RNA that can be resistant to exonuclease- mediated degradation. However, challenges exist in generating and producing circRNAs. There remains a need for efficient method for RNA circularization, particularly for large RNA molecules, and for reducing contamination with linear RNA that can result in immunogenicity and subsequently reduced expression of the insert gene product. Provided herein are embodiments that meet such needs.

Summary

[0005] Provided herein are ribonucleic acid (RNA) molecules, combinations thereof, reaction intermediates or reaction products thereof, including linear RNA, cleaved linear RNA and circular RNA (circRNA). In some of any embodiments, the provided RNA molecules and various reaction intermediates or reaction products thereof are for generating circRNA. Also provided are methods for generating any of the RNA molecules, combinations thereof, reaction intermediates or reaction products thereof and compositions comprising any of the foregoing. Also provided are methods and uses of any of the RNA molecules, combinations thereof, reaction intermediates or reaction products thereof, or compositions thereof, including therapeutic and prophylactic uses.

[0006] Provided herein are ribonucleic acid (RNA) molecules comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme.

[0007] In some of any embodiments, the 5' substrate sequence comprises a 5' overhang sequence, and the 3' substrate sequence comprises a 3' overhang sequence In some of any embodiments, the 5' ribozyme and the 3' ribozyme together are capable of cleaving the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence; the insert sequence; and the 3' overhang sequence.

[0008] In some of any embodiments, the RNA molecule further comprises a 5' homology region and 3' homology region. In some of any embodiments, the 5' homology region is located 3' of the 5' ribozyme. In some of any embodiments, the 3' homology region is located 3' of the insert sequence.

[0009] Also provided herein are cleaved RNA molecules comprising, in 5' to 3' order: a 5' overhang sequence of a Twister- Sister ribozyme; a 5' homology region; an insert sequence; a 3’ homology region; and a 3' overhang sequence of a Twister ribozyme. In some of any embodiments, at least a portion of the 5' homology region and at least a portion of 3' homology region are complementary and are capable of forming a stem structure.

[0010] In some of any embodiments, the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus. In some of any embodiments, the hydroxyl group at the 5' terminus and the 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase to generate a circular RNA molecule.

[0011] In some of any embodiments, the RNA ligase is a tRNA splicing ligase. In some of any embodiments, the RNA ligase is an RtcB ligase. In some of any embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

[0012] Also provided herein are circular RNA molecules comprising: a 5' overhang sequence of a Twister-Sister ribozyme; a 5' homology region; an insert sequence; a 3' homology region; and a 3' overhang sequence of a Twister ribozyme.

[0013] In some of any embodiments, the Twister-Sister ribozyme comprises a Twister-Sister- 1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme. In some of any embodiments, the Twister- Sister ribozyme comprises a TS-1 ribozyme.

[0014] In some of any embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6. In some of any embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6.

[0015] In some of any embodiments, the catalytic sequence of a Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:4. In some of any embodiments, the catalytic sequence of a Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

[0016] In some of any embodiments, the Twister ribozyme comprises a type Pl Twister ribozyme. In some of any embodiments, the Twister ribozyme comprises a Nasonia vitripennis Type Pl Twister ribozyme.

[0017] In some of any embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

[0018] In some of any embodiments, the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:23. In some of any embodiments, the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO: 23.

[0019] In some of any embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 10. In some of any embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10. In some of any embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 19. In some of any embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO: 19.

[0020] In some of any embodiments, the insert sequence comprises a translation initiation element. In some of any embodiments, the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element. In some of any embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 14. In some of any embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

[0021] In some of any embodiments, the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules. In some of any embodiments, the one or more exogenous molecule is selected from among a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

[0022] In some of any embodiments, the one or more exogenous molecule comprises a vaccine antigen. In some of any embodiments, the vaccine antigen comprises a viral vaccine antigen. In some of any embodiments, the vaccine antigen comprises a cancer antigen.

[0023] In some of any embodiments, the one or more exogenous molecule comprises a sequence-specific nuclease. In some of any embodiments, the sequence-specific nuclease is a Cas nuclease. In some of any embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Casl2 nuclease, or a Casl3 nuclease.

[0024] In some of any embodiments, the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

[0025] In some of any embodiments, the one or more exogenous molecule comprises an immunomodulatory polypeptide. In some of any embodiments, the immunomodulatory polypeptide comprises a cytokine. [0026] In some of any embodiments, the one or more exogenous molecule comprises a transcription factor.

[0027] In some of any embodiments, the one or more exogenous molecule comprises a reporter molecule. In some of any embodiments, the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP). In some of any embodiments, the reporter molecule comprises a firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16.

[0028] In some of any embodiments, the insert sequence is at least about 500 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

[0029] In some of any embodiments, among a population of the RNA molecules, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some of any embodiments, among a population of the RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are cleaved RNA molecules.

[0030] In some of any embodiments, among a population of the RNA molecules, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase. In some of any embodiments, among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

[0031] In some of any embodiments, the RNA molecule comprises a modified nucleoside. In some of any embodiments, the modified nucleoside comprises a pseudouridine or a Nl- methylmethylpseudouridine.

[0032] In some of any embodiments, the RNA molecule leads to reduced activation of one or more toll-like receptors (TLRs) or evades detection by one or more TLRs when incubated with a cell comprising the TLR.

[0033] Also provided are combinations of RNA molecules, the combination comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme.

[00341 Iⁿ some of any embodiments, the 5' substrate sequence comprises a 5' overhang sequence. In some of any embodiments, the 5' ribozyme of the first RNA molecule and the transacting ribozyme of the second RNA molecule together are capable of cleaving the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence; the insert sequence; and the 3' overhang sequence.

[0035] In some of any embodiments, the cleaved RNA molecule further comprises a 5' homology region and 3' homology region. In some of any embodiments, the 5' homology region is located 3' of the 5' ribozyme. In some of any embodiments, the 3' homology region is located 3' of the insert sequence. In some of any embodiments, at least a portion of the 5' homology region and at least a portion of 3' homology region are complementary and are capable of forming a stem structure.

[0036] In some of any embodiments, the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus. In some of any embodiments, the hydroxyl group at the 5' terminus and the 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase to generate a circular RNA molecule.

[0037] In some of any embodiments, the RNA ligase is a tRNA splicing ligase. In some of any embodiments, the RNA ligase is an RtcB ligase. In some of any embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

[0038] In some of any embodiments, the Twister-Sister ribozyme comprises a Twister-Sister- 1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme. In some of any embodiments, the Twister- Sister ribozyme comprises a TS-1 ribozyme.

[0039] In some of any embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6. In some of any embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6.

[0040] In some of any embodiments, the catalytic sequence of a Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NOT, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:4. In some of any embodiments, the catalytic sequence of a Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

[0041] In some of any embodiments, the Twister ribozyme comprises a type Pl Twister ribozyme. In some of any embodiments, the Twister ribozyme comprises a Nasonia vitripennis Type Pl Twister ribozyme.

[0042] In some of any embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:21. In some of any embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

[0043] In some of any embodiments, the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:23. In some of any embodiments, the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO: 23.

[0044] In some of any embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NOTO. In some of any embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NOTO. In some of any embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO T9. In some of any embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO: 19.

[0045] In some of any embodiments, the insert sequence comprises a translation initiation element. In some of any embodiments, the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element. In some of any embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 14. In some of any embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14. [0046] In some of any embodiments, the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules. In some of any embodiments, the one or more exogenous molecule is selected from among a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

[0047] In some of any embodiments, the one or more exogenous molecule comprises a vaccine antigen. In some of any embodiments, the vaccine antigen comprises a viral vaccine antigen. In some of any embodiments, the vaccine antigen comprises a cancer antigen.

[0048] In some of any embodiments, the one or more exogenous molecule comprises a sequence-specific nuclease. In some of any embodiments, the sequence-specific nuclease is a Cas nuclease. In some of any embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Casl2 nuclease, or a Casl3 nuclease.

[0049] In some of any embodiments, the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

[0050] In some of any embodiments, the one or more exogenous molecule comprises an immunomodulatory polypeptide. In some of any embodiments, the immunomodulatory polypeptide comprises a cytokine.

[0051] In some of any embodiments, the one or more exogenous molecule comprises a transcription factor.

[0052] In some of any embodiments, the one or more exogenous molecule comprises a reporter molecule. In some of any embodiments, the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP). In some of any embodiments, the reporter molecule comprises a firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16.

[0053] In some of any embodiments, the insert sequence is at least about 500 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

[0054] In some of any embodiments, among a population of the RNA molecules, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some of any embodiments, among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

[0055] In some of any embodiments, among a population of the RNA molecules, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase. In some of any embodiments, among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

[0056] In some of any embodiments, the RNA molecule comprises a modified nucleoside. In some of any embodiments, the modified nucleoside comprises a pseudouridine or a Nl- methylmethylpseudouridine.

[0057] In some of any embodiments, the RNA molecule leads to reduced activation of one or more toll-like receptors (TLRs) or evades detection by one or more TLRs when incubated with a cell comprising the TLR.

[0058] Also provided herein are deoxyribonucleic acid (DNA) molecules. In some of any embodiments, the DNA molecule encodes any of the provided RNA molecules, the first RNA molecule of any of the provided combinations, the second RNA molecule of any of the provided combinations, or the first RNA molecule and the second RNA molecule of any of the provided combinations.

[0059] Also provided are systems for generating a circular RNA molecule. In some of any embodiments, the system comprises any of the provided RNA molecules, or any of the provided combinations.

[0060] Also provided are systems for generating a circular RNA molecule. In some of any embodiments, the system comprises any of the provided DNA molecules, and a reagent for in vitro transcription.

[0061] In some of any embodiments, the system further comprises an RNA ligase. In some of any embodiments, the RNA ligase is a tRNA splicing ligase. In some of any embodiments, the RNA ligase is an RtcB ligase. In some of any embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

[0062] Also provided herein are methods for generating a ribonucleic acid (RNA) molecule, that involve producing any of the provided RNA molecules.

[0063] Also provided herein are methods for generating a ribonucleic acid (RNA) molecule, that involve producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme.

[0064] Also provided herein are methods for generating a combination of ribonucleic acid (RNA) molecules, that involve producing any of the provided combinations.

[0065] Also provided herein are methods for generating a combination of ribonucleic acid (RNA) molecules, that involve producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme.

[0066] In some of any embodiments, the methods also involve incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0067] Also provided herein are methods for generating a cleaved RNA molecule, that involve: (1) producing any of the provided RNA molecules; and (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0068] Also provided herein are methods for generating a cleaved RNA molecule, that involve:

(1) producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme; and

(2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0069] Also provided herein are methods for generating a cleaved RNA molecule, that involve: (1) producing any of the provided combinations; and (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0070] Also provided herein are methods for generating a cleaved RNA molecule, that involve: (1) producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme; and (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[00711 Iⁿ some of any embodiments, the methods also involve incubating the RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

[0072] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing any of the provided RNA molecules; (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

[0073] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme; (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

[0074] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing any of the provided combinations; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

[0075] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

[0076] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing any of the provided RNA molecules; (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) administering the cleaved RNA molecule to a subject, thereby generating a circular RNA molecule.

[0077] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme; (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) administering the cleaved RNA molecule to a subject, thereby generating a circular RNA molecule.

[0078] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing any of the provided combinations; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) administering the cleaved RNA molecule to a subject, thereby generating a circular RNA molecule.

[0079] Also provided herein are methods for generating a circular RNA molecule, that involve: (1) producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) administering the cleaved RNA molecule to a subject, thereby generating a circular RNA molecule.

[0080] In some of any embodiments, the circular RNA is generated by an RNA ligase endogenously present in the subject.

[0081] In some of any embodiments, the RNA molecule or the first RNA molecule or the second RNA molecule of the combination is produced by in vitro transcription.

[0082] In some of any embodiments, the RNA molecule or the first RNA molecule or the second RNA molecule of the combination is produced by RNA synthesis.

[0083] In some of any embodiments, the incubation in step (2) generates a cleaved RNA molecule after cleavage of the 5' substrate sequence and the 3' substrate sequence by the catalytic sequences. In some of any embodiments, the incubation in step (2) generates a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule.

[0084] In some of any embodiments, the solution comprises sodium acetate (Na2OAc), magnesium acetate (MgOAc ) or both. In some of any embodiments, the solution does not comprise potassium chloride (KC1) or magnesium chloride (MgCk).

[0085] In some of any embodiments, the solution comprises cyclic di-guanosine monophosphate (c-di-GMP). In some of any embodiments, the solution comprises c-di-GMP at a concentration of between about 0.5 mM and about 10 mM. In some of any embodiments, the solution comprises c-di-GMP at a concentration of about 5 mM.

[0086] In some of any embodiments, the solution comprises distilled water (DW). In some of any embodiments, the solution consists of distilled water (DW).

[0087] In some of any embodiments, the solution comprises Tris-EDTA (TE) buffer, optionally TE buffer pH 7.0 or TE buffer pH 8.0.

[0088] In some of any embodiments, the methods also involve a denaturation step and a renaturation step. In some of any embodiments, the denaturation step is performed between about 60°C and about 85 °C, or about 65 °C and about 80 °C, or at about 65 °C, or at about 80 °C. In some of any embodiments, the renaturation step comprises incubating at ambient temperature or 4 °C after the denaturation step.

[0089] In some of any embodiments, the denaturation step and the renaturation step are performed in the solution for generating the cleaved RNA molecule. In some of any embodiments, the solution comprises Tris-EDTA (TE) buffer, optionally TE buffer pH 7.0 or TE buffer pH 8.0.

[0090] In some of any embodiments, among a population of the RNA molecules generated by the method, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some of any embodiments, among a population of the RNA molecules generated by the method, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

[0091] In some of any embodiments, the hydroxyl group at the 5' terminus and the 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule is capable of being ligated in the presence of an RNA ligase to generate a circular RNA molecule.

[0092] In some of any embodiments, the incubating with the RNA ligase in step (3) is performed for between about 5 and about 60 minutes, about 10 and about 30 minutes, about 15 and about 25 minutes, or about 10 and about 20 minutes. In some of any embodiments, the incubating with the RNA ligase in step (3) is performed for about 20 minutes.

[0093] In some of any embodiments, the incubating with the RNA ligase in step (3) is performed between about 30°C and about 40 °C, about 35 °C and about 39 °C, or about 36 °C and about 38 °C. In some of any embodiments, the incubating with the RNA ligase in step (3) is performed at about 37°C.

[00941 Iⁿ some of any embodiments, the incubating with the RNA ligase in step (3) is performed in a buffer that comprises Mg²⁺. In some of any embodiments, the incubating with the RNA ligase in step (3) is performed in a buffer that comprises Tris-HCl, KC1, MgCh, and DTT. In some of any embodiments, the incubating with the RNA ligase in step (3) is performed in a buffer that comprises 50 mM Tris-HCl, 75 mM KC1, 3 mM MgCh, and 10 mM DTT.

[0095] In some of any embodiments, the incubating with the RNA ligase in step (3) is performed in a buffer that does not comprise Mg²⁺.

[0096] In some of any embodiments, the RNA ligase is a tRNA splicing ligase. In some of any embodiments, the RNA ligase is an RtcB ligase. In some of any embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

[0097] In some of any embodiments, the methods also involve purifying the cleaved linear RNA molecule or the circular RNA molecule. In some of any embodiments, the purifying is carried out by chromatography. In some of any embodiments, the chromatography comprises high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEC), or size exclusion chromatography-high performance liquid chromatography (SEC-HPLC).

[0098] In some of any embodiments, the methods also involve enriching for the circular RNA molecules. In some of any embodiments, the enriching is carried out by incubation with a kinase. In some of any embodiments, the kinase comprises a polynucleotide kinase (PNK). In some of any embodiments, the enriching is carried out by incubation with a phosphatase.

[0099] In some of any embodiments, the methods also involve analyzing the circular RNA molecules. In some of any embodiments, the analyzing is performed in part by running the circular RNA on a gel, for example a agarose gel. In some of any embodiments, the gel comprising the circular RNA is run at 4°C. In some of any embodiments, after the gel is run, the gel is cooled on ice. As used herein, “running” a gel includes applying an electrophoretic field to the gel to cause movement of charged particles loaded into the well, as understood by a person of ordinary skill in the art. [0100] In some of any embodiments, the enriching is carried out by incubation with one or more ribonucleases. In some of any embodiments, ribonucleases comprises an RNase R and/or a 5' phosphate-dependent exonuclease.

[0101] In some of any embodiments, among a population of the RNA molecule generated by the method, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase. In some of any embodiments, among a population of the RNA molecule generated by the method, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

[0102] In some of any embodiments, the RNA molecule comprises a modified nucleoside. In some of any embodiments, the modified nucleoside comprises a pseudouridine or a Nl- methylmethylpseudouridine.

[0103] Also provided are RNA molecules generated by any of the provided methods.

[0104] Also provided are cleaved RNA molecules generated by any of the provided methods. In some of any embodiments, the RNA molecule is a linear RNA molecule.

[0105] Also provided are circular RNA molecules generated by any of the provided methods.

[0106] Also provided are compositions comprising any of the provided RNA molecules.

[0107] Also provided are compositions comprising any of the provided cleaved RNA molecules.

[0108] Also provided are compositions comprising any of the provided circular RNA molecules.

[0109] Also provided are compositions comprising any of the provided combinations.

[0110] In some of any embodiments, the composition is a pharmaceutical composition. In some of any embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0111] In some of any embodiments, the composition comprises a lipid nanoparticle (LNP).

[0112] Also provided herein are methods of vaccinating a subject, the method comprising administering any of the provided RNA molecules, any of the provided combinations, an RNA molecule generated by any of the provided methods, or any of the provided compositions.

[0113] Also provided herein are methods of treating a disease or disorder in a subject, the method comprising administering any of the provided RNA molecules, any of the provided combinations, an RNA molecule generated by any of the provided methods, or any of the provided compositions. [0114] Also provided are any of the provided RNA molecules, any of the provided combinations, an RNA molecule generated by any of the provided methods, or any of the provided compositions for use in vaccinating a subject, wherein the RNA molecule or the composition is administered to the subject.

[0115] Also provided are any of the provided RNA molecules, any of the provided combinations, an RNA molecule generated by any of the provided methods, or any of the provided compositions for use in treating a disease or disorder in a subject, wherein the RNA molecule or the composition is administered to the subject.

[0116] Also provided are uses of any of the provided RNA molecules, any of the provided combinations, an RNA molecule generated by any of the provided methods, or any of the provided compositions in the manufacture of medicament for vaccinating a subject, wherein the medicament is administered to the subject.

[0117] Also provided are uses of any of the provided RNA molecules, any of the provided combinations, an RNA molecule generated by any of the provided methods, or any of the provided compositions in the manufacture of medicament for treating a disease or disorder in a subject, wherein the medicament is administered to the subject.

Brief Description of the Drawings

[0118] FIGS. 1A-1D show schematics representing exemplary circular RNA (circRNA) synthesis using a unimolecular approach with a 5' Twister- Sister ribozyme and a 3' Twister ribozyme (FIG. 1A); or a bimolecular approach with a 5' Twister-Sister ribozyme and a trans-acting Twister ribozyme (FIG. IB); and ligation/circularization using an RNA ligase. FIG. 1A shows the design of exemplary Construct Ul, which comprises: 5' Twister- Si ster-1 (TS-1) ribozyme and 3' Twister ribozyme of N. vitripermis, both of which flank an internal ribosome entry site (IRES) sequence and an exemplary insert (firefly luciferase coding sequence). FIG. IB shows the design of exemplary Construct Bl, which comprises the substrate portion of the 3' Twister ribozyme at the 3' end; which can be cleaved by Construct B2, which contains a trans-acting Twister ribozyme. Once cleavage of the 5' and 3' ends occur, the generated linear pre-circRNA comprising a 5' hydroxyl group and a 2', 3 '-cyclic phosphate at the 3' end, can be ligated and circularized by a tRNA splicing ligase such as the RtcB ligase, generating a circRNA.

[0119] FIG. 1C shows RNAFold predictions of precursor RNA secondary structure for Construct U1 (SEQ ID NO: 29) before and after 5' and 3' ribozyme cleavage. Arrows indicate the cleavage site of 5' Twister-Sister ribozyme and 3' Twister ribozyme, respectively. The insets show portions of Construct U1 corresponding to SEQ ID NOS 119, 116-118, and 120-121, respectively, from left to right. FIG. ID shows RNAFold predictions of precursor RNA secondary structure for Constructs Bl (SEQ ID NO: 37) and B2 (SEQ ID NO: 35) before and after 5' ribozyme cleavage and 3' cleavage by the trans-acting ribozyme. Arrows indicate the cleavage site of 5' TS-1 and the trans-acting Twister ribozyme, respectively. The small box with dotted lines represents RNAFold predictions of Construct B2 secondary structure of the trans-acting Twister ribozyme specific for 3' substrate in Construct Bl. The insets show portions of Constructs Bl and B2 corresponding to SEQ ID NOS 122-123, 35, and 120-121, respectively, from left to right.

[0120] FIGS. 2A-2C show electrophoresis gel profiles depicting circularization efficiency of various constructs employing various 5' and 3' ribozymes. FIG. 2A shows an electrophoresis profile of RNA preparations, depicting cleavage intermediates of Construct 3 comprising 5' P3 Twister U2A ribozyme and 3' type Pl Twister ribozyme, after in vitro transcription and with or without circularization by RtcB and enrichment by RNase R. Band a: linear RNA, including uncleaved precursor RNA, semi-cleaved (single cleavage) RNA, and 5' and 3' cleaved pre- circRNA; band b: circular RNA; band c: cleaved 5' ribozyme; band d: cleaved 3' ribozyme. FIG. 2B shows an electrophoresis profile depicting cleavage activity of various constructs comprising 5' Twister- Sister ribozyme or Twister ribozyme, and various 3' ribozymes. Lane 1, 3, 5, 7, 9: RNA samples before the cleavage reaction. Lane 2, 4, 6, 8, 10: RNA samples after the cleavage reaction in Cleavage Buffer 1. FIG. 2C shows the relative intensities of the cleaved 5' ribozyme band after a 1 hour cleavage reaction, plotted as a bar graph.

[0121] FIGS. 3A-3F show electrophoresis profiles depicting circularization efficiency under different cleavage buffer and ligase buffer conditions. The percentage of circularization was quantitated by band densitometry. FIG. 3A shows the percentage of circRNA generated after cleavage in different cleavage buffer conditions. Lanes 2-6: in vitro transcription-generated precursor RNA cleaved in commercial T7 RNA polymerase buffers from Thermo Fisher (Lane 2), Promega (Lane 3), Roche (Lane 4), and MEGAscript (Lane 6), and Cleavage Buffer 1 (Lane 5). Lane 1 : no cleavage reaction (negative control). FIG. 3B shows the percentage of circRNA generated after cleavage in in cleavage buffers containing different additions or buffer compositions. Lane 1 : Cleavage Buffer 1. Lanes 2 and 3: Cleavage Buffer 1 with addition of cyclic- di-GMP (c-di-GMP) at low (0.5 mM) and high (5mM) concentrations, respectively. Lanes 4 and 5: Cleavage Buffer 2 and Cleavage Buffer 3. FIG. 3C compares the percentage of circRNA generated after cleavage in Cleavage Buffer 1 or Cleavage Buffer 2 with (+) or without (-) the addition of c- di-GMP (5mM). FIG. 3D shows the effects of magnesium ion (Mg²⁺) on the stability of generated circRNA, and the generation of nicked circular RNA, at various temperatures. FIG. 3E shows the effect of the amount of RtcB ligase in the ligation reaction on circularization efficiency. FIG. 3F shows the effect of the length of time of the RtcB ligation reaction on circularization efficiency.

[0122] FIGS. 4A-4D show the function of circRNAs and linear intermediate RNAs generated using exemplary unimolecular Construct Ul, introduced into mammalian cells, and the expression of encoded gene product. FIG. 4A shows linear the electrophoresis profile of in vitro transcribed RNA from Construct Ul (Lane 1; “IVT”), linear RNA transcribed in vitro from Construct Ul and cleaved (Lane 2; “CLV”), and linear RNA transcribed in vitro from Construct Ul that have been cleaved, circularized with RtcB, and enriched with RNase R treatment (Lane 3; “RtcB+RR”) in transfected HEK 293 T cells. FIG. 4B shows circRNA production measured by qPCR using primers that target the junctional sites of circRNA 24 hrs post-transfection. Average expression was calculated as mean. P values were calculated by One-way ANOVA. N.S: not significant **P < 0.005. FIG. 4C shows the luciferase activities at 24, 48, 72, 96, and 120 hrs in HEK 293T cells transfected with the various RNA preparations from Construct Ul (IVT, CLV, RtcB+RR). FIG. 4D shows the kinetics of luciferase activity over 120 hours in HEK 293T cells transfected with the various RNA preparations from Construct Ul (IVT, CLV, RtcB+RR) compared to HEK 293T transfected with linear mRNA encoding firefly luciferase, comprising unmodified uridine (U) or a modified pseudouridine (pU; N1 -methylpseudouridine).

[0123] FIG. 5 shows cleavage activity of Construct Bl by trans-acting Twister ribozyme in Construct B2, in Cleavage Buffer 1 or the MEGAscript T7 RNA transcription buffer for the cleavage reaction, and with (+) or without (-) a ligation reaction using RtcB, and/or enrichment using RNase R. The efficiency of circularization was measured by band densitometry.

[0124] FIG. 6A shows gel electrophoresis of RT-PCR products using junctional-specific (upper panels) and non-junctional specific (internal) primers (lower panels) on circular RNAs generated using exemplary unimolecular Construct Ul and bimolecular Constructs Bl /B2 pair, with (+) or without (-) RtcB ligase treatment. FIG. 6B shows Sanger sequencing results of the exact predicted sequences of the junctional site of the circRNA based on ribozyme cleavage and ligation sites. Also included is a diagram illustrating the relative position and directionality of the RT-PCR primer pairs to amplify the internal region or the circRNA junctional site. Figure discloses SEQ ID NO: 124.

[01251 FIGS. 7A-7D show the function of circRNAs and linear intermediate RNAs generated using exemplary bimol ecular Construct Bl /B2 pair, the conversion of linear RNA to circRNA in mammalian cells, and the expression of encoded gene product.

[0126] FIG. 7A shows gel electrophoresis of RT-PCR products using junctional-specific (upper panels) and non-junctional specific (internal) primers (lower panels) on RNA extracted from HEK 293 T cells 24 hours after transfection with linear RNA transcribed in vitro from Construct Bl (“IVT”), Construct Bl cleaved by incubation with trans-acting ribozyme of Construct B2 (“CLV”), and linear RNA transcribed in vitro from Construct Bl that have been cleaved by incubation with Construct B2, circularized with RtcB, and enriched with RNase R treatment ( “RtcB+RR”), or control cells. FIG. 7B (semi-quantitative PCR) and FIG. 7C (quantitative PCR) shows the relative amount of amplified junctional site (indicative of circRNA formation) from HEK 293T cells transfected with Construct B1/B2 CLV and RtcB+RR preparations, plotted as a bar graph. FIG. 7D shows the luciferase activities at 24 hrs in HEK 293T cells transfected with the various RNA preparations from Construct B1/B2 (IVT, CLV, RtcB+RR).

[0127] FIG. 8 shows electrophoresis profiles depicting circRNA generation efficiency using the exemplary unimolecular Construct U1 or the Group I intro-PIE system, with RNA molecules comprising unmodified uridine residues (unmodified U) or a modified pseudouridine (pseudo U; N1 -methylpseudouridine), with (+) or without incubation with RtcB ligase and/or RNase R enrichment.

[0128] FIGS. 9A-9D show HPLC profiles of cleaved linear RNA preparation (FIG. 9A for RNA containing unmodified uridine (U) and FIG. 9C for RNA containing N1 -methylpseudouridine (ml )) or RNA preparations generated after ligation by RtcB treatment, containing a mixture of circRNA and linear RNA (FIG. 9B for RNA containing unmodified uridine and FIG. 9D for RNA containing ml'P) using the exemplary unimolecular Construct Ul. FIG. 9E shows gel electrophoresis profile each RNA preparation after HPLC (FIG. 9E).

[0129] FIGS 10 -10C show SEAP reporter activity from culture supernatants of HEK-Blue TLR-3 (FIG. 10A), TLR-7 (FIG. 10B), or TLR-8 (FIG. 10C) reporter cells after incubation with cleaved linear RNA preparation or RNA preparations generated after ligation by RtcB treatment containing a mixture of circRNA and linear RNA, containing unmodified uridine (U) or Nl- methylpseudouridine (ml ) using the exemplary unimolecular Construct Ul. Linear mRNA encoding firefly luciferase, poly I:C (TLR3 ligand), or R848 (TLR7/8 ligand) were used as controls. Averages were calculated as means. P values were calculated by One-way ANOVA. ***P < 0.001, and ****P < 0.0001.

[0130] FIGS. 11A-11B show a schematic depicting various purification and enrichment steps that were used to purify and enrich for circRNA in the preparation, including serial treatments with T4 polynucleotide kinase (PNK), 5' phosphate-dependent exonuclease, and RNase R. FIG. 11A depicts a schematic showing the reactions that PNK, 5' phosphate-dependent exonuclease, and RNase R catalyze, to which circRNA is resistant to. After the serial treatment, the preparation is enriched for circRNA. FIG. 11B shows the electrophoresis profile and percentage of circRNA after treatment with various enzymes for enrichment. The cleaved RNAs were purified by HPLC and serially treated with the following enzymes: Lane 1 : RtcB only; Lane 2: RtcB followed by PNK, then 5' phosphate-dependent exonuclease; Lane 3: RtcB followed by PNK, then RNase R; Lane 4: RtcB followed by PNK, then 5' phosphate-dependent exonuclease and RNase R.

[0131] FIGS. 12A-12B show schematics depicting conserved features of Twister ribozymes and Twister-Sister ribozymes. FIG. 12A shows consensus sequences and secondary structure models for Twister ribozymes (left panel, including secondary structure diagrams for Type Pl, P3 and P5 Twister ribozymes) and Twister-Sister ribozymes (right panel). FIG. 12B shows a bimolecular construct structure derived from the TS-1 ribozyme (see Roth et al. Nat Chem Biol, 10(1), 56-60 (2014); Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015)). Figure discloses SEQ ID NO: 125.

[0132] FIGS. 13A-13B show the cleavage efficiency of the 3 'Twister ribozyme in the generation of circRNA. FIG. 13A shows a schematic depicting an exemplary Construct Ul modified with an extended 3' end sequence of variable length, named Constructs U2, U3, and U4. FIG. 13B shows the electrophoresis profile of the RNA species after of cleavage and ligation of the various constructs with 3' end extensions of variable length.

[0133] FIGS. 14A-14F show the gel electrophoresis profiles depicting circRNA generation efficiency under various cleavage and ligation conditions. FIG. 14A shows the gel electrophoresis profile and percentage of circRNA generation (circularization efficiency) after no separate cleavage reaction ("No cleavage (DW)”) or a separate cleavage reaction in a cleavage buffer (“Cleavage”). FIG. 14B shows the gel electrophoresis profile and percentage of circRNA after ligation in buffers of variable concentrations of MnCh. FIG. 14C shows the gel electrophoresis profile and percentage of circRNA after ligation in RtcB buffers of variable pH. FIG. 14D show the gel electrophoresis profile and percentage of circRNA after ligation in solutions containing individual components of RtcB buffer. FIG. 14E shows the gel electrophoresis profile and percentage of circRNA following denaturation/renaturation in TE buffers with various pH. FIG. 14F shows the gel electrophoresis profile and percentage of circRNA of the various exemplary constructs (Constructs Ul, U2, U3, and U4) following denaturation and renaturation in TE buffer and ligation in RtcB reaction buffer.

[0134] FIGS. 15A-15C show cleavage of circular RNA due to heat generated during gel running at room temperature. FIG. 15A shows that circular RNA (+RtcB) is cleaved when running a gel at room temperature. FIG. 15B shows that when running the gel at 4°C, the same circular RNA (+RtcB) used in A is not cleaved. Linear RNA (-RtcB) was used as a control to determine if circular RNA is cleaved. FIG. 15C shows that when circular RNA (+RtcB) is exposed to a denaturation condition (65°C for 3 min) and the sample is gel run at 4°C, the circular RNA is cleaved. However, circular RNA that has not been exposed to the same denaturation conditions is not cleaved and remains circular status.

[0135] FIGs 16A-16B show cleavage of circular RNA due to the heat generated at 4°C when running gels for relatively long periods. FIG. 16A shows that when circular RNA (+RtcB) is run on a gel at 4°C for a relatively long time (10 min), circular RNA is cleaved. FIG 16B shows that running the gel at 4°C for 3 minutes and cooling it on ice for 10 minutes, repeated at least three times, prevents the cleavage of circular RNA.

[0136] FIGS. 17A-17B shows the circularization efficiency of genes with different lengths. FIG. 17A shows that RNA expressing EGFP (720 bp), human erythropoietin (hEPO, 582 bp), firefly luciferase (FLuc, 1653 bp), and Cas9 (3270 bp) in LVT14 format were circularized with RtcB. After the reaction, circularized RNAs (+RtcB) or linear RNAs (-RtcB) were run in the gel by using the cooling conditions described in FIGS. 16A-16B. FIG. 17B shows that the circularization efficiency of each gene with indicated gene size was quantified using Image Lab software (BioRad).

[0137] FIGS. 18A-18C shows the functional comparison of IRES from different viruses based on Coxsackie virus (CVB3)’ IRES. FIG. A shows a schematic illustration showing the CVB3 IRES structure that drives expression of FLuc and the domains to which the major translational factors eIF4G and eIF4A bind. FIG. B shows that the schematic illustration shows the IRES of CVB3 and other viruses expressing FLuc. FIG. C shows that firefly luciferase (FLuc) activity was measured 24 hours after HEK293T cells were transfected with circRNAs containing the indicated IRES of CVB3 and other viruses. FLuc activity was normalized by dividing the FLuc activity of each sample by the renilla luciferase activity used for co-transfection. Relative folds of FLuc activity are calculated by dividing the normalized FLuc acitvity of each IRES by the CVB3 value. Data are mean ± s.e.m. for n = 4 biological replicates.

[0138] FIG. 19A-19B shows enhanced FLuc activity through CVB3 IRES engineering using poly(A)-binding protein (PABP) and/or eIF4G-binding aptamers. FIG. 19A shows a schematic representation of the genetically engineered CVB3 IRES, showing the location of PABP and eIF4G-binding aptamers in LVT14. FIG. 19B shows the relative FLuc activity expressed by each genetically engineered CVB3 IRES compared to the activity of the CVB3 IRES (LVT14) was calculated. Data are mean ± s.e.m. for n = 3 biological replicates.

[0139] FIGS. 20A-20G shows circRNAs that can avoid detection by TLRs, RIG-I, and MDA5. FIG. 20A shows that linear RNA or circular RNA was purified by HPLC, and the purification was confirmed by gel running using cooling conditions described in Figure 16. B-G. HEK reporter cell lines expressing human TLR-3 (FIG. 20B), -7 (FIG. 20C), -8 (FIG. 20D), or RIG1 (FIG. 20E) and A549 cells knocked out (KO) of RIG1 (FIG. 20F) or MDA5 (FIG. 20G) were transfected with purified linear RNA, circular RNA, mRNA containing 5-methyl-pseudouridine (5MoU) and agonists of the corresponding receptors (3-phpRNA, Poly I: C, R848) respectively. The secreted alkaline phosphatase (SEAP) or lucia-luciferase activity expressed by each stimulus was measured in the corresponding cell lines 24 h after stimulation. Data are mean ± s.e.m. for n = 4 biological replicates.

[0140] FIGS. 21A-21C show immunogenicity assessment of circular RNA. FIGS 21A to 21C. Twenty-four hours after stimulation of A549 cells with purified linear RNA, circular RNA, mRNA containing 5-methyl-pseudouridine (5MoU) and agonists of the corresponding receptors (3- phpRNA, Poly LC, R848) respectively, IL-6 (FIG. 21A), IFN- (FIG. 21B) or RANTES (FIG. 21C) was measured by ELISA in supernatants obtained from cell lines treated with each stimulant. Data are mean ± s.e.m. for n = 3 biological replicates.

[0141] FIGS. 22A-22C show circular RNA with a 3-fold longer half-life and 45-fold higher FLuc activity compared to mRNA (5 MoU) ex vivo. 293 T cells were treated with FLuc mRNA (5MoU) or circular RNA encapsulated with lipid nanoparticle (LNP) and FLuc activity was measured for 7 days after treatment. FIG. 22A shows that FLuc activity is normalized by dividing the FLuc activity measured at each time point by the FLuc activity at 24 hours after transfection. Based on the normalized FLuc activity, the half-life was calculated. FIG. 22B shows that the absolute FLuc activity by mRNA and circular RNA was measured daily for 7 days after transfection. FIG. 22C shows that FLuc activity of mRNA and circular RNA accumulated over 7 days are shown.

[0142] FIGS. 23A-23C show circular RNA with 7.6-fold higher FLuc activity compared to mRNA (5 MoU) in vivo. FIG. 23A shows that FLuc activity was measured in mice treated with FLuc mRNA (5MoU) or circRNA FLuc encapsulated in lipid nanoparticles (LNPs) via in vivo luminescence images at each time point indicated for 9 days after administration. FIG. 23B shows that FLuc activity in each group (n=5) injected with FLuc mRNA (5MoU) or circRNA was measured by in vivo luminescence imaging at each indicated time point for 9 days after administration. FIG. 23C shows that FLuc activity of mRNA or circular RNA accumulated over 9 days was measured in each group.

Detailed Description

[0143] Provided herein are compositions and methods for producing circular RNA (circRNA). In some aspects, the compositions include nucleic acid molecules, such as RNA molecules, including linear RNA molecules, cleaved linear RNA molecules, and circRNA molecules. In some aspects, the provided RNA molecules comprise ribozymes, such as self-cleaving ribozymes, on either or both ends of the nucleic acid molecule. In some aspects, also provided are combinations of nucleic acid molecules, such as combination of RNA molecules, which include a first RNA molecule comprising a ribozyme, and a second RNA molecule comprising a trans-acting ribozyme. In some aspects, the ribozymes present in the provided RNA molecules or combinations thereof are cleaved, generating a cleaved linear RNA molecule. In some aspects, in the presence of a ligase, such as an RNA ligase, the cleaved linear RNA molecules are circularized, to generate circRNA. In some aspects, the cleaved RNA molecule is circularized in the presence of an endogenous ligase, for example, of a mammalian cell. In some aspects, the generated circRNA molecule can be used to deliver and express gene products encoded by an insert sequence present in the RNA molecule, in cells, such as mammalian cells, and subjects such as mammals or humans. In some aspects, the provided embodiments permit efficient generation of circRNA, particularly for large RNA molecules, and consistent and stable expression of the encoded gene product in cells or subjects.

[0144] Also provided are methods for producing the provided RNA molecules, combinations, intermediates, reaction products, and circRNA. In some contexts, the provided methods, including particular reaction conditions and components, result in increased efficiency and yield of the generated RNA molecules, combinations, intermediates, reaction products, and circRNA, and can lead to improved efficiency of generation of circRNA, improved purity and stability of the generated circRNA compositions, improved gene product expression, and/or reduced immunogenicity. Also provided are methods and uses of the provided RNA molecules, combinations, intermediates, reaction products, and circRNA, such as in therapeutic and prophylactic uses, such as in treating a disease or disorder or vaccination of a subject. The methods and uses provided herein may be applicable for treating various patients with different diseases, developing therapeutics, and/or developing vaccines against various diseases, including infectious diseases and cancers. Also provided are generated RNA molecules combinations, intermediates, reaction products, circRNA, and kits for use in generation of the RNA molecules and/or the methods provided herein.

[0145] In some aspects, reduced immunogenicity includes reduced detection by pattern recognition receptors. In some aspects, reduced immunogenicity includes reduced detection by TLRs, RIG-1, or MDA5.

[0146] In some aspects, the provided embodiments offer various advantages and improvements in producing RNA molecules for delivery of genes and gene products, for example gene products for therapy and/or vaccination, into a cell or a subject. In some aspects, the provided method and compositions for producing circRNA permit stable protein expression in a cell or a subject. circRNA is a single-stranded RNA that is resistant to exonuclease-mediated degradation. In some aspects, the structure of circRNA confers many benefits including extended half-life, enhanced gene product expression, functional stability, and reduced immunogenicity compared to linear mRNA. In some of the provided embodiments, circRNA is generated using self-cleaving ribozymes, including Twister ribozymes and Twister- Sister ribozymes.

[0147] In some embodiments, the circRNA shows or permits higher protein expression compared to a non-circular RNA counterpart. As used herein, a non-circular RNA counterpart is an non-circular RNA molecule that has substantially the same components (i.e. substantially the same promotor, coding region, and regulatory elements) as the circRNA that it is being compared to. The non-circular RNA counterpart may be an mRNA molecule. In some aspects, the circRNA show higher protein expression to the non-circular mRNA counterpart.

[01481 Existing approaches for generation of circRNA have various limitations. First, methods such permuted intron-exon (PIE) splicing has a critical limitation in purifying circRNA, because of the contamination by nicked circRNAs consistently produced during circularization reactions. Another problem with the existing approaches is that circRNA generation efficiently is reduced for larger insert sizes, indicating an inverse relationship between insert size and circRNA generation efficiency. Further, existing approaches such as the PIE splicing approach may not allow the use of modified nucleosides in the RNA molecule, such as modified uridines, without substantially affecting efficiency of circRNA generation. Accordingly, there is a need for methods and compositions that can be used to efficiently generate circRNA and to produce a pure composition of circRNA without contaminating species such as nicked circRNAs, and also to increase the efficiency of circRNA generation particularly for larger inserts. As demonstrated in the Examples herein, the provided embodiments meet such needs.

[0149] As observed and described in the Examples provided herein, exemplary molecules generated in accordance with the provided embodiments, result in efficient cleavage of the RNA molecules, efficient ligation of cleaved RNA molecules, and stable expression of the gene product in a cell that was introduced with various forms of the RNA molecules, including cleaved linear RNA or circRNA. In some aspects, the provided embodiments permit direct delivery of cleaved linear RNA molecules into cells, which can be circularized by an endogenous ligase present in a cell to generate circRNA in the cell. In some contexts, the provided embodiments could reduce the need for an additional ligation reaction to generate circRNA. In some aspects, the provided embodiments are based on an observation that exemplary molecules generated in accordance with the provided embodiments, result in improvements at each steps of cleavage, circularization and gene product expression, particularly for RNA molecules containing large inserts, and also lead to reduced immunogenicity of the RNA molecule, resulting in improved gene product expression.

[0150] The provided embodiments also lead to improved purity of the generated compositions containing circRNAs, reducing the proportion of linear RNAs or undesired species such as nicked circRNAs, and increasing the yield of circRNAs. In aspects, the provided RNA molecules were observed to reduce or eliminate undesired immune responses, for example, as evidenced by reduced activation of one or more toll-like receptors (TLRs), or evading detection by one or more TLRs when incubated with cells expressing TLRs. Tn addition, the provided embodiments lead to improved purity and improved efficiency for large insert sizes, eventually improving efficient translation and durable expression of gene products in cells, such as mammalian cells. Further, as described in the Examples, the provided embodiments permit the use of modified nucleosides such as pseudouridines in the RNA molecules, without substantially affecting efficiency of circRNA generation. The resulting RNA molecules, including circRNA molecules or compositions comprising the RNA molecules can be used in various therapeutic and prophylactic uses, including for example infectious disease vaccines, cancer vaccines, immunotherapy, gene therapy, gene editing and others.

[0151] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0152] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. NUCLEIC ACID MOLECULES FOR GENERATION OF CIRCULAR RNA (CircRNA)

[0153] Provided are nucleic acid molecules, such as RNA molecules, combinations thereof, reaction intermediates or reaction products thereof, including linear RNA, cleaved linear RNA and circular RNA (circRNA), and methods, kits and systems for generation of circRNA. In some aspects, the provided embodiments lead to improved efficiency in generation of circRNA and improved purity and stability of compositions comprising circRNAs, resulting in improved expression of the gene product (e.g., encoded by the insert present in the nucleic acid molecules) in a cell or a subject.

[0154] In some aspects, the provided embodiments include utilizing a unimolecular approach, e.g., using one RNA molecule that comprises two different ribozymes at the 5' and 3' ends, to generate circRNA.

[0155] In some aspects, the provided embodiments include utilizing a bimolecular approach, e.g., using two separate RNA molecules, one that comprises a 5' ribozyme and a 3' substrate sequence, and a separate RNA molecule that comprises a trans-acting ribozyme, to generate circRNA.

[01561 Iⁿ some aspects, also provided are methods, such as methods involving particular cleavage reactions and ligation/circularization reactions, for generating circRNA, and improving the efficiency of the steps for generating circRNA.

A. Circular RNA (CircRNA)

[0157] Provided herein are compositions, systems and methods for generating circular RNA (circRNA). CircRNA is a covalently closed continuous loop of single-stranded RNA. CircRNA can be divided into four categories including exonic circRNA (ecircRNA), circular intronic RNA (ciRNA), exon-intron circRNA (ElciRNA), and intergenic circRNA.

[0158] For delivery of RNA-encoded genes into cells and expression of the gene products, the stability of RNA is one of the critical factors in enhancing the gene product expression. Strategies have been developed to overcome the lack of stability and potential immunogenicity of linear mRNAs. Developed approaches include using untranslated regions (UTRs) such as those present in the native beta-globin mRNA, using methylguanosine cap analogs to protect the mRNA from decapping enzymes, nucleoside modification, and codon optimization. However, only modest improvements have been observed.

[0159] The configuration of circRNA results in several advantages compared to linear mRNA, including resistance to exonuclease-mediated degradation, increased stability, extended half-life, increased protein expression, and reduced immunogenicity (Chen, RNA Biol, 12(4):381 -388 (2015); Wesselhoeft et al., Nat Commun, 9(1):2629 (2018)). circRNA generally has a longer halflife compared to their linear mRNA counterparts (Wesselhoeft et al., Nat Commun, 9(1):2629 (2018)). Accordingly, circRNA improves protein expression (e.g., of the encoded gene product) over its lifetime compared to linear mRNAs.

[0160] Existing approaches to synthesize circRNA include generating a linear RNA precursor followed by ligation of 5' and 3' ends to produce a covalently closed loop. CircRNA can be synthesized in vitro by chemical, enzymatic, and ribozymatic approaches. One approach, using permuted intron-exon (PIE) splicing to result in RNA circularization, has been developed to express gene products from circRNA. PIE splicing systems based on Group I introns that are naturally found in the rRNA, tRNA, and mRNA genes of bacteria and non-metazoan eukaryotes can produce circRNA by self-splicing. However, this approach has a critical limitation in purifying circRNA, because of the contamination by nicked circRNAs consistently produced during circularization reactions. Because nicked circRNA and intact circRNA are equal in molecular weight, separating the two species by methods such as high performance liquid chromatography (HPLC) is very difficult. In addition, the nicked circRNAs induce immunogenicity when introduced to a subject, by stimulating innate immunity, and this frequently results in reduction in translation and production of the desired gene product in the subject. Also, another problem with the existing approaches is that circRNA generation efficiently is reduced for larger insert sizes, indicating an inverse relationship between insert size and circRNA generation efficiency. Further, existing approaches such as the PIE splicing approach may not allow the use of modified nucleosides in the RNA molecule, such as modified uridines, without substantially affecting efficiency of circRNA generation.

[0161] The provided embodiments include a unimolecular approach, e.g., using one RNA molecule that comprises two different ribozymes at the 5' and 3' ends. The provided embodiments also include a bimolecular approach, e g., using two separate RNA molecules, one that comprises a 5' ribozyme and a 3' substrate sequence, and a separate RNA molecule that comprises a trans-acting ribozyme, to generate circRNA.

B. Unimolecular Ribonucleic Acid (RNA) Approach

[0162] Provided herein are ribonucleic acid (RNA) molecules comprising one or more ribozyme sequences, for example at the 5' end and the 3' end of the molecule. In some aspects, provided are unimolecular RNA molecules, that comprise two ribozymes, one at the 5' end and one at the 3' end of the molecule. In some embodiments, the unimolecular RNA molecule is used to generate circular RNA (circRNA).

[0163] In some aspects, the unimolecular approach for generating circRNA involves generating one linear RNA molecule (in some aspects also referred to as a “precursor RNA”, before cleavage) that comprises two ribozymes, one at the 5' end and one at the 3' end of the molecule, allowing cleavage of the ribozymes at the 5' end and 3' end to generate a cleaved RNA molecule (in some aspects also referred to as a “pre-circRNA”), then allowing ligation of the ends of the cleaved RNA molecule to generate a circular RNA (circRNA) (see, e.g., FIG. 1A).

[0164] In some embodiments, the ribozyme sequences at the 5' end and the 3' end of the molecule are different. In some aspects, the unimolecular RNA molecule comprises a 5' Twister- Sister ribozyme. In some aspects, the unimolecular RNA molecule comprises a 3' Twister ribozyme. In some embodiments, the RNA molecule comprises an insert sequence encoding one or more exogenous molecules. Tn some embodiments, the RNA molecule comprises other sequences, such as one or more homology regions and/or one or more spacer sequences.

[01651 In some embodiments, the RNA molecule, for example the unimolecular RNA molecule, comprises, in 5' to 3' order: a 5' ribozyme comprising a Twister- Si st er ribozyme, an insert sequence, and a 3' ribozyme comprising a Twister ribozyme. In some embodiments, the RNA molecule, for example the unimolecular RNA molecule, comprises, in 5' to 3' order: a 5' ribozyme comprising a Twister- Sister ribozyme comprising a 5' substrate sequence, an insert sequence, and a 3' ribozyme comprising a Twister ribozyme comprising a 3' substrate sequence. In some embodiments, the 5' ribozyme comprises a 5' substrate sequence. In some embodiments, the 5' substrate sequence comprises a 5' overhang sequence. In some embodiments, the 3' ribozyme comprises a 3' substrate sequence. In some embodiments, the 3' substrate sequence comprises a 3' overhang sequence.

[0166] In some embodiments, the 5' ribozyme is capable of a cleavage reaction to cleave the 5' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence. In some embodiments, the 3' ribozyme is capable of a cleavage reaction to cleave the 3' substrate sequence to generate a cleaved RNA molecule comprising the 3' overhang sequence. In some embodiments, the 5' ribozyme and the 3' ribozyme are capable of a cleavage reaction to cleave the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence, the insert sequence and the 3' cleaved 3' sequence.

1. Twister-Sister Ribozyme

[0167] In some aspects, the provided RNA molecules comprise a Twister-Sister ribozyme on the 5' end. In some of any of the provided embodiments, the 5' ribozyme comprises a Twister-Sister ribozyme. In some of any of the provided embodiments, the 5' ribozyme is a Twister- Sister ribozyme. In some aspects, the provided RNA molecules comprise a 5' ribozyme comprising a Twister- Sister ribozyme comprising a 5' substrate sequence. In some aspects, the provided RNA molecule comprises a Twister- Sister ribozyme on the 5' end. In some aspects, the provided RNA molecule comprises a Twister-Sister substrate sequence on the 5' end. In some aspects, the provided RNA molecule comprises a Twister-Sister 5' overhang sequence on the 5' end. In some aspects, the provided RNA molecule comprises TS-1 ribozyme on the 5' end. In some aspects, the provided RNA molecule comprises TS-1 substrate sequence on the 5' end. In some aspects, the provided RNA molecule comprises TS-1 5' overhang sequence. In some aspects, the provided circRNA molecule comprises TS-1 5' overhang sequence. [0168] In some aspects, Twister-Sister ribozymes (also known as RNAs Associated with Genes Associated With Twister and Hammerhead-3 or RAGATH-3) are in a distinct class of self-cleaving catalytic RNA that have some similarities in sequence and secondary structure to the Twister ribozymes (see FIG. 12A; Roth et al. Nat Chem Biol, 10(1), 56-60 (2014); Weinberg et al., Nat Chem Bio, 11 (8):606-610 (2015)). The Twister- Sister ribozymes were identified based on a conserved RNA structural sequence search, and were identified as appearing near genetic elements described in pfam06252 and pfam09299 (Weinberg et al., (2015) Nat Chem Biol, 11(8), 606-610). The Twister-Sister ribozymes have Pl through P5 stems in an arrangement similar to that of Twister class of ribozymes, and similarities in the nucleotides in the P4 terminal loop. But, a pseudo-knot formation via Watson-Crick base-pairing, which occurs in all known Twister ribozyme, may be absent in Twister-Sister ribozymes. Further, there is poor correspondence among many of the most highly conserved nucleotides in each of the two motifs. Cleavage of Twister-Sister ribozymes is dependent on the presence of the catalytic RNA sequence and Mg²⁺, and is on the right side of the internal loop linking Pl and P2. The cleavage site between nucleotide C13 and A14 for the Twister- Sister ribozymes is on the opposite side relative to the internal loop cleaved by Twister (Weinberg et al., (2015) Nat Chem Biol, 11(8), 606-610).

[0169] Twister- Sister ribozymes mainly include two co-axial stacked helical sections connected by a three-way junction and two tertiary contacts (Liu et al., Nat Chem Biol, (2017), 13 (5): 508- 513). Five divalent metal ions are directly coordinated to RNA substrate, and the scissile phosphate lies in a quasi-helical loop region organized by a network of hydrogen bonding. A divalent metal ion is directly bound to the nucleobase 5' to the scissile phosphate, with an inner-sphere water molecule positioned to interact with the 02' nucleophile. The metal ion is thought to be the general base for the cleavage reaction, and the rate of ribozyme cleavage correlates in a log-linear manner with divalent metal ion pKa, consistent with proton transfer in the transition state (Liu et al., Nat Chem Biol, (2017), 13(5):508-513). In some contexts, the catalytic activity of Twister-Sister increases with pH and depends on divalent metal ion, such as Mg²⁺. In some contexts, nucleolytic ribozymes cleave a specific phosphodiester linkage by SN2 mechanism. The 02' acts as a nucleophile to attack the adjacent P, with 05' as a leaving group. The catalytic products comprise a cyclic 2', 3' phosphate and a 5'-hydroxyl (Ren et al., Curr Opin Chem Biol. 2017 Dec; 41 : 71-83). In some aspects, after cleavage of the Twister-Sister substrate sequence on the 5' end, the RNA molecule comprises a 5' hydroxyl group. [0170] Exemplary Twister- Sister ribozymes employed in any of the provided embodiments include a Twister-Sister- 1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, and a TS-4 ribozyme. Exemplary Twister- Sister ribozymes also include those described in, for example, Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015), Liu et al., Nat Chem Biol, (2017), 13(5):508- 513, and Ren et al., Curr Opin Chem Biol. 2017 Dec; 41 : 71-83. The TS-1 ribozyme, engineered based on a microbial metagenomic DNA source, comprises biomolecular strands including a 5' enzyme strand and a 3' substrate strand and P1-P5 stem regions (see FIG. 12B; Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015)). In some embodiments, the Twister- Sister ribozyme comprises TS-1. In some embodiments, the Twister-Sister ribozyme comprises TS-2. In some embodiments, the Twister-Sister ribozyme comprises TS-3. In some embodiments, the Twister-Sister ribozyme comprises TS-4.

[0171] In some embodiments, the RNA molecule comprises an overhang sequence of a Twister- Sister ribozyme. In some embodiments, the 5' ribozyme comprises an overhang sequence of a Twister-Sister ribozyme. In some embodiments, the RNA molecule comprises an overhang sequence of TS-1. In some embodiments, the 5' ribozyme comprises an overhang sequence of TS-1.

[0172] In some aspects, the “overhang sequence” (in some cases also called “cleaved sequence”) refers to the portion of the substrate sequence that remain attached to the RNA molecule comprising the insert sequence (“cleaved RNA” or “pre-circRNA” after cleavage), after cleavage by the ribozyme. In some aspects, as used herein, an overhang sequence is a part of a substrate sequence, and a substrate sequence comprises an overhang sequence.

[0173] In some embodiments, the RNA molecules provided herein comprise a 5’ overhang sequence and a 3' overhang sequence. In some embodiments, the 5' overhang sequence is between 2 and 15 nucleotides (nt) in length, such as between 3 and 10 nt, between 4 and 8 nt, or about 3, 4, 5, 6, 7, 8, 9, or 10 nt in length. In some embodiments, 5' overhang sequence is less than 10 nt in length. In some embodiments, the 5' overhang sequence is 6 nt in length. In some embodiments, the 5' overhang sequence and the 3' overhang sequence together can form a loop structure recognized by an RNA ligase such as RtcB.

[0174] In some embodiments, the 5' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:6. In some embodiments, the 5' ribozyme comprises SEQ ID NO:6. In some embodiments, the 5' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:5. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO: 5.

[01751 Iⁿ some embodiments, the 5' overhang sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:6. In some embodiments, the 5' overhang sequence comprises SEQ ID NO:6. In some embodiments, the 5’ overhang sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 5. In some embodiments, the 5' overhang sequence comprises a sequence encoded by SEQ ID NO:5.

[0176] In some embodiments, the RNA molecule comprises the substrate sequence of a 5' ribozyme. In some embodiments, the 5' ribozyme comprises the substrate sequence of a 5' ribozyme. In some embodiments, the RNA molecule comprises the substrate sequence of TS-1. In some embodiments, the 5' ribozyme comprises the substrate sequence of TS-1.

[0177] In some aspects, the “substrate sequence” refers to the portion of the ribozyme sequence that is cleaved by the catalytic portion of the ribozyme. In some aspects, before cleavage, the substrate sequence of the ribozyme can be contained in the same RNA molecule as the catalytic sequence of the ribozyme (unimolecular or cz -acting ribozyme). In some aspects, before cleavage, the substrate sequence of the ribozyme can be contained in a distinct RNA molecule compared to the catalytic sequence of the ribozyme (bimolecular or /ra/rs-acting ribozyme). In some aspects, the substrate sequence comprises an overhang sequence which remains attached to the cleaved RNA molecule comprising the insert sequence, after cleavage by the ribozyme.

[0178] In some embodiments, the 5' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:45. In some embodiments, the 5' ribozyme comprises SEQ ID NO:45. In some embodiments, the 5' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:44. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO: 44.

[0179] In some embodiments, the 5' substrate sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:45. In some embodiments, the 5' substrate sequence comprises SEQ ID NO:45. In some embodiments, the 5' substrate sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:44. In some embodiments, the 5' substrate sequence comprises a sequence encoded by SEQ ID NO:44.

[0180] In some embodiments, the 5' ribozyme comprises the catalytic sequence of a 5' ribozyme. In some embodiments, the 5' ribozyme comprises the catalytic sequence of TS-1.

[0181] In some aspects, the “catalytic sequence” refers to the portion of the ribozyme sequence that catalyzes the cleavage of the substrate sequence. In some aspects, the catalytic sequence cleaves the substrate sequence at or after the overhang sequence such that after cleavage, the overhang sequence remains attached to the cleaved RNA molecule comprising the insert sequence, after cleavage by the ribozyme.

[0182] In some embodiments, the 5' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the 5' ribozyme comprises SEQ ID NO:4. In some embodiments, the 5' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:3.

[0183] In some embodiments, the 5' Twister- Sister catalytic sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the 5' Twister-Sister catalytic sequence comprises SEQ ID NO:4. In some embodiments, the 5' Twister- Si st er catalytic sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the 5' Twister-Sister catalytic sequence comprises a sequence encoded by SEQ ID NO:3.

[0184] In some embodiments, the Twister-Sister ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the Twister- Sister ribozyme comprises SEQ ID NO:4. In some embodiments, the Twister-Sister ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the Twister- Sister ribozyme comprises a sequence encoded by SEQ ID NO:3.

[0185] In some embodiments, the 5' ribozyme comprises the catalytic sequence and substrate sequence of a Twister- Sister ribozyme. In some embodiments, the 5' ribozyme comprises the catalytic sequence and substrate sequence of TS-1.

[0186] In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:47. In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) comprises SEQ ID NO:47. In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:46. In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) comprises a sequence encoded by SEQ ID NO: 46.

2. Twister Ribozyme

[0187] In some aspects, the provided RNA molecules, for example a unimolecular RNA molecule, comprise a Twister ribozyme on the 3' end. In some of any of the provided embodiments, the 3' ribozyme comprises a Twister ribozyme. In some of any of the provided embodiments, the 3' ribozyme is a Twister ribozyme. In some aspects, the provided RNA molecules comprise a 3' ribozyme comprising a Twister ribozyme comprising a 3' substrate sequence. In some aspects, the provided RNA molecule comprises a Twister ribozyme on the 3' end. In some aspects, the provided RNA molecule comprises a Twister substrate sequence on the 3' end. In some aspects, the provided RNA molecule comprises a Twister 3' overhang sequence on the 3' end. In some aspects, the provided RNA molecule comprises Nasonia vitripennis type Pl Twister ribozyme on the 3' end. In some aspects, the provided RNA molecule comprises Nasonia vitripennis type Pl Twister substrate sequence on the 3' end. In some aspects, the provided RNA molecule comprises Nasonia vitripennis type Pl Twister 3' overhang sequence. In some aspects, the provided circRNA molecule comprises Nasonia vitripennis type Pl Twister 3' overhang sequence.

[0188] In some aspects, Twister ribozymes are in a class of self-cleaving catalytic RNA with a doubly pseudo-knotted RNA structure (see FIG. 12A; Roth et al. Nat Chem Biol, 10(1), 56-60 (2014); Weinberg et al., Nat Chem Bio, 11 (8):606-610 (2015)). The Twister ribozymes were identified based from a small, highly conserved RNA motif that occurs commonly in the bacterial class Clostridia as well as in diverse eukaryotic species (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)), with a consensus secondary structure that exhibits three stems cojoined by internal and terminal loops. In certain instances, examples of the twister motif are circularly permuted, with the termini for a given representative contained within stem Pl (type Pl), stem P3 (type P3) or stem P5 (type P5) (see FIG. 12A). Cleavage of Twister ribozymes is dependent on the presence of the catalytic RNA sequence and Mg²⁺ (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)), and cleavage is by internal phosphoester transfer from attack of the 2' oxygen of U5 on the adjacent phosphorus atom, with subsequent departure of the 5' oxygen of A6 (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)). In some contexts, the catalytic activity of Twister increases with pH and depends on divalent metal ion, such as Mg²⁺. In some contexts, nucleolytic ribozymes cleave a specific phosphodiester linkage by SN2 mechanism. Cleavage for Twister ribozyme is based on a general- acid-base catalysis involving highly conserved adenine (Al) and guanine (G33) bases, where N3 of Al acts as a proton donor and G33 the general base. The catalytic products comprise a cyclic 2', 3' phosphate and a 5'-hydroxyl (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)). In some aspects, after cleavage of the Twister substrate sequence on the 3' end, the RNA molecule comprises a cyclic 2', 3' phosphate at the 3' end.

[0189] Exemplary Twister ribozymes employed in any of the provided embodiments include those described in, for example, Roth et al. Nat Chem Biol, 10(1), 56-60 (2014), Liu et al., Nat Chem Biol. 10 (9): 739-744 (2014), and Eiler et al., Proceedings of the National Academy of Sciences. I l l (36): 13028-13033 (2014). Nearly 2,700 Twister ribozymes have been identified with a shared consensus secondary structure that exhibits three stems cojoined by internal and terminal loops. Exemplary Twister ribozymes include type Pl Twister ribozymes, type P3 Twister ribozymes, and type P5 Twister ribozymes. Exemplary Twister ribozymes also include Twister ribozymes from Nasonia vitripennis, Clostridium bolteae, Oryza Sativa, Schistosoma mansoni Twister env22, and Twister env9. In some embodiments, the Twister ribozyme comprises N. vitripennis type Pl Twister ribozyme. In some embodiments, the Twister ribozyme comprises a C. bolteae Twister ribozyme. In some embodiments, the Twister ribozyme comprises a O. Sativa Twister ribozyme. In some embodiments, the Twister ribozyme comprises a S. mansoni Twister ribozyme.

[0190] In some embodiments, the RNA molecule comprises an overhang sequence of a Twister ribozyme. In some embodiments, the 3' ribozyme comprises an overhang sequence of a Twister ribozyme. In some embodiments, the RNA molecule comprises an overhang sequence of N. vitripennis type Pl Twister ribozyme. In some embodiments, the 3' ribozyme comprises an overhang sequence of N. vitripennis type Pl Twister ribozyme.

[0191] In some embodiments, the RNA molecules provided herein comprise a 5' overhang sequence and a 3' overhang sequence. In some embodiments, the 3' overhang sequence is between 2 and 15 nucleotides (nt) in length, such as between 3 and 10 nt, between 4 and 8 nt, or about 3, 4, 5, 6, 7, 8, 9, or 10 nt in length. Tn some embodiments, 3' overhang sequence is less than 10 nt in length. In some embodiments, the 3' overhang sequence is 4 nt in length. In some embodiments, the 5' overhang sequence and the 3' overhang sequence together can form a loop structure recognized by an RNA ligase such as RtcB.

[0192] In some embodiments, the 3' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:21. In some embodiments, the 3' ribozyme comprises SEQ ID NO:21. In some embodiments, the 3' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:20. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO:20.

[0193] In some embodiments, the 3' overhang sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:21. In some embodiments, the 3' overhang sequence comprises SEQ ID NO:21. In some embodiments, the 3' overhang sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:20. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO:20.

[0194] In some embodiments, the 3' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:52. In some embodiments, the 3' ribozyme comprises SEQ ID NO:52. In some embodiments, the 3' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:51. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO : 51.

[0195] In some embodiments, the 3' overhang sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:52. In some embodiments, the 3' overhang sequence comprises SEQ ID NO:52. In some embodiments, the 3' overhang sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:51. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO:51.

[0196] In some embodiments, the RNA molecule comprises the substrate sequence of a 3' ribozyme. In some embodiments, the 3' ribozyme comprises the substrate sequence of a 3' ribozyme. In some embodiments, the RNA molecule comprises the substrate sequence of N. vitripennis type Pl Twister ribozyme. In some embodiments, the 3' ribozyme comprises the substrate sequence of N. vitripennis type Pl Twister ribozyme.

[01971 Iⁿ some aspects, before cleavage, the substrate sequence of the ribozyme can be contained in the same RNA molecule as the catalytic sequence of the ribozyme (unimolecular or cz -acting ribozyme). In some aspects, before cleavage, the substrate sequence of the ribozyme can be contained in a distinct RNA molecule compared to the catalytic sequence of the ribozyme (bimolecular or Zra/zs-acting ribozyme). In some aspects, the substrate sequence comprises an overhang sequence which remains attached to the cleaved RNA molecule comprising the insert sequence, after cleavage by the ribozyme.

[0198] In some embodiments, the 3' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:33. In some embodiments, the 3' ribozyme comprises SEQ ID NO:33. In some embodiments, the 3' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:32. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO:32.

[0199] In some embodiments, the 3' substrate sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:33. In some embodiments, the 3' substrate sequence comprises SEQ ID NO:33. In some embodiments, the 3' substrate sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:32. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO:32.

[02001 Iⁿ some embodiments, the 3' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:31. In some embodiments, the 3' ribozyme is comprises SEQ ID NO:31. In some embodiments, the 3' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:30. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO: 30.

[0201] In some embodiments, the 3' substrate sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:31. In some embodiments, the 3' substrate sequence comprises SEQ ID NO:31. In some embodiments, the 3' substrate sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:30. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO:30.

[02021 Iⁿ some embodiments, the 3' ribozyme comprises the catalytic sequence of a Twister ribozyme. In some embodiments, the 3' ribozyme comprises the catalytic sequence of N. vitripennis type Pl Twister ribozyme. In some aspects, in a unimolecular approach (for example, as described in Section I.B herein), the catalytic sequence is comprised in the same molecule (e.g., unimolecular RNA) as the substrate sequence of the same ribozyme. In some aspects, in a bimolecular approach (for example, as described in Section I.C herein), the catalytic sequence is comprised in a separate RNA molecule as the substrate sequence for the ribozyme.

[0203] In some embodiments, the 3' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:23. In some embodiments, the 3' ribozyme comprises SEQ ID NO:23. In some embodiments, the 3' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:22. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO:22.

[0204] In some embodiments, the 3' Twister catalytic sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:23. In some embodiments, the 3' Twister catalytic sequence comprises SEQ ID NO:23. In some embodiments, the 3' Twister catalytic sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:22. In some embodiments, the 3' Twister catalytic sequence comprises a sequence encoded by SEQ ID NO:22.

[02051 Iⁿ some embodiments, the Twister ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:23. In some embodiments, the Twister ribozyme comprises SEQ ID NO:23. In some embodiments, the Twister ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:22. In some embodiments, the Twister ribozyme comprises a sequence encoded by SEQ ID NO:22.

[0206] In some embodiments, the 3' ribozyme comprises the catalytic sequence and substrate sequence of a Twister ribozyme. In some embodiments, the 3' ribozyme comprises the catalytic sequence and substrate sequence of N. vitripennis type Pl Twister ribozyme.

[0207] In some embodiments, the 3' ribozyme (including the catalytic sequence and the substrate sequence) comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:47. In some embodiments, the 3' ribozyme (including the catalytic sequence and the substrate sequence) comprises SEQ ID NO:47. In some embodiments, the 3' ribozyme (including the catalytic sequence and the substrate sequence) is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:46. In some embodiments, the 3' ribozyme (including the catalytic sequence and the substrate sequence) comprises a sequence encoded by SEQ ID NO: 46.

3. Exemplary Molecules

[0208] In some embodiments, an exemplary unimolecular RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises in 5' to 3' order: a 5' coverage sequence, a 5' Twister-Sister ribozyme catalytic sequence, a 5' Twister- Sister ribozyme substrate sequence (including the 5' overhang sequence), a 5' homology region, a 5' spacer sequence, an IRES sequence, an exogenous molecule coding sequence, a poly AC sequence, a 3' homology region, a 3' Twister ribozyme substrate sequence (including the 3' overhang sequence), a 3' Twister ribozyme catalytic sequence, and a 3' coverage sequence.

[0209] In some embodiments, an exemplary unimolecular RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises in 5' to 3' order: a 5' coverage sequence, a 5' TS-1 catalytic sequence, a 5' TS-1 substrate sequence (including the 5' overhang sequence), a 5' homology region, a 5' spacer sequence, a Coxsackievirus B3 IRES sequence, a Firefly luciferase (FLuc) coding sequence, a poly AC sequence, a 3' homology region, a 3' N. vitripennis type Pl Twister substrate sequence (including the 3' overhang sequence), a 3' A. vitripennis type Pl Twister catalytic sequence, and a 3' coverage sequence.

[0210] In some embodiments, the provided unimolecular RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:29. In some embodiments, the unimolecular RNA molecule, before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO:29. In some aspects, provided are exemplary RNA molecules designated Construct U1 in the Examples provided herein. In some embodiments, the unimolecular RNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO:28. In some embodiments, the unimolecular RNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO:28.

C. Bimolecular Ribonucleic Acid (RNA) Approach and Combinations

[0211] Provided herein are ribonucleic acid (RNA) molecules for bimolecular approaches for circRNA generation, combinations of RNA molecules and systems for generating circRNA. In some aspects, the bimolecular RNA molecules comprise two different molecules, which act together to cleave the 5' and 3' ends of one of the RNA molecules for circRNA generation. In some aspects, the combinations or systems of RNA molecules for bimolecular circRNA generation comprise: a first RNA molecule that comprises a 5' ribozyme and a substrate sequence of a ribozyme at the 3' end; and a second RNA molecule comprising a trans-acting ribozyme that can cleave the substrate sequence present at the 3' end of the first RNA molecule. In some aspects of the bimolecular approach, the catalytic sequence of the ribozyme that cleaves the 3' substrate sequence on the first RNA molecule is present as a distinct, second RNA molecule that comprises a transacting ribozyme.

[0212] In some aspects, the bimolecular approach for generating circRNA involves generating a first RNA molecule which is a linear RNA molecule (in some aspects also referred to as a “precursor RNA”, before cleavage) that comprises the cleavage and substrate sequences of one ribozyme at the 5' end, and only the substrate sequence of a ribozyme at the 3' end. A second RNA molecule comprises a trans-acting ribozyme that is capable of cleaving the 3' substrate sequence of the first RNA molecule. The 5' ribozyme at the 5' end of the first RNA molecule is also cleaved, to generate a cleaved RNA molecule (in some aspects also referred to as a “pre-circRNA”), then ligation of the ends of the cleaved RNA molecule generates a circular RNA (circRNA) (see, e.g., FIG. IB)

[0213] In some embodiments, the ribozyme sequences at the 5' end of the first RNA molecule and the trans-acting ribozyme are different. In some aspects, the first RNA molecule comprises a 5' Twister- Sister ribozyme, such as any Twister-Sister ribozyme described in Section I.B.l herein. In some aspects, the second RNA molecule comprises the catalytic sequence of a trans-acting Twister ribozyme, such as any Twister ribozyme described in Section I.B.2 herein. In some embodiments, the first RNA molecule comprises an insert sequence encoding one or more exogenous molecules. In some embodiments, the first RNA molecule comprises other sequences, such as one or more homology regions and/or one or more spacer sequences.

[0214] In some aspects, bimolecular approach of circRNA generation utilizes a trans-acting ribozyme comprising the catalytic sequence, which cleaves the substrate sequence at the 3' end of the first RNA molecule at or after the overhang sequence such that after cleavage, the overhang sequence remains attached to the cleaved RNA molecule comprising the insert sequence, after cleavage by the trans-acting ribozyme.

[0215] In some aspects, the second RNA molecule comprises a trans-acting ribozyme. In some aspects, the trans-acting ribozyme is or comprises a Twister ribozyme.

[0216] Also provided herein is a combination of RNA molecules. In some embodiments, the combination comprises a first RNA molecule, such as any first RNA molecule described herein, for example, in Section I.C.l, and a second RNA molecule, such as any second RNA molecule described herein, for example, in Section I.C.2. In some embodiments, the combination comprises a plurality of first RNA molecules and a plurality of second RNA molecules.

1. First RNA Molecule - 5 ' Ribozyme and 3 ' Substrate Sequence

[0217] In some embodiments, the first RNA molecule comprises a 5' ribozyme comprising a 5' substrate sequence near the 5' end and a 3' substrate sequence of a ribozyme near the 3' end.

[0218] In some of any of the provided embodiments, the 5' ribozyme of the first RNA molecule comprises a Twister- Sister ribozyme. In some of any of the provided embodiments, the 5' ribozyme of the first RNA molecule is a Twister- Sister ribozyme. In some of any of the provided embodiments, the 3' substrate sequence of the first RNA molecule comprises a substrate sequence of a Twister ribozyme. In some of any of the provided embodiments, the 3' substrate sequence of the first RNA molecule is a substrate sequence of a Twister ribozyme.

[0219] In some embodiments, the first RNA molecule comprises a 5' Twister- Sister ribozyme near the 5' end and a 3' substrate sequence of a Twister ribozyme near the 3' end. In some aspects, the first RNA molecule comprises an insert sequence flanked on the 5' side by a 5' ribozyme, and on the 3' side by a 3' substrate sequence. In some aspects, the 3' substrate sequence of the first RNA molecule is cleaved in the presence of a trans-acting ribozyme, such as a trans-acting ribozyme of the second RNA molecule. In some embodiments, the trans-acting ribozyme is a Twister ribozyme. In some embodiments, the first RNA molecule and the second RNA molecule in combination generates a cleaved RNA molecule, in which the 5' substrate sequence and the 3' substrate sequence are cleaved. In some aspects, after cleavage, the cleaved RNA molecule comprises a 5' overhang sequence, an insert and a 3' overhang sequence.

[0220] In some embodiments, the 5' Twister-Sister ribozyme is or comprises any of the Twister- Sister ribozymes described herein, for example, in Section I.B.l . In some embodiments, the 5' Twister- Sister ribozyme is or comprises a TS-1 ribozyme. In some aspects, the first RNA molecule is generally similar in structure as the unimolecular precursor RNA molecule in general, with the exception that at the 3' end, only the substrate sequence of the 3' ribozyme and not the catalytic sequence of the 3' ribozyme is present in the same molecule. In the bimolecular approach, the catalytic sequence of the ribozyme that cleaves the 3' substrate is present in the second RNA molecule as a trans-acting ribozyme.

[0221] In some embodiments, the 5' ribozyme comprises an overhang sequence. In some embodiments, the 5' ribozyme comprises a 5' overhang sequence of a TS-1 ribozyme. In some embodiments, the 5' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:6. In some embodiments, the 5' ribozyme comprises SEQ ID NO:6. In some embodiments, the 5' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:5. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:5.

[0222] In some embodiments, the 5' ribozyme comprises a substrate sequence. In some embodiments, the 5' ribozyme comprises a 5' substrate sequence of a TS-1 ribozyme. In some embodiments, the 5' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:45. In some embodiments, the 5' ribozyme comprises SEQ ID NO:45. In some embodiments, the 5' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:44. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:44.

[0223] In some embodiments, the 5' ribozyme comprises a catalytic sequence. In some embodiments, the 5' ribozyme comprises a catalytic sequence of a TS-1 ribozyme. In some embodiments, the 5' ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the 5' ribozyme comprises SEQ ID NO:4. In some embodiments, the 5' ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:3 [0224] In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:47. In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) comprises SEQ ID NO:47. In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:46. In some embodiments, the 5' ribozyme (including the catalytic sequence and the substrate sequence) comprises a sequence encoded by SEQ ID NO: 46.

[0225] In some embodiments, the first RNA molecule comprises a 3' overhang sequence of a ribozyme. In some aspects, the 3' overhang sequence is or comprises a overhang sequence of a Twister ribozyme. In some embodiments, the Twister ribozyme is or comprises any of the Twister ribozymes described herein, for example, in Section I B.2. In some embodiments, the 3' overhang sequence is a Twister overhang sequence of N. vitripennis type Pl Twister ribozyme. In some embodiments, the first RNA molecule comprises a 3' overhang sequence of N. vitripennis type Pl Twister ribozyme. In some aspects, after cleavage of the 3' substrate sequence present in the first RNA molecule by the trans-acting ribozyme of the second RNA molecule, the 3' overhang sequence remains as part of the cleaved RNA molecule which comprises an insert sequence.

[0226] In some embodiments, the 3' overhang sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:21. In some embodiments, the 3' overhang sequence comprises SEQ ID NO:21. In some embodiments, the 3' overhang sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:20. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO: 20.

[0227] In some embodiments, the 3' overhang sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:52. In some embodiments, the 3' overhang sequence comprises SEQ ID NO:52. In some embodiments, the 3' overhang sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:51. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO:51.

[0228] In some embodiments, the first RNA molecule comprises a 3' substrate sequence of a ribozyme. In some aspects, the 3' substrate sequence is or comprises a substrate sequence of a Twister ribozyme. In some embodiments, the Twister ribozyme is or comprises any of the Twister ribozymes described herein, for example, in Section I.B.2. In some embodiments, the 3' substrate sequence is a Twister substrate sequence of N. vitripennis type Pl Twister ribozyme. In some embodiments, the first RNA molecule comprises a 3' substrate sequence of N. vitripennis type Pl Twister ribozyme. In some aspects, a 3' overhang sequence is a portion of a 3' substrate sequence, and after cleavage of the 3' substrate sequence present in the first RNA molecule by the trans-acting ribozyme of the second RNA molecule, the 3 ' overhang sequence remains as part of the cleaved RNA molecule which comprises an insert sequence.

[0229] In some embodiments, the first RNA molecule comprises a 3' substrate sequence at the 3' end. In some aspects, the 3' substrate sequence is or comprises a substrate sequence of a Twister ribozyme. In some embodiments, the first RNA molecule comprises a 3' substrate sequence of N. vitripennis type Pl Twister ribozyme.

[0230] In some embodiments, the 3' substrate sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:33. In some embodiments, the 3' substrate sequence comprises SEQ ID NO:33. In some embodiments, the 3' substrate sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:32. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO:32.

[0231] In some embodiments, the 3' substrate sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:31. In some embodiments, the 3' substrate sequence comprises SEQ ID NO:31. In some embodiments, the 3' substrate sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:30. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO:30.

2. Second RNA Molecule — Trans-acting Ribozyme

[0232] In some embodiments, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a ribozyme. In some embodiments, the trans-acting ribozyme of the second RNA molecule comprises a Twister ribozyme. In some embodiments, the trans-acting ribozyme of the second RNA molecule is a Twister ribozyme. In some embodiments, the second RNA molecule comprises the catalytic sequence of a Twister ribozyme. In some aspects, the 3' substrate sequence of the first RNA molecule is cleaved in the presence of the trans-acting ribozyme of the second RNA molecule.

[0233] In some embodiments, the second RNA molecule comprises a trans-acting ribozyme. In some embodiments, the trans-acting ribozyme comprises a Twister ribozyme, such as any Twister ribozyme described herein, for example in Section I.B.2. In some aspects, the trans-acting ribozyme comprises a catalytic sequence from a Twister ribozyme.

[0234] In some embodiments, the Twister catalytic sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:23. In some embodiments, the Twister catalytic sequence comprises SEQ ID NO:23. In some embodiments, the Twister catalytic sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:22. In some embodiments, the Twister catalytic sequence comprises a sequence encoded by SEQ ID NO:22.

[0235] In some embodiments, the Twister catalytic sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:35. In some embodiments, the Twister catalytic sequence comprises SEQ ID NO:35. In some embodiments, the Twister catalytic sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:34. In some embodiments, the Twister catalytic sequence comprises a sequence encoded by SEQ ID NO:34.

[0236] In some embodiments, the trans-acting ribozyme comprises the catalytic sequence of a Twister ribozyme. In some embodiments, the trans-acting ribozyme comprises the catalytic sequence of N. vitripennis type Pl Twister ribozyme. In some aspects, in a bimolecular approach (for example, as described in Section I.C herein), the catalytic sequence is comprised in a separate molecule (e.g., second RNA molecule) compared to the substrate sequence of the same ribozyme, which is comprised at the 3 ' end of the first RNA molecule.

[0237] In some embodiments, the trans-acting ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:23. In some embodiments, the trans-acting ribozyme comprises SEQ ID NO:23. In some embodiments, the trans-acting ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:22. In some embodiments, the transacting ribozyme comprises a sequence encoded by SEQ ID NO:22.

[0238] In some aspects, the trans-acting ribozyme comprises one or more non-native guanosine residues at the 5' end. In some aspects, the trans-acting ribozyme comprises two non-native guanosine residues at the 5' end. In some embodiments, the trans-acting ribozyme comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:35. In some embodiments, the trans-acting ribozyme comprises SEQ ID NO:35. In some embodiments, the trans-acting ribozyme is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:34. In some embodiments, the trans-acting ribozyme comprises a sequence encoded by SEQ ID NO:34. In some aspects, triphosphate is present on the 5' end of the trans-acting ribozyme or the second RNA molecule.

3. Exemplary Molecules

[0239] In some embodiments, an exemplary combination of RNA molecules for generating a circRNA for the bimolecular approach, comprises a first RNA molecule that comprises a 5' ribozyme and a substrate sequence of a ribozyme at the 3' end; and a second RNA molecule comprising a trans-acting ribozyme that can cleave the substrate sequence present at the 3' end of the first RNA molecule. In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises in 5’ to 3' order: a 5' coverage sequence, a 5' Twister-Sister ribozyme catalytic sequence, a 5' Twister- Sister ribozyme substrate sequence (including the 5' overhang sequence), a 5' homology region, a 5' spacer sequence, an IRES sequence, an exogenous molecule coding sequence, a poly AC sequence, a 3' homology region, and a 3' Twister ribozyme substrate sequence (including the 3' overhang sequence). In some aspects, an exemplary second RNA molecule comprises a Twister ribozyme catalytic sequence.

[0240] In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e g., precursor RNA molecule, comprises in 5' to 3' order: a 5' coverage sequence, a 5' TS-1 catalytic sequence, a 5' TS-1 substrate sequence (including the 5' overhang sequence), a 5' homology region, a 5' spacer sequence, a Coxsackievirus B3 IRES sequence, a Firefly luciferase (FLuc) coding sequence, a polyAC sequence, a 3' homology region, and a 3' N. vitripennis type Pl Twister substrate sequence (including the 3' overhang sequence). In some aspects, an exemplary second RNA molecule comprises a N. vitripennis type Pl Twister catalytic sequence.

[0241] In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:37. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO:37. In some aspects, provided are exemplary first RNA molecules designated Construct Bl in the Examples provided herein. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO:36. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO:36.

[0242] In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 109. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 109. In some aspects, provided are exemplary first RNA molecules designated LVT-18 in the Examples provided herein. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 108. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 108.

[0243] In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:111. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 111. In some aspects, provided are exemplary first RNA molecules designated LVT-20 in the Examples provided herein. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 110. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 110.

[0244] In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:113. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 113. In some aspects, provided are exemplary first RNA molecules designated LVT-22 in the Examples provided herein. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 112. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 112.

[0245] In some aspects, an exemplary first RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:115. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 115. In some aspects, provided are exemplary first RNA molecules designated LVT14/eIF4G in the Examples provided herein. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 114. In some embodiments, the first DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 114.

[0246] In some aspects, an exemplary second RNA molecule comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:23. In some embodiments, the second RNA molecule comprises SEQ ID NO:23. In some aspects, provided are exemplary second RNA molecules designated Construct Bl in the Examples provided herein. In some embodiments, the second RNA molecule comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO:22. In some embodiments, the second RNA molecule comprises a sequence encoded by SEQ ID NO:22.

[0247] In some aspects, an exemplary second RNA molecule comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:35. In some embodiments, the second RNA molecule comprises SEQ ID NO:35. In some aspects, provided are exemplary second RNA molecules designated Construct Bl in the Examples provided herein. In some embodiments, the second RNA molecule comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO:34. In some embodiments, the second RNA molecule comprises a sequence encoded by SEQ ID NO:34.

[02481 Iⁿ some aspects, an exemplary second RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 109. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 109. In some aspects, provided are exemplary second RNA molecules designated LVT-18 in the Examples provided herein. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 108. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 108.

[0249] In some aspects, an exemplary second RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:111. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 111. In some aspects, provided are exemplary second RNA molecules designated LVT-20 in the Examples provided herein. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 110. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 110.

[0250] In some aspects, an exemplary second RNA molecule before cleavage of the ribozyme substrate sequences, e g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 113. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 113. In some aspects, provided are exemplary second RNA molecules designated LVT-22 in the Examples provided herein. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 112. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 112.

[0251] In some aspects, an exemplary second RNA molecule before cleavage of the ribozyme substrate sequences, e.g., precursor RNA molecule, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:115. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequences, comprises SEQ ID NO: 115. In some aspects, provided are exemplary second RNA molecules designated LVT14/eIF4G in the Examples provided herein. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO: 114. In some embodiments, the second DNA molecule, before cleavage of the ribozyme substrate sequences, comprises a sequence encoded by SEQ ID NO: 114.

D. Other Sequences

[0252] In some embodiments, the RNA molecules, including the unimolecular RNA precursor molecule, first RNA molecule, second RNA molecule, cleaved RNA molecule (pre-circRNA) or a circRNA molecule, provided herein further comprise additional sequences and/or additional components. In some aspects, exemplary configuration and designation of the sequences present in the RNA molecules are depicted in FIG. 1A (unimolecular approach) and FIG. IB (bimolecular approach).

1. Homology Regions, Coverage Sequence and Extension Sequence

[0253] In some embodiments, the RNA molecule further comprises a 5' homology region and a 3' homology region. In some aspects, the 5' homology region and the 3' homology region are capable of binding to or hybridizing to each other to form a stem structure (see FIGS. 1A and IB). In some aspects, the sequences of the 5' homology region and the sequences of the 3' homology region are at least partially complementary to each other. In some aspects, the sequences of the 5' homology region and the sequences of the 3' homology region contain sequences that are complementary to each other.

[0254] In some aspects, in a cleaved RNA molecule, the 5' homology region and 3' homology region is capable of forming a stem structure. In some contexts, in a cleaved RNA molecule (i.e., pre-circRNA), the binding or hybridization of the 5' homology region and 3' homology region with each other (e g., the formation of the stem structure) can facilitate bringing the hydroxyl group at the 5' terminus and the 2'3'-cyclic phosphate at the 3' terminus of the cleaved RNA molecule in proximity to each other, for the ligase to ligate the two termini to generate a circRNA. In some aspects, in the precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) or a cleaved RNA molecule, the 5' homology region is located 3' of the 5' ribozyme. In some aspects, in the precursor RNA molecule or a cleaved RNA molecule, the 3 ' homology region is located 3 ' of the insert sequence. In some aspects, in a circRNA molecule, the 5' homology region and 3' homology region are still present. In some aspects, the homology region is between 5 and 50 nucleotides (nt) in length, such as between 8 and 45 nt, between 10 and 30 nt, between 14 and 25 nt, between 15 and 20 nt, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nt in length. In some aspects, the homology region is 19 nt in length.

[0255] In some embodiments, the 5' homology region comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 10. In some embodiments, the 5' homology region comprises SEQ ID NO: 10. In some embodiments, the 5' homology region is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:9. In some embodiments, the 5' homology region comprises a sequence encoded by SEQ ID NO:9.

[0256] In some embodiments, the 3' homology region comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 19. In some embodiments, the 3' homology region comprises SEQ ID NO: 19. In some embodiments, the 3' homology region is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 18. In some embodiments, the 3' homology region comprises a sequence encoded by SEQ ID NO: 18.

[0257] In some aspects, a precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) comprises a 5' coverage sequence and/or a 3' coverage sequence. In some aspects, a precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) comprises a 5' coverage sequence. In some aspects, a precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) comprises a 3' coverage sequence. In some aspects, the 5' coverage sequence is capable of binding to or hybridizing to the 5' homology sequence, before cleavage by the 5' ribozyme. In some aspects, the 3' coverage sequence is capable of binding to or hybridizing to the 3' homology sequence, before cleavage by the 3' ribozyme or the trans-acting ribozyme. In some aspects, in a precursor RNA molecule before cleavage, the 5' coverage sequence is placed at or near the 5' terminus of the precursor RNA molecule. In some aspects, in a precursor RNA molecule before cleavage, the 3' coverage sequence is placed at or near the 3' terminus of the precursor RNA molecule. In some aspects, the first RNA molecule in a bimolecular combination does not comprise a 3' coverage sequence.

[0258] In some aspects, the coverage sequence is between 5 and 50 nucleotides (nt) in length, such as between 8 and 45 nt, between 10 and 30 nt, between 14 and 25 nt, between 15 and 20 nt, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nt in length. In some aspects, the coverage sequence is 8 nt in length. In some aspects, the coverage sequence is 14 nt in length.

[0259] In some embodiments, the 5' coverage sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:2. In some embodiments, the 5' coverage sequence comprises SEQ ID NO:2. In some embodiments, the 5' coverage sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the 5' coverage sequence comprises a sequence encoded by SEQ ID NO:1.

[0260] In some embodiments, the 3' coverage sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:27. In some embodiments, the 3' coverage sequence comprises SEQ ID NO:27. In some embodiments, the 3' coverage sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:26. In some embodiments, the 3' coverage sequence comprises a sequence encoded by SEQ ID NO: 26.

[0261] In some embodiments, the RNA molecules provided herein further comprise a spacer sequence. In some embodiments, the spacer sequence is located near the 5' end of the RNA molecule (i.e., 5' spacer sequence). In some embodiments, the spacer sequence is located near the 3' end of the RNA molecule (i.e., 3' spacer sequence). In some aspects, the spacer sequence can be present between different sequence components present in the RNA molecule, for example, between the 5' homology region and the translation initiation element, and/or between the coding sequence for the exogenous molecule and the 3' homology region.

[0262] In some embodiments, the 5' spacer sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 12. In some embodiments, the 5' spacer sequence comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the 5' spacer sequence is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:11. In some embodiments, the 5' spacer sequence comprises a sequence encoded by SEQ ID NO:11.

[0263] In some aspects, a precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) comprises a 5' extension sequence and/or a 3' extension sequence. In some aspects, a precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) comprises a 5' extension sequence. In some aspects, a precursor RNA molecule (e.g., unimolecular RNA or first RNA molecule) comprises a 3' extension sequence. In some aspects, in a precursor RNA molecule before cleavage, the 5' extension sequence is placed at or near the 5' terminus of the precursor RNA molecule. In some aspects, in a precursor RNA molecule before cleavage, the 3' extension sequence is placed at or near the 3' terminus of the precursor RNA molecule.

[0264] In some aspects, the 3' extension sequence is located 3' of the substrate sequence of a 3' ribozyme. In some aspects, before cleavage, the extension sequence of the ribozyme can be contained in the same RNA molecule as the catalytic sequence of the ribozyme (unimolecular or cz -acting ribozyme). In some aspects, before cleavage, the extension sequence of the ribozyme can be contained in a distinct RNA molecule compared to the catalytic sequence of the ribozyme (bimolecular or /ra/z.s-acting ribozyme). In some embodiments, the RNA molecule comprises an 3' extension sequence, located 3' of the cleavage site of the 3' ribozyme.

[0265] In some aspects, the extension sequence is between 50 and 5000 nucleotides (nt) in length, such as between 80 and 4000 nt, between 100 and 3000 nt, between 200 and 2000 nt, between 300 and 1000 nt, or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 2000 or 3000 nt in length. In some aspects, the extension sequence is 312 nt in length. In some aspects, the extension sequence is 544 nt in length. In some aspects, the extension sequence is about 1000 nt in length.

[0266] In some embodiments, the 3' extension sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOS:61-63. In some embodiments, the 3' extension sequence comprises SEQ ID NO:61. In some embodiments, the 3' extension sequence comprises SEQ ID NO:62. In some embodiments, the 3' extension sequence comprises SEQ ID NO:63.

2. Insert [0267] In some embodiments, the RNA molecules provided herein, including cleaved RNA molecules (i.e., pre-circRNA) and circRNA molecules, comprise an insert sequence. In some aspects, the insert sequence comprises sequences encoding one or more exogenous molecules. In some aspects, the insert sequence is retained after cleavage and circularization. In some aspects, the insert sequence comprises a coding sequence for a gene product (e.g., a protein) and the provided RNA molecules and/or circRNA molecules comprising the insert sequence is delivered to a cell or a subject. In some aspects, RNA molecules and/or circRNA molecules are used to deliver the insert sequence to a cell or a subject, and to express the exogenous molecules (e.g., gene product encoded by a coding sequence in the insert) in the cell or the subject. In some aspects, the insert sequence comprises gene coding sequences and/or other elements that may be required for transcription, translation, and/or expression of the encoded gene product.

[0268] In some aspects, the insert sequence can refer to the sequences located between the 5' overhang sequence and 3' overhang sequence present in the provided RNA molecules and/or circRNA molecules. In some aspects, the insert sequence can refer to the sequences located between the 5' homology region and 3' homology region present in the provided RNA molecules and/or circRNA molecules.

[0269] In some embodiments, the insert sequence is at least about 50 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length. In some embodiments, the insert sequence is at least about 2000 nt in length. In some embodiments, the insert sequence is at least about 3000 nt in length. In some embodiments, the insert sequence is at least about 4000 nt in length. In some embodiments, the insert sequence is at least about 5000 nt in length. a. Exogenous Molecule

[0270] In some aspects, the RNA molecules provided herein, including cleaved RNA molecules (i.e., pre-circRNA) and circRNA molecules, comprise an insert sequence that includes a sequence encoding one or more exogenous molecules. In some aspects, the one or more exogenous molecules (e.g., gene product encoded by a coding sequence in the insert) includes one or more of: a vaccine antigen, a cancer antigen, a CRISPR system, including a nuclease and/or a guide RNA (gRNA), a nuclease, a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

[0271] In some embodiments, the exogenous molecule comprises a vaccine antigen. In some embodiments, the vaccine antigen comprises a viral vaccine antigen. In some aspects, the viral vaccine antigen comprises an antigen encoded by a virus. In some aspects, the viral vaccine antigen is from a virus that is a member of Coronaviridae. In some embodiments, the viral vaccine antigen is from an alphacoronavirus, a betacoronavirus, a deltacoronavirus, or a gammacoronavirus. In some embodiments, the virus is a human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUl), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV- NL63), Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the virus is SARS-CoV-2. In some aspects, the viral vaccine antigen is from a SARS-CoV-2 virus.

[0272] In some embodiments, the vaccine antigen comprises a cancer antigen. In some aspects, the cancer antigen is a tumor-associated antigens (TAAs) or a tumor-specific antigens (TSAs). Exemplary cancer antigens, TAAs or TSAs include, but are not limited to, those described in, for example, Liu et al., Journal of Hematology & Oncology 15:28 (2022); Buonaguro et al., Vaccines (Basel). 2020 Dec; 8(4): 615; and Zhao et al., Vaccines (Basel). 2021 Feb; 9(2): 85.

[0273] In some embodiments, the exogenous molecule comprises one or more components of a CRISPR system. In some aspects, the term “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, and various nucleic acid sequences associated with a Cas nuclease, such as a guide RNA (gRNA), and/or other sequences and transcripts from a CRISPR locus. In some embodiments, the CRISPR system includes a sequence-specific nuclease. In some embodiments, the sequence-specific nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Casl2 nuclease or a Casl3 nuclease. In some aspects, the CRISPR system includes a non-coding guide RNA (gRNA), which sequence- specifically binds to DNA, and a Cas nuclease (e.g., Cas9, CasX, Casl2, or Casl3), with a sequence-specific nuclease functionality. In some aspects, a gRNA is a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas nuclease complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. In some embodiments, the exogenous molecule comprises a guide RNA (gRNA). In some aspects, the exogenous molecule encoded by sequences in the insert sequences include a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease. In some embodiments, the Cas nuclease is a CasX nuclease. In some embodiments, the Cas nuclease is a Casl2 nuclease. In some embodiments, the Cas nuclease is a Casl 3 nuclease. Exemplary Cas nucleases that can be encoded in the provided RNA molecules, including circRNA molecules, include, but are not limited to, those described in, for example, Jinek et al., Science, 343(6176): 1247997, 2014; Nishimasu et al., Cell, 156:935-949, 2014; Cong et al., Science 2013, 399(6121):819-823; Wang et al., Cell 2013, 153(4):910-918; Mali et al., Science 2013, 399(6121):823-826; Cebrian-Serrano et al., Mamm Genome. 2017; 28(7): 247-261; Collias et al., Nature Communications 12:555 (2021); and Chen et al., Innovation (Camb). 2022 Jul 12; 3(4): 100264.

[0274] In some embodiments, the exogenous molecule comprises an antibody or an antigenbinding fragment thereof. In some aspects, exemplary antibody or antigen-binding fragment thereof that can be encoded in the provided RNA molecules, including circRNA molecules, include any known therapeutic antibodies, for example, those described in The Therapeutic Structural Antibody Database (Thera-SAbDab); Raybould et al., Nucleic Acids Research, 2020, 48(D1): D383-D388,; http : //opig . stats . ox . ac . uk/web app s/therasab dab .

[0275] In some embodiments, the exogenous molecule comprises an exogenous molecule comprises an immunomodulatory polypeptide. In some aspects, the immunomodulatory polypeptide is selected from among an adjuvant, an immune checkpoint inhibitor, a cytokine or any combination thereof. In some embodiments, the immunomodulatory polypeptide comprises a cytokine. In some embodiments, the immunomodulatory polypeptide is selected from among IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, interferon (IFN)-a, IFN-p, IFN-y, tumor necrosis factor (TNF)-a, TNF-P, human growth hormone, N-methionyl human growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor (FGF), prolactin, placental lactogen, tumor necrosis factor-a and -P, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor (VEGF), integrin, thrombopoietin (TPO), nerve growth factors (NGF)-P, platelet-growth factor, transforming growth factor (TGF)-u, TGF-P, insulin-like growth factor (IGF)-l, IGF-2, erythropoietin (EPO), osteoinductive factors, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), leukemia inhibitory factor (LIF), kit ligand (KL) and/or a portion and/or combination thereof. [0276] In some embodiments, the exogenous molecule comprises a transcription factor. In some aspects, exemplary transcription factors that can be encoded in the provided RNA molecules, including circRNA molecules, include those described in, for example, Becskei et al., Molecules. 2020 Apr; 25(8): 1902 or Pandelakis et al., Cell Systems (2020) 10(1): 1-14; and/or zinc finger protein (ZFP) or transcription activator-like effectors (TALEs) proteins that can be engineered to target specific sequences, for example, as described in US 6,140,081; US 6,453,242; US 6,534,261; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; WO 03/016496; US 20110301073; and Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405.

[0277] In some embodiments, the exogenous molecule comprises a reporter molecule. In some embodiments, the reporter molecule is a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, codon-optimized, stabilized and/or enhanced variants of the fluorescent proteins. In some aspects, the reporter molecule is an enzyme, such as a luciferase, including a firefly luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter molecules include luciferase (luc), firefly luciferase P-galactosidase, chloramphenicol acetyltransferase (CAT), P-glucuronidase (GUS) or variants thereof. In some aspects, expression of the enzyme can be detected by addition of a substrate that can be detected upon the expression and functional activity of the enzyme. In some embodiments, the reporter molecule comprises an eGFP. In some embodiments, the reporter molecule comprises an RFP.

[0278] In some embodiments, the reporter molecule comprises a firefly luciferase. In some embodiments, the firefly luciferase RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16. In some embodiments, the firefly luciferase RNA sequence comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the firefly luciferase is encoded by a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the firefly luciferase is encoded by the sequence set forth in SEQ ID NO: 15. h. Regulatory Sequences and Translation Initiation Elements

[0279] In some embodiments, the RNA molecules provided herein comprise one or more translation initiation elements. In some embodiments, the insert sequence comprises a translation initiation element. In some embodiments, the insert sequences comprise a translation initiation element, for example, located 5' of the sequence encoding an exogenous molecule, and the translation initiation element facilitates the translation of the coding sequence of the exogenous molecule.

[0280] In some embodiments, the translation initiation element is or comprises an internal ribosome entry site (IRES). In some embodiments, the IRES is located near the 5' end of the RNA molecules. In some embodiments, the IRES is located 3' of the 5' overhang sequence, the 5' homology region, and/or the 5' spacer sequence.

[0281] In some embodiments, the IRES sequence comprises an IRES sequence from Coxackie B3 virus (SEQ ID NO: 14), coxackie Bl virus (CVB1, SEQ ID NO: 71), encephalomyocarditis virus (EMCV, SEQ ID NO: 73), Epstein-Barr nuclear antigen 1 (EBNA1, SEQ ID NO: 75), enterovirus serotype EV-B107 (SEQ ID NO: 77), enterovirus serotype EV-D94 (SEQ ID NO: 79), Echovirus El 1 (EchoVl 1, SEQ ID NO: 81), Coronavirus disease 19 (Covidl9, SEQ ID NO: 83), coxsackievirus A20 (CVA20, SEQ ID NO: 85), poliovirus serotype 3 (PV3, SEQ ID NO: 87), Simian V4 (SEQ ID NO: 89), Human Rhinovirus Al (HRV-A1, SEQ ID NO: 91), Hepatitus C virus (HCV, SEQ ID NO: 93), Human Rhinovirus A21 (HRV-A21, SEQ ID NO: 95), Human Rhinovirus B17 (HRV-B17, SEQ ID NO: 97), Human Rhinovirus (HRV-A100, SEQ ID NO: 99), Human Rhinovirus B37 (HRV-B37, SEQ ID NO: 101), Human Rhinovirus B92 (HRV-B92, SEQ ID NO: 103), Human Rhinovirus B3 (HRV-B3, SEQ ID NO: 105), or Human Rhinovirus C54 (HRV-C54, SEQ ID NO: 107).

[0282] In some embodiments, the IRES comprises a Coxsackievirus B3 internal ribosome entry site (CVB3 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 14. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 13. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 13. [0283] In some embodiments, the IRES comprises a Coxsackievirus Bl internal ribosome entry site (CVB1 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:71. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:71. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:70. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:70.

[0284] In some embodiments, the IRES comprises an encephalomyocarditis virus internal ribosome entry site (EMCV IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:73. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:73. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:72. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:72.

[0285] In some embodiments, the IRES comprises a Eptesin-Barr nuclear antigen 1 internal ribosome entry site (EBNA1 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 75. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:75. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:74. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:74.

[0286] In some embodiments, the IRES comprises a enterovirus serotype EV-B107 internal ribosome entry site (EV-B107 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:77. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:77. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:76. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:76.

[0287] In some embodiments, the IRES comprises a enterovirus serotype EV-D94 internal ribosome entry site (EV-D94 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:79. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:79. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:78. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:78.

[0288] In some embodiments, the IRES comprises an Echovirus El l internal ribosome entry site (EchoVl 1 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:81. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:81. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:80. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 80.

[0289] In some embodiments, the IRES comprises a Coronavirus disease 19 internal ribosome entry site (Covidl9 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 83. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 83. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:82. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 82.

[0290] In some embodiments, the IRES comprises a coxsackievirus A20 internal ribosome entry site (CVA20 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:85. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 85. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:84. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 84.

[02911 In some embodiments, the IRES comprises a poliovirus serotype 3 internal ribosome entry site (PV3 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:87. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:86. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 86.

[0292] In some embodiments, the IRES comprises a Simian V4 internal ribosome entry site (Simian V4 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:89. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:89. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:88. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:88.

[0293] In some embodiments, the IRES comprises a Human Rhinovirus Al internal ribosome entry site (HRV-A1 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:91. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NONE In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:90. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:90.

[0294] In some embodiments, the IRES comprises a Hepatitus C virus internal ribosome entry site (HCV IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:93. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:93. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:92. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:92.

[0295] In some embodiments, the IRES comprises a Human Rhinovirus A21 internal ribosome entry site (HRV-A21 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 95. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:95. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:94. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 94.

[0296] In some embodiments, the IRES comprises a Human Rhinovirus B17 internal ribosome entry site (HRV-B17 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:97. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:97. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:96. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 96.

[0297] In some embodiments, the IRES comprises a Human Rhinovirus A100 internal ribosome entry site (HRV-A100 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:99. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO:99. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:98. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO:98.

[0298] In some embodiments, the IRES comprises a Human Rhinovirus B37 internal ribosome entry site (HRV-B37 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 101. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 100. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 100.

[0299] In some embodiments, the IRES comprises a Human Rhinovirus B92 internal ribosome entry site (HRV-B92 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 103. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 103. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 102. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 102.

[0300] In some embodiments, the IRES comprises a Human Rhinovirus B3 internal ribosome entry site (HRV-B3 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 105. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 105. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 104. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 104.

[0301] In some embodiments, the IRES comprises a Human Rhinovirus C54 internal ribosome entry site (HRV-C54 IRES) sequence. In some embodiments, the IRES RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 107. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, the IRES DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 106. In some embodiments, the IRES DNA sequence comprises the sequence set forth in SEQ ID NO: 106.

[0302] In some embodiments, the translation initiation element comprises a Translation Initiator of Short 5' UTR (TISU) element. TISU is a regulatory element that controls both transcription and translation initiation of genes with basic cellular functions. [0303] In some cases, when the insert sequence contains sequences encoding multiple exogenous molecules, a multicistronic element can be used, typically placed between the different coding sequences, to express multiple polypeptide gene products. Exemplary multicistronic elements include 2A elements that result in the separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2: 13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)).

[0304] In some embodiments, the RNA molecules provided herein comprises a poly adenine cytosine (poly AC) sequence. In some aspects, the poly AC sequence can be located 3' of the coding sequence for the exogenous molecule. In some embodiments, the poly AC RNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 50. In some embodiments, the polyAC RNA sequence comprises the sequence set forth in SEQ ID NO:50. In some embodiments, the polyAC DNA sequence comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polyAC DNA sequence comprises the sequence set forth in SEQ ID NO: 17.

IL METHODS FOR GENERATION OF circRNA

[0305] Provided herein are methods of generating or producing circular RNA (circRNA). In some aspects, the provided methods involve the use of any of the provided RNA molecules, combinations thereof, reaction intermediates or reaction products thereof, including linear RNA, cleaved linear RNA and circular RNA (circRNA). In some aspects, the provided methods improve the efficiency in generation of circRNA and improved purity and stability of compositions comprising circRNAs, resulting in improved expression of the gene product (e.g., encoded by the insert present in the nucleic acid molecules) in a cell or a subject.

[0306] In some aspects, the methods involve generation of any of the described RNA molecules, such as a precursor RNA (including the unimolecular precursor RNA molecule or the first RNA molecule). In some embodiments, the methods involve incubating the precursor RNA molecule under conditions that favor cleavage of the ribozyme substrates by the ribozymes (e.g., cleavage reaction) to generate a cleaved RNA molecule (also referred to as “pre-circRNA” in some cases). In some cases, the methods involve incubating the cleaved RNA molecule under conditions that favor ligation and circularization of the cleaved RNA molecule (e.g., ligation reaction, in the presence of an RNA ligase) to generate a circRNA. In some aspects, the methods do not involve a separate ligation reaction, and the cleaved RNA molecule can be circularized in a cell or in vivo in a subject. In some aspects, the methods also involve purifying and enriching preparations or reaction samples that contain comprising the RNA molecules to achieve a highly pure composition of any of the provided RNA molecules, including circRNA molecules or cleaved RNA molecules.

[0307] In some aspects, provided herein is a method for generating a cleaved RNA molecule, the method comprising: (1) producing any of the precursor RNA molecules provided herein; and (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0308] In some aspects, provided herein is a method for generating a combination of RNA molecules provided herein. In some embodiments, the method also involves incubating the RNA molecule in a solution, thereby generating a cleaved molecule.

[0309] In some aspects, provided herein is a method for generating a cleaved RNA molecule, the method comprising: (1) producing any of the precursor RNA molecules described herein; and (2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0310] In some aspects, provided herein is a method for generating a cleaved RNA molecule, the method comprising: (1) producing any of the combination of RNA molecules provided herein; and (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule.

[0311] In some aspects, also provided are methods for generating a circRNA molecule. In some embodiments, the method also involves incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circRNA.

[0312] In some aspects, provided herein is a method for generating a circRNA molecule, the method comprising: (1) producing any of the precursor RNA molecules provided herein; (2) incubating the precursor RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circRNA molecule

[0313] In some aspects, provided herein is a method for generating a circRNA molecule, the method comprising: (1) producing the combination of RNA molecules provided herein; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circRNA molecule.

[0314] In some aspects, provided herein is a method for generating a circRNA molecule, the method comprising: (1) producing any of the combination of RNA molecules comprising a first RNA molecule and a second RNA molecule described herein; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and (3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circRNA molecule.

[0315] In some aspects, the methods also involve a step for purification. In some aspects, the methods also involve a step for enrichment of circular RNA.

A. Generation of RNA Molecules

[0316] In some of aspects, any of the provided RNA molecules, in particular unimolecular precursor RNA molecule, or the first RNA molecule and the second RNA molecule in the combination, are produced. In some aspects, provided herein are precursor RNA molecules that can be cleaved at one or both of the 5' and 3' ends, and methods to generate the precursor RNA molecules. In some embodiments, the methods involve producing any of the provided RNA molecules, including known methods such as in vitro transcription, chemical synthesis, RNA synthesis, or oligonucleotide synthesis and assembly. In some aspects, larger RNA molecules, such as a unimolecular precursor RNA molecule or a first RNA molecule in the combination, which can comprise a large insert sequence, can be produced by in vitro transcription. In some aspects, smaller RNA molecules, such as a second RNA molecule in the combination, can be generated by chemical synthesis. Any known method to generate RNA sequences and molecules can be used.

[0317] In some embodiments, the RNA molecule is produced by in vitro transcription. In some embodiments, the RNA molecule is produced by RNA synthesis.

[0318] In some aspects, when generating the RNA molecules, modified nucleosides, such as pseudouridines, for example N1 -methylpseudouridine (ml T), can be employed to generate a precursor RNA molecule that contains the modified nucleoside.

B. Cleavage of RNA Molecules

[0319] In some embodiments, the provided methods for generating circRNA involve incubating the RNA molecule in particular conditions to facilitate cleavage of the ribozyme substrates present in the molecules, thereby generating a cleaved RNA molecule. In some aspects, the methods involve a separate cleavage reaction step. In some aspects, the methods involve incubating the RNA molecule in a solution. 1. Cleavage Reaction

[0320] In some embodiments, the provided methods involve a cleavage reaction, for example, by incubating the RNA molecule in a solution after production of the precursor RNA molecule. In some embodiments, the ribozymes of the precursor RNA molecules undergo a cleavage reaction. In some embodiments, the cleavage reaction is a spontaneous cleavage reaction. In some embodiments, the cleavage reaction is a self-cleavage reaction. In some embodiments, the cleavage reaction occurs in a solution. In some aspects, the cleavage reaction occurs in a solution that is different from the solution used for generation of the RNA molecule (e.g., by in vitro transcription). In some aspects, the cleavage reaction occurs in a solution that is the same as the solution used for generation of the RNA molecule (e.g., by in vitro transcription), but under different conditions or in a different vessel.

[0321] In some embodiments, the solution comprises sodium acetate (Na2OAc), magnesium acetate (MgOAc2) or both.

[0322] In some embodiments, the solution does not comprise potassium chloride (KC1). In some embodiments, the solution does not comprise magnesium chloride (MgCh). In some embodiments, the solution does not comprise potassium chloride (KC1) or magnesium chloride (MgCh). In some embodiments, the solution comprises potassium chloride (KC1) or magnesium chloride (MgCh). In some aspects, the solution comprises a solution used for in vitro transcription. In some aspects, the solution comprises 40 mM HEPES pH 7.5, 40 mM DTT, 10 mM NaOAc, 75 mM MgOAc2, and 0.2 mM spermidine. In some aspects, the solution comprises 30 mM HEPES pH 7.5, 100 mM KC1, 20 mM MgCh. In some aspects, the solution comprises 100 mM HEPES pH 7.5, 10 mM MgCh, 2 mM spermidine, 40 mM DTT, and 0.1 mg/ml BSA.

[0323] In some embodiments, the solution comprises cyclic di-guanosine monophosphate (c-di- GMP). In some embodiments, the solution comprises c-di-GMP at a concentration of between about 0.5 mM and about 10 mM. In some embodiments, the solution comprises c-di-GMP at a concentration of about 5 mM. In some aspects, the addition of c-di-GMP was reported to exhibit picomolar or nanomolar dissociation constant (KD) for riboswitches, which is more than three orders of magnitude higher binding affinity compared to those of guanine analogs (Lee et al. Science, 329(5993), 845-848, Sudarsan et al., Science, 321(5887), 411-413). This strong binding affinity potentially allows c-di-GMP access the catalytic sites of ribozymes to enhance cleavage activity by providing a more nucleophilic environment at the sites. In some aspects, the cleavage reaction is performed in the presence of c-di-GMP.

[03241 Iⁿ some embodiments, the solution comprises distilled water (DW). In some embodiments, the solution consists of distilled water (DW).

[0325] In some embodiments, the solution comprises Tris-EDTA (TE) buffer, optionally TE buffer pH 7.0 or TE buffer pH 8.0.

[0326] In some embodiments, in a population of RNA molecules, the cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some embodiments, in a population of RNA molecules, the 5' cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some embodiments, in a population of RNA molecules, the 3' cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some embodiments, in a population of RNA molecules, the 5' cleavage reaction and the 3' cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. In some embodiments, in a population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules are cleaved linear RNA molecules, with at least the 5' cleaved. In some embodiments, in a population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules are cleaved linear RNA molecules, with at least the 3' cleaved. In some embodiments, in a population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules are cleaved linear RNA molecules, with both 5' and 3' ends cleaved.

2. Cleaved RNA Molecules

[0327] In some aspects, provided are cleaved RNA molecules (also called pre-circRNA), generated after cleavage of the ribozyme substrates present at the 5' and 3' ends of the precursor RNA molecules. In some aspects, the cleavage reaction results in generation of the cleaved RNA molecules.

[0328] In some aspects, a “cleaved RNA molecule” also refers to a pre-circRNA (see FIG. 1A and FIG. IB). In some embodiments, the cleaved RNA molecule is produced by a method provided herein. In some embodiments, after cleavage, the cleaved RNA molecule lacks a 5' Twister-Sister catalytic sequence. In some embodiments, the cleaved RNA molecule lacks a Twister catalytic sequence. In some embodiments, the cleaved RNA molecule lacks a 5' Twister-Sister catalytic sequence and a Twister catalytic sequence. [0329] In some aspects, after cleavage, both the unimolecular approach and the bimolecular approach can produce the same cleaved RNA molecule. In some embodiments, even though the ribozyme that cleaves the 3' substrate sequence are located in different locations (3' end of the precursor RNA molecule in the unimolecular approach, and as a trans-acting ribozyme in the bimolecular approach), if the same ribozyme substrate sequences and catalytic sequences are utilized and the insert sequences are the same, the resulting cleaved RNA (pre-circRNA) can be identical after cleavage.

[0330] In some aspects, provided herein is a cleaved RNA comprising in 5’ to 3' order: a 5' overhang sequence of a Twister- Si st er ribozyme; a 5' homology region; an insert sequence; a 3' homology region; and a 3' overhang sequence of a Twister ribozyme.

[0331] In some embodiments, the cleaved RNA molecule comprises one or more overhang sequences. In some embodiments, the cleaved RNA molecule comprises a 5' overhang sequence, e.g., of a Twister- Sister ribozyme. In some embodiments, the cleaved RNA molecule comprises a 3' overhang sequence, e.g., of a Twister ribozyme.

[0332] In some aspects, the cleaved RNA molecule comprises an insert sequence, flanked by 5' and 3' homology regions. In some aspects, the cleaved RNA molecule comprises an insert sequence, flanked by 5' overhang sequences and homology region and 3' overhang sequences and homology region.

[0333] After cleavage, in some aspects, the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus. In some embodiments, the cleaved RNA molecule comprises a 2',3'-cyclic phosphate at the 3' terminus. In some embodiments, the hydroxyl group at the 5' terminus and the 2'3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase, such as RtcB ligase.

[0334] In some embodiments, an exemplary cleaved RNA molecule (e.g., pre-circRNA) comprises in 5' to 3' order: a 5' Twister-Sister ribozyme overhang sequence, a 5' homology region, a 5' spacer sequence, an IRES sequence, an exogenous molecule coding sequence, a polyAC sequence, a 3' homology region, and a 3' Twister ribozyme overhang sequence. In some aspects, the exemplary cleaved RNA molecule (e.g., pre-circRNA) comprises hydroxyl group at the 5’ terminus and the 2 '3 '-cyclic phosphate at the 3' terminus.

[0335] In some embodiments, an exemplary cleaved RNA molecule (e.g., pre-circRNA) comprises in 5' to 3' order: a 5' TS-1 overhang sequence, a 5' homology region, a 5' spacer sequence, a Coxsackievirus B3 IRES sequence, a Firefly luciferase (FLuc) coding sequence, a poly AC sequence, a 3' homology region, and a 3' A. vitripennis type Pl Twister overhang sequence. In some aspects, the exemplary cleaved RNA molecule (e.g., pre-circRNA) comprises hydroxyl group at the 5' terminus and the 2'3 '-cyclic phosphate at the 3' terminus.

[0336] In some embodiments, the provided cleaved RNA molecule (e.g., pre-circRNA) comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:54. In some embodiments, the provided cleaved RNA molecule (e.g., pre-circRNA) comprises SEQ ID NO:54. In some embodiments, the provided cleaved RNA molecule (e.g., pre-circRNA) comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO:53. In some embodiments, the provided cleaved RNA molecule (e.g., pre-circRNA) comprises a sequence encoded by SEQ ID NO: 53.

C. Denaturation/Renaturation

[0337] In some embodiments, the methods and uses also involve a denaturation step and a renaturation step. In some embodiments, the denaturation step is performed after incubating the RNA molecule for a cleavage reaction. In some embodiments, the renaturation step is performed after the denaturation step.

[0338] In some embodiments, the denaturation step is performed in the same solution as the cleavage reaction. In some embodiments, the denaturation step is performed in a different solution as the cleavage reaction. In some embodiments, the renaturation step is performed in the same solution as the denaturation step and/or the cleavage reaction. In some embodiments, the renaturation step is performed in a different solution as the denaturation step and/or the cleavage reaction.

[0339] In some embodiments, the denaturation step is performed at a sufficiently high temperature to denature and inhibit stem-loop structures in the RNA molecule. In some embodiments, the denaturation step is performed at between about 60°C and about 85 °C, or about 65 °C and about 80 °C. In some embodiments, the denaturation step is performed at about 65 °C, or at about 80 °C. In some embodiments, the denaturation step is at about 65 °C.

[0340] In some embodiments, the denaturation step is performed for a particular amount of time. In some aspects, the denaturation step is performed for between 1 minute and 20 minutes, such as for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes or 20 minutes. In some embodiments, the denaturation step is performed for no more than 5 minutes.

[0341] In some embodiments, the renaturation step is performed at a sufficiently low temperature to renature the RNA molecule. In some embodiments, the renaturation step is performed at between about 0 °C and about 30 °C, or about 4 °C and about 25 °C. In some embodiments, the renaturation step is performed at ambient temperature. In some embodiments, the renaturation step is performed at about 25 °C. In some embodiments, the renaturation step is performed at about 4 °C.

[0342] In some embodiments, the renaturation step is performed for a particular amount of time. In some aspects, the renaturation step is performed for between 1 minute and 20 minutes, such as for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes or 20 minutes. In some embodiments, the renaturation step is performed for no more than 5 minutes.

[0343] In some embodiments, the denaturation step and the renaturation step are performed in the solution for generating the cleaved RNA molecule. In some embodiments, the solution comprises Tris-EDTA (TE) buffer. In some embodiments, the solution comprises TE buffer pH 7.0 or TE buffer pH 8.0. In some embodiments, the solution comprises TE buffer pH 7.0.

D. Ligation/Circularization of RNA molecules

[0344] In some embodiments, the provided methods for generating circRNA involve incubating the cleaved RNA molecules (pre-circRNA) in particular conditions to facilitate the ligation of the cleaved 5' and 3' ends, thereby generating a circular RNA (circRNA) molecule. In some aspects, the methods involve a separate ligation reaction step. In some aspects, the methods involve incubating the cleaved RNA molecule with an RNA ligase, such as a tRNA splicing ligase, for example, RtcB ligase.

[0345] In some embodiments, the methods involve a separate in vitro ligation reaction by incubating the generated cleaved RNA molecule with a ligase. In some embodiments, the methods do not involve a separate in vitro ligation reaction. In some instances, the 5' and 3' ends of the cleaved RNA molecule (pre-circRNA) are ligated together when introduced into a cell or a subject (i.e., in vivo). In some aspects, an endogenous RNA ligase, such as an endogenous RtcB ligase in the cell or the subject, catalyzes the ligation and generation of circRNA in a cell or in vivo.

1. Ligation/Circularization Reaction

[0346] In some embodiments, the provided methods involve a ligation reaction. In some aspects, the ligation reaction involves incubating the RNA molecule with an RNA ligase, thereby generating a circular RNA molecule. In some aspects, the RNA ligase catalyzes the ligation between hydroxyl group at the 5' terminus and the 2'3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule, thereby generating a circRNA. In some aspects, the ligation reaction involves incubating the generated cleaved RNA molecule with a tRNA splicing ligase. a. tRNA Splicing Ligases

[0347] In some embodiments, the ligase is an RNA ligase. In some embodiments, the RNA ligase is a tRNA splicing ligase. In some embodiments, the RNA ligase is an RtcB ligase.

[0348] tRNA splicing ligases are ligases that play essential roles in tRNA splicing (exon-exon ligation during tRNA biogenesis), unfolded protein response, and RNA repair. tRNA splicing ligases are GTP-dependent enzymes that catalyze the direct joining of RNA strands with terminal 2'3 '-cyclic phosphate and 5' hydroxyl group. tRNA splicing ligases incorporate the substrate- derived 2'3 ’-cyclic phosphate into the resulting 3 ',5 '-phosphodiester bond, and are generally conserved throughout many domains of life (see, e.g., Tanaka et al., J Biol Chem 2011 Sep 2;286(35):30253-30257; Chakravarty et al., PNAS (2012) 109 (16) 6072-6077; Kroupova et al., eLife 10 :e71656). In mammals, RtcB ligase (also called HSPC117 or FAAP) is involved in tRNA splicing and is present endogenously in a mammalian cell.

[0349] In some aspects, after cleavage of the 5' and 3' substrate sequences to form a cleaved linear RNA, the 5' and 3' overhang sequences can form a loop structure (see, e g., pre-circRNA in FIG. 1A and FIG. IB and as described in Sections I.B.l and I.B.2). In some aspects, after cleavage, the 5' and 3' homology regions can form a stem structure to facilitate the generation of the loop structure. The stem and loop structure resembles the physiological tRNA substrate for tRNA ligases such as RtcB. For tRNAs, the 5' and 3' ends are presented at the ends of a base-paired stem. The stem and loop structures of the cleaved RNA molecules facilitate formation of a structure similar to the endogenous substrate for tRNA ligases such as RtcB, with the hydroxyl group at the 5' terminus and the 2'3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule in proximity to each other, and the ligation of the cleaved RNA termini to generate a circRNA.

[0350] In some aspects, the ligase used for the ligation reaction involves incubating the cleaved RNA molecule with an RNA ligase. In some aspects, the RNA ligase is an RtcB ligase. In some aspects, the 5' terminus and the 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule is capable of being ligated in the presence of an RNA ligase. [0351] In some embodiments, the RNA ligase comprises a sequence that has at least at or about 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO:43. In some embodiments, the RNA ligase is encoded by a sequence that has at least at or about 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:42. In some embodiments, the RNA ligase is encoded by the sequence set forth in SEQ ID NO:42.

[0352] In some aspects, the ligation occurs in the presence of an endogenous tRNA ligase, such as RtcB ligase, in a cell or in a subject. b. Ligation Conditions

[0353] In some aspects, the methods involve a ligation reaction (e.g., incubation with a ligase). The ligation reaction can be performed at a range of temperatures. In some embodiments, incubating with the RNA ligase is performed between about 30°C and about 40 °C, about 35 °C and about 39 °C, or about 36 °C and about 38 °C. In some embodiments, the incubating with the RNA ligase is performed at about 37°C. In some embodiments, the ligation reaction is performed at 37°C. In some embodiments, the ligation reaction is performed at 30°C. In some embodiments, the ligation reaction is performed at 25°C. In some embodiments, the ligation reaction is performed at or near room temperature.

[0354] In some embodiments, the incubating with the RNA ligase is performed in a buffer that does not comprise Mg²⁺.

[0355] In some aspects, the ligation reaction is performed for a particular amount of time. In some aspects, the ligation reaction is performed for between 1 minute and 120 minutes, such as for 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes or 90 minutes. In some embodiments, ligation is performed for no more than 10 minutes. In some embodiments, ligation is performed for no more than 20 minutes. In some embodiments, the ligation reaction is performed for no more than 30 minutes. In some embodiments, incubating with the RNA ligase is performed for between about 5 and 60 minutes, about 10 and about 30 minutes, about 15 and about 25 minutes, or about 10 and about 20 minutes. In some embodiments, the incubating with the RNA ligase is performed for 20 minutes.

[0356] In some aspects, as described herein, extension of the ligation reaction for more than 30 minutes reduced the efficiency of circRNA generation, and incubation for 20 minutes exhibited the highest circRNA generation efficiency. In some embodiments, ligation is performed at 37°C for no more than 10 minutes. In some embodiments, ligation is performed at 37°C for no more than 20 minutes. In some embodiments, the ligation reaction is performed at 37°C for no more than 30 minutes.

[0357] In some embodiments, the hydroxyl group at the 5' terminus and the 2', 3 -cyclic phosphate at the 3' terminus of the cleaved RNA molecule is capable of being ligated in the presence of an RNA ligase.

2. Circular RNA (circRNA) Molecules

[0358] In some embodiments, provided herein are circular RNA (circRNA) molecules. In some embodiments, a circular RNA molecule is generated by any of the methods or systems provided herein.

[0359] In some embodiments, the circRNA molecule is generated after ligation of the 5' and 3' termini of the cleaved RNA (pre-circRNA), for example, by an RtcB ligase (see, e.g., circRNA in FIG. 1A and FIG. IB) In some aspects, the circRNA molecule comprises the 5' overhang sequences, 5' homology region, 3' homology region and 3' overhang sequence. In some embodiments, a circular RNA molecule comprises a 5' overhang sequence of a Twister-Sister ribozyme; a 5' homology region; an insert sequence; a 3' homology region; and a 3' overhang sequence of a Twister ribozyme.

[0360] In some aspects, the exemplary circRNA molecule comprises the same sequence as the cleaved RNA molecules described herein, except that the molecule is in a closed circular loop and do not contain 5' and 3' termini.

[0361] In some embodiments, an exemplary circRNA molecule comprises: a 5' Twister- Si st er ribozyme overhang sequence, a 5' homology region, a 5' spacer sequence, an IRES sequence, an exogenous molecule coding sequence, a poly AC sequence, a 3' homology region, and a 3' Twister ribozyme overhang sequence.

[0362] In some embodiments, an exemplary circRNA molecule comprises: a 5' TS-1 overhang sequence, a 5' homology region, a 5' spacer sequence, a Coxsackievirus B3 IRES sequence, a Firefly luciferase (FLuc) coding sequence, a polyAC sequence, a 3' homology region, and a 3' N. vitripennis type Pl Twister overhang sequence.

[0363] In some embodiments, the provided circRNA molecule comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:54, and is a closed circular loop. In some embodiments, the provided circRNA molecule comprises SEQ ID NO:54, and is a closed circular loop. In some embodiments, the provided circRNA molecule comprises a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence encoded by SEQ ID NO:53, and is a closed circular loop. In some embodiments, the provided circRNA molecule comprises a sequence encoded by SEQ ID NO:53, and is a closed circular loop.

E. Methods for Purification

[0364] In some embodiments, provided herein are methods for purifying any of the RNA molecules described herein, including precursor RNA molecules, cleaved RNA molecules or circRNA molecules.

[0365] In some aspects, purification steps can be used to remove undesired byproducts (e.g., ribozyme catalytic sequences, cleaved small sequences, etc.) or undesired contaminating species. In some aspects, the purity of circRNA is a critical factor for maximizing protein production from circRNA and for avoiding innate cellular immune responses associated with contaminated nicked circRNA. Contamination of circRNA compositions with nicked circRNAs presents difficulties in purifying circRNAs. In some aspects, because nicked circRNA is resistant to RNase R treatment and has the same molecular weight, challenges exist in generating a circRNA compositions that reduces or minimizes the contamination by nicked circRNAs.

[0366] In some embodiments, the method of purifying is carried out by chromatography. In some embodiments, chromatography comprises high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEC) or size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). In some embodiments, the method of purifying comprises HPLC.

F. Methods for Enrichment

[0367] In some embodiments, provided are methods for enriching the circRNA molecules provided herein.

[0368] In some aspects, enzymes that can modify and/or degrade types of undesired or contaminating species of RNA molecules can be incubated with the RNA molecule preparations to enrich the composition for the desired circRNA molecules.

[0369] In some embodiments, enriching is carried out by incubation with a kinase. In some embodiments, the kinase comprises a polynucleotide kinase (PNK). In some embodiments, the enriching is carried out by incubation with a phosphatase.

[03701 Iⁿ some embodiments, the kinase comprises a polynucleotide kinase (PNK). In some embodiments, the PNK comprises T4 PNK, which is a bifunctional enzyme with 5 '-kinase and 3'- phosphatase activities that play key roles in RNA and DNA repair. In some aspects, the physiological substrate for PNK phosphatase is an RNA 2',3'-cyclic phosphate end (Das et al., Nucleic Acids Res, 2013, 41(l):355-365.

[0371] In some cases, treatment with a PNK results in phosphorylation of the 5' end of an RNA species that has not been circularized (for example, a semi-cleaved RNA molecule in which only the 5' ribozyme has been cleaved, thus cannot be circularized; see FIG. 11 A, left side). In some aspects, such RNA species with a phosphorylated 5' end can be degraded by a 5' phosphatedependent exonuclease (see FIG. 11 A, left side). In some aspects, the methods involve incubation of the RNA molecule preparation with a PNK. In some embodiments, the methods involve incubation of the RNA molecule preparation with a 5' phosphate-dependent exonuclease.

[0372] In some cases, treatment with a PNK results in removal of the 2'3 '-cyclic phosphate from the 5' end of an RNA species that has not been circularized (for example, a semi-cleaved RNA molecule in which only the 3' ribozyme has been cleaved, thus cannot be circularized; see FIG.

11 A, right side). In some aspects, such RNA species with a 3' hydroxyl group (after removal of the 2'3 '-cyclic phosphate) can be degraded by a specific nuclease, such as an RNase R (see FIG. 11A, right side). In some aspects, the methods involve incubation of the RNA molecule preparation with a PNK. In some embodiments, the methods involve incubation of the RNA molecule preparation with an RNase R.

[0373] In some embodiments, the enriching is carried out by one or more ribonucleases (RNases). In some embodiments, exemplary ribonucleases that can be used for degradation of undesired or contaminating species include RNase A, RNase B, RNase C, RNase E, RNase H, RNase HI, RNase HII, RNase II, RNase III, RNase Fl, RNase L, RNase M, RNase Ms, RNase N, RNase P, RNase PhyM, RNase R, RNase Sa, RNase St, RNase Tl, RNase T2, RNase U2, RNase IV, RNase V, RNase E, RNase E, polynucleotide phosphorylase (PNPase), RNase PH, RNase, RNase BN, RNase D, RNase T, RNase 1, exonuclease, oligoribonuclease, exoribonuclease I, or exoribonuclease II. In some embodiments, the one or more RNases comprise RNase R. In some embodiments, the one or more RNases comprise a 5' phosphate-dependent exonuclease. [0374] In some embodiments, the enriching is carried out by a restriction enzyme. Exemplary restriction enzymes may include but are not limited to EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, HinFI, Sau3AI, PvuII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl, PstI, SacI, Sall, Seal, Spel, SphI, Stul, and Xbal.

[0375] In some embodiments, the methods of enrichment provided herein enriches circRNA molecules by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to circRNA preparations that have not been subject to an enrichment step. In some embodiments, among a population of the RNA molecule molecules generated by the method, at least 70%, 80%, 90% or 95% of the RNA molecule molecules in the population are circular RNA molecules.

III. SYSTEMS FOR GENERATING CIRCULAR RIBONUCLEIC ACID (RNA) MOLECULES

[0376] Provided herein are systems, kits, compositions, and articles of manufacture, for generating any one of the RNA molecules provided herein, for example, precursor RNA molecules, cleaved RNA molecules or circRNA molecules. In some aspects, the systems, kits, compositions, and articles of manufacture include one or more components that are required for generating any of the RNA molecules provided herein, for example, precursor RNA molecules, cleaved RNA molecules or circRNA molecules or compositions comprising any of the provided RNA molecules. In some aspects, the systems, kits, compositions, and articles of manufacture also include instructions for carrying out any of the methods described herein, and/or instructions for uses.

[0377] In some aspects, the systems, kits, compositions, and articles of manufacture can include one or more of: a deoxyribonucleic acid (DNA) molecule or a vector containing the DNA molecule for generating a precursor RNA molecule or a combination of RNA molecules; a system for in vitro transcription and/or a system for RNA synthesis; an RNA ligase; one or more solutions and components for a cleavage reaction; one or more ligases; one or more components for purification; and/or one or more enzymes for enrichment. In some aspects, the systems, kits, compositions, and articles of manufacture can include any of the components required for carrying out the methods or uses described herein, for example in Sections I, II and IV. In some aspects, the systems, kits, compositions, and articles of manufacture can include instructions for carrying out the methods or uses described herein, for example in Sections I, II and IV.

[0378] The components for the systems, kits, compositions, and articles of manufacture can be selected in accordance with the particular RNA molecules and methods elected for circRNA generation.

A. Deoxyribonucleic Acid (DNA) Molecules and Vectors

[0379] Provided herein are deoxyribonucleic acid (DNA) molecules encoding any one of the RNA molecules provided herein. In some embodiments, the provided DNA molecules comprise the any of the sequences described herein that encode one or more of the RNA molecules described herein or a portion or a combination thereof. Also provided herein are vectors comprising any of the DNA molecules described herein. In some embodiments, the DNA molecules can be inserted into a nucleic acid vector. As used herein, the term “nucleic acid vector” is intended to mean any nucleic acid that functions to carry, harbor or express a nucleic acid of interest. Nucleic acid vectors can have specialized functions such as expression, packaging, pseudotyping, transduction or sequencing, for example. Nucleic acid vectors also can have, for example, manipulatory functions such as a cloning or shuttle vector. The structure of the vector can include any desired form that is feasible to make and desirable for a particular use. Such forms include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms. A nucleic acid vector can be composed of, for example, DNA or RNA, as well as contain partially or fully, nucleotide derivatives, analogs and mimetics. Such nucleic acid vectors can be obtained from natural sources, produced recombinantly or chemically synthesized.

[0380] In some embodiments, the vectors include vectors for in vitro transcription of the encoded RNA molecules, such as any of the precursor RNA molecules, first RNA molecules and/or second RNA molecules described herein. In some aspects, the vectors are suitable for in vitro transcription and contain elements (such as regulatory elements) required for in vitro transcription, for example to produce any of the precursor RNA molecules, first RNA molecules and/or second RNA molecules described herein.

[0381] In some aspects, the provided systems and kits include components required for generation of the RNA molecules, such as any of the precursor RNA molecules, first RNA molecules and/or second RNA molecules described herein, such as RNA polymerases, solutions, buffers and co-factors for in vitro transcription.

B. Components for Cleavage Reaction

[0382] In some aspects, the provided systems and kits include one or more solutions and components for the cleavage reaction. In some aspects, the solutions and components include any described herein, for example, in Section II.B.l , or in the Examples. In some aspects, the solution comprises sodium acetate (Na20Ac), magnesium acetate (M OAc2) or both. In some embodiments, the solution comprises cyclic di-guanosine monophosphate (c-di-GMP).

C. Components for Ligation Reaction

[0383] In some aspects, the provided systems and kits include an RNA ligase, and components for the ligation reaction. In some aspects, the RNA ligase and components include any described herein, for example, in Section II.C.1, or in the Examples. In some aspects, the RNA ligase comprises an RtcB ligase. In some embodiments, the RNA ligase comprises a sequence that has at least at or about 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO:43. In some embodiments, the RNA ligase is encoded by a sequence that has at least at or about 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:42. In some embodiments, the RNA ligase is encoded by the sequence set forth in SEQ ID NO:42.

D. Components for Purification and Enrichment

[0384] In some aspects, the provided systems and kits include components for the purification step and/or enrichment step. In some aspects, the components include any described herein, for example, in Section II. D and Section TLE, or in the Examples. In some aspects, the systems and kits include a kinase, a phosphatase and/or a ribonuclease.

IV. THERAPEUTIC AND PROPHYLACTIC METHODS AND USES

[0385] Provided herein are methods of vaccination and/or treatment, e.g., including administering any of the RNA molecules described herein, for example, circRNA or pre-circRNA, or a composition comprising the same, to a subject who is at risk of having a disease or disorder, or has a disease or disorder. In some aspects, also provided are methods of administering any of the RNA molecules described herein, for example, circRNA or pre-circRNA, or a composition comprising the same, to a subject, such as a subject who is at risk of having a disease or disorder, or has a disease or disorder. The described RNA molecules, for example, circRNA or pre-circRNA, or a composition comprising the same, are useful in treating a variety of diseases and disorders in a subject, or vaccinating a subject against a potential disease or disorder. Such methods and uses include therapeutic methods and uses, for example, involving administration of the RNA molecules or a composition comprising the same, to a subject having a disease or disorder. Such methods and uses also include prophylactic methods and uses, for example, to prevent a disease or disorder in a subject, that involves administration of the RNA molecules or a composition comprising the same, to a subject. Exemplary prophylactic uses include vaccination, such as against infectious disease antigens or cancer antigens. In some embodiments, the RNA molecules or a composition comprising the same, are administered in an effective amount to treat or prevent the disease or disorder. Uses include uses of the RNA molecules or a composition comprising the same, in such methods, treatments and prophylaxis, and in the preparation of a medicament in order to carry out such methods. In some embodiments, the methods are carried out by administering the RNA molecules or a composition comprising the same, to the subject having or suspected of having the disease or disorder. In some embodiments, the methods thereby treat the disease or disorder in the subject.

A. Administration

[0386] In some embodiments, the provided methods and uses involve administration of any of the RNA molecules described herein or compositions comprising the same, to a subject. In some embodiments, the RNA molecules described herein that can be administered include cleaved RNA molecules and circRNA molecules.

[0387] In some aspects, any of the provided circRNA molecules or cleaved RNA molecules (pre-circRNA) are administered to a subject to vaccinate a subject. In some aspects, vaccination includes vaccination against infectious diseases, such as viral infections, using insert sequences encoding viral antigens. In some aspects, vaccination includes vaccination against cancer, s using insert sequences encoding cancer antigens. In some aspects, any of the provided circRNA molecules or cleaved RNA molecules (pre-circRNA) are administered to treat a disease or disorder.

[0388] In some aspects, circRNA molecules or a composition comprising circRNA molecules is administered to the subject.

[0389] In some aspects, cleaved RNA molecules (i.e., pre-circRNA) or a composition comprising cleaved RNA molecules is administered to the subject. In some aspects, the cleaved RNA molecule (pre-circRNA), when administered to the subject, is circularized in vivo in the subject by the endogenous tRNA splicing enzymes, such as endogenous RtcB ligases. B. Delivery Vectors

[0390] In some embodiments, any of the RNA molecules described herein or compositions comprising the same, are compatible with viral or non-viral vector delivery methods. Exemplary non-viral vector delivery methods include physical or chemical means of delivery. For example, a physical non-viral delivery method includes electroporation. Exemplary chemical non-viral delivery vectors include nanoparticles, fat molecules (e.g., lipids) or polymers. In some embodiments, the any of the RNA molecules described herein or compositions comprising the same, are delivered via nanoparticle. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

V. DEFINITIONS

[0391] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0392] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0393] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0394] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[03951 Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0396] The terms “nucleic acid” and “nucleotide” include naturally-occurring species or functional analogs thereof, or variants thereof. A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are established in the field. A nucleic acid can include native or non-native nucleosides. In this regard, a native ribonucleic acid (RNA) can have one or more nucleosides selected from the group consisting of uridine (U), adenosine (A), cytidine (C), or guanosine (G). Useful non-native nucleosides are established in the field, such as a pseudouridine in place of a uridine.

[0397] The terms “hybridizing,” or “hybridize” refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another.

[0398] In some aspects, “precursor RNA” can refer to an RNA molecule that has not undergone cleavage of the ribozyme substrate sequences.

[0399] In some aspects, “cleaved RNA” or “pre-circRNA” can refer to an RNA molecule that has undergone cleavage of the ribozyme substrate sequences present at the 5' and 3' termini of the molecule.

[0400] In some aspects, “circular RNA” or “circRNA” can refer to a circularized RNA molecule that is generated from ligation of the 5' and 3' termini of the cleaved RNA (or pre- circRNA) molecule. [0401] In some aspects, the “overhang sequence” (in some cases also called “cleaved sequence”) of a ribozyme can refer to the portion of the substrate sequence that remain attached to the RNA molecule comprising the insert sequence (“cleaved RNA” or “pre-circRNA” after cleavage), after cleavage by the ribozyme. In some aspects, as used herein, an overhang sequence is a part of a substrate sequence, and a substrate sequence comprises an overhang sequence.

[0402] In some aspects, the “substrate sequence” of a ribozyme can refer to the portion of the ribozyme sequence that is cleaved by the catalytic portion of the ribozyme. In some aspects, before cleavage, the substrate sequence of the ribozyme can be contained in the same RNA molecule as the catalytic sequence of the ribozyme (unimolecular or cz -acting ribozyme). In some aspects, before cleavage, the substrate sequence of the ribozyme can be contained in a distinct RNA molecule compared to the catalytic sequence of the ribozyme (bimolecular or /raz/.s-acting ribozyme). In some aspects, the substrate sequence comprises an overhang sequence which remains attached to the cleaved RNA molecule comprising the insert sequence, after cleavage by the ribozyme.

[0403] In some aspects, the “catalytic sequence” of a ribozyme can refer to the portion of the ribozyme sequence that catalyzes the cleavage of the substrate sequence. In some aspects, the catalytic sequence cleaves the substrate sequence at or after the overhang sequence such that after cleavage, the overhang sequence remains attached to the cleaved RNA molecule comprising the insert sequence, after cleavage by the ribozyme.

VI. EXEMPLARY EMBODIMENTS

[0404] Among the provided embodiments are:

1. A ribonucleic acid (RNA) molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme.

2. The RNA molecule of embodiment 1, wherein the 5' substrate sequence comprises a 5' overhang sequence, and the 3' substrate sequence comprises a 3' overhang sequence

3. The RNA molecule of embodiment 1 or 2, wherein the 5' ribozyme and the 3' ribozyme together are capable of cleaving the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence; the insert sequence; and the 3' overhang sequence.

4. The RNA molecule of any of embodiments 1-3, further comprising a 5' homology region and 3' homology region.

5. The RNA molecule of embodiment 4, wherein the 5' homology region is located 3' of the 5' ribozyme.

6. The RNA molecule of embodiment 4 or 5, wherein the 3' homology region is located 3' of the insert sequence.

7. A cleaved ribonucleic acid (RNA) molecule comprising, in 5' to 3 ' order: a 5' overhang sequence of a Twister-Sister ribozyme; a 5' homology region; an insert sequence; a 3' homology region; and a 3' overhang sequence of a Twister ribozyme.

8. The RNA molecule of any of embodiments 4-7, wherein at least a portion of the 5' homology region and at least a portion of 3' homology region are complementary and are capable of forming a stem structure.

9. The RNA molecule of any of embodiments 3-8, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus.

10. The RNA molecule of embodiment 9, wherein the hydroxyl group at the 5' terminus and the 2',3'-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase to generate a circular RNA molecule.

11. The RNA molecule of embodiment 10, wherein the RNA ligase is a tRNA splicing ligase.

12. The RNA molecule of embodiment 10 or 11, wherein the RNA ligase is an RtcB ligase.

13. The RNA molecule of any of embodiments 10-12, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

14. A circular ribonucleic acid (RNA) molecule comprising: a 5' overhang sequence of a Twister-Sister ribozyme; a 5' homology region; an insert sequence; a 3' homology region; and a 3' overhang sequence of a Twister ribozyme.

15. The RNA molecule of any of embodiments 1-14, wherein the Twister- Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme.

16. The RNA molecule of any of embodiments 1-15, wherein the Twister- Sister ribozyme comprises a TS-1 ribozyme.

17. The RNA molecule of any of embodiments 1-16, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6.

18. The RNA molecule of any of embodiments 1-17, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6.

19. The RNA molecule of any of embodiments 1-18, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:4.

20. The RNA molecule of any of embodiments 1-19, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

21. The RNA molecule of any of embodiments 1-20, wherein the Twister ribozyme comprises a type Pl Twister ribozyme.

22. The RNA molecule of any of embodiments 1-21, wherein the Twister ribozyme comprises aNasonia vitripennis Type Pl Twister ribozyme.

23. The RNA molecule of any of embodiments 1-22, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:21.

24. The RNA molecule of any of embodiments 1-23, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

25. The RNA molecule of any of embodiments 1-24, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:23.

26. The RNA molecule of any of embodiments 1-25, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23.

27. The RNA molecule of any of embodiments 1-26, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:10.

28. The RNA molecule of any of embodiments 1-27, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10.

29. The RNA molecule of any of embodiments 1-28, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:19.

30. The RNA molecule of any of embodiments 1-29, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19.

31. The RNA molecule of any of embodiments 1-30, wherein the insert sequence comprises a translation initiation element.

32. The RNA molecule of embodiment 31, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element.

33. The RNA molecule of embodiment 31 or 32, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 14.

34. The RNA molecule of any of embodiments 31-33, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

35. The RNA molecule of any of embodiments 1-34, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules.

36. The RNA molecule of embodiment 35, wherein the one or more exogenous molecule is selected from among a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

37. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecule comprises a vaccine antigen.

38. The RNA molecule of embodiment 37, wherein the vaccine antigen comprises a viral vaccine antigen.

39. The RNA molecule of embodiment 38, wherein the vaccine antigen comprises a cancer antigen, optionally a cancer neoantigen.

40. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecule comprises a sequence-specific nuclease.

41. The RNA molecule of embodiment 40, wherein the sequence-specific nuclease is a Cas nuclease.

42. The RNA molecule of embodiment 41, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas 12 nuclease, or a Cas 13 nuclease.

43. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

44. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecule comprises an immunomodulatory polypeptide.

45. The RNA molecule of embodiment 44, wherein the immunomodulatory polypeptide comprises a cytokine.

46. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecule comprises a transcription factor.

47. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecule comprises a reporter molecule.

48. The RNA molecule of embodiment 47, wherein the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP).

49. The RNA molecule of embodiment 48, wherein the reporter molecule comprises a firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16.

50. The RNA molecule of any of embodiments 1-49, wherein the insert sequence is at least about 500 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

51. The RNA molecule of any of embodiments 1-50, wherein among a population of the RNA molecules, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

52. The RNA molecule of any of embodiments 3-13 and 15-51, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

53. The RNA molecule of any of embodiments 1-52, wherein among a population of the RNA molecules, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase.

54. The RNA molecule of any of embodiments 10-53, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

55. The RNA molecule of any of embodiments 1-54, wherein the RNA molecule comprises a modified nucleoside.

56. The RNA molecule of embodiment 55, wherein the modified nucleoside comprises a pseudouridine or a N1 -methylmethylpseudouridine.

57. The RNA molecule of any of embodiments 1-56, wherein the RNA molecule leads to reduced activation of one or more toll-like receptors (TLRs) or evades detection by one or more TLRs when incubated with a cell comprising the TLR.

58. A combination of ribonucleic acid (RNA) molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme.

59. The combination of embodiment 58, wherein the 5' substrate sequence comprises a 5' overhang sequence.

60. The combination of embodiment 58 or 59, wherein the 5' ribozyme of the first RNA molecule and the trans-acting ribozyme of the second RNA molecule together are capable of cleaving the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence; the insert sequence; and the 3' overhang sequence.

61. The combination of embodiment 60, wherein the cleaved RNA molecule further comprises a 5' homology region and 3' homology region.

62. The combination of embodiment 61, wherein the 5' homology region is located 3' of the 5' ribozyme.

63. The combination of embodiment 61 or 62, wherein the 3' homology region is located 3 ' of the insert sequence.

64. The combination of any of embodiments 61-63, wherein at least a portion of the 5' homology region and at least a portion of 3' homology region are complementary and are capable of forming a stem structure.

65. The combination of any of embodiments 60-64, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus.

66. The combination of embodiment 65, wherein the hydroxyl group at the 5' terminus and the 2 ',3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase to generate a circular RNA molecule.

67. The combination of embodiment 66, wherein the RNA ligase is a tRNA splicing ligase.

68. The combination of embodiment 66 or 67, wherein the RNA ligase is an RtcB ligase.

69. The combination of any of embodiments 66-68, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

70. The combination of any of embodiments 58-69, wherein the Twister- Si st er ribozyme comprises a Twister- Si ster-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme.

71. The combination of any of embodiments 58-70, wherein the Twister- Si st er ribozyme comprises a TS-1 ribozyme.

72. The combination of any of embodiments 58-71, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6.

73. The combination of any of embodiments 58-72, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6.

74. The combination of any of embodiments 58-73, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:4.

75. The combination of any of embodiments 58-74, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

76. The combination of any of embodiments 58-75, wherein the Twister ribozyme comprises a type Pl Twister ribozyme.

77. The combination of any of embodiments 58-76, wherein the Twister ribozyme comprises Nasonia vitripennis Type Pl Twister ribozyme.

78. The combination of any of embodiments 58-77, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:21.

79. The combination of any of embodiments 58-78, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

80. The combination of any of embodiments 58-79, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO 23.

81. The combination of any of embodiments 58-80, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23.

82. The combination of any of embodiments 58-81, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NOTO.

83. The combination of any of embodiments 58-82, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10.

84. The combination of any of embodiments 58-83, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence that has at least at or about 90%, 95%>, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:19.

85. The combination of any of embodiments 58-84, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19.

86. The combination of any of embodiments 58-85, wherein the insert sequence comprises a translation initiation element.

87. The combination of embodiment 86, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element.

88. The combination of embodiment 86 or 87, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence that has at least at or about 90%, 95%o, 96%o, 97%o, 98%o, 99%o, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:14.

89. The combination of any of embodiments 86-88, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

90. The combination of any of embodiments 58-89, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules.

91. The combination of embodiment 90, wherein the one or more exogenous molecule is selected from among a vaccine antigen, a cancer antigen, optionally a cancer neoantigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

92. The combination of embodiment 90 or 91, wherein the one or more exogenous molecule comprises a vaccine antigen.

93. The combination of embodiment 92, wherein the vaccine antigen comprises a viral vaccine antigen.

94. The combination of embodiment 92, wherein the vaccine antigen comprises a cancer antigen, optionally a cancer neoantigen.

95. The combination of embodiment 90 or 91, wherein the one or more exogenous molecule comprises a sequence-specific nuclease. 96. The combination of embodiment 95, wherein the sequence-specific nuclease is a Cas nuclease.

97. The combination of embodiment 96, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas 12 nuclease, or a Cas 13 nuclease.

98. The combination of embodiment 90 or 91, wherein the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

99. The combination of embodiment 90 or 91, wherein the one or more exogenous molecule comprises an immunomodulatory polypeptide.

100. The combination of embodiment 99, wherein the immunomodulatory polypeptide comprises a cytokine.

101. The combination of embodiment 90 or 91, wherein the one or more exogenous molecule comprises a transcription factor.

102. The combination of embodiment 90 or 91, wherein the one or more exogenous molecule comprises a reporter molecule.

103. The combination of embodiment 102, wherein the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP).

104. The combination of embodiment 103, wherein the reporter molecule comprises a firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16.

105. The combination of any of embodiments 58-104, wherein the insert sequence is at least about 500 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

106. The combination of any of embodiments 58-105, wherein among a population of the RNA molecules, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

107. The combination of any of embodiments 60-106, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

108. The combination of any of embodiments 58-107, wherein among a population of the RNA molecules, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase.

109. The combination of any of embodiments 66-108, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

110. The combination of any of embodiments 58-109, wherein the RNA molecule comprises a modified nucleoside.

111. The combination of embodiment 110, wherein the modified nucleoside comprises a pseudouridine or a N1 -methylmethylpseudouridine.

112. The combination of any of embodiments 58-111, wherein the RNA molecule leads to reduced activation of one or more toll-like receptors (TLRs) or evades detection by one or more TLRs when incubated with a cell comprising the TLR.

113. A deoxyribonucleic acid (DNA) molecule encoding the RNA molecule of any of embodiments 1-57, the first RNA molecule of the combination of any of embodiments 58-112, the second RNA molecule of the combination of any of embodiments 58-112, or the first RNA molecule and the second RNA molecule of the combination of any of embodiments 58-112.

114. A system for generating a circular RNA molecule comprising the RNA molecule of any of embodiments 1-57, or the combination of any of embodiments 58-112.

115. A system for generating a circular RNA molecule comprising the DNA molecule of embodiment 113, and a reagent for in vitro transcription.

116. The system of embodiment 114 or 115, further comprising an RNA ligase.

117. The system of embodiment 116, wherein the RNA ligase is a tRNA splicing ligase.

118. The system of embodiment 116 or 117, wherein the RNA ligase is an RtcB ligase.

119. The system of any of embodiments 116-118, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

120. A method for generating a ribonucleic acid (RNA) molecule, the method comprising producing the RNA molecule of any of embodiments 1-57.

121. A method for generating a ribonucleic acid (RNA) molecule, the method comprising producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme.

122. A method for generating a combination of ribonucleic acid (RNA) molecules, the method comprising producing the combination of any of embodiments 58-112.

123. A method for generating a combination of ribonucleic acid (RNA) molecules, the method comprising producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a

Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme.

124. The method of any of embodiments 120-123, further comprising incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

125. A method for generating a cleaved RNA molecule, the method comprising:

(1) producing the RNA molecule of any of embodiments 1-57; and

126. A method for generating a cleaved RNA molecule, the method comprising:

(1) producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme; and

127. A method for generating a cleaved RNA molecule, the method comprising:

(1) producing the combination of any of embodiments 58-112; and

(2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule.

128. A method for generating a cleaved RNA molecule, the method comprising:

(1) producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme; and

129. The method of any of embodiments 124-128, further comprising incubating the RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

130. A method for generating a circular RNA molecule, the method comprising:

(1) producing the RNA molecule of any of embodiments 1-57;

(2) incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule; and

(3) incubating the cleaved RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

131. A method for generating a circular RNA molecule, the method comprising:

(1) producing an RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme;

132. A method for generating a circular RNA molecule, the method comprising:

(1) producing the combination of any of embodiments 58-112;

(2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and

133. A method for generating a circular RNA molecule, the method comprising:

(1) producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a

Twister- Si st er ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme;

134. A method for generating a circular RNA molecule, the method comprising:

(1) producing the RNA molecule of any of embodiments 1-57;

(3) administering the cleaved RNA molecule to a subject, thereby generating a circular RNA molecule.

135. A method for generating a circular RNA molecule, the method comprising:

136. A method for generating a circular RNA molecule, the method comprising:

(1) producing the combination of any of embodiments 58-112;

137. A method for generating a circular RNA molecule, the method comprising:

Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme;

138. The method of any of embodiments 134-137, wherein the circular RNA is generated by an RNA ligase endogenously present in the subject.

139. The method of any of embodiments 120-138, wherein the RNA molecule or the first RNA molecule or the second RNA molecule of the combination is produced by in vitro transcription.

140. The method of any of embodiments 120-139, wherein the RNA molecule or the first RNA molecule or the second RNA molecule of the combination is produced by RNA synthesis.

141. The method of any of embodiments 120-140, wherein the incubation in step (2) generates a cleaved RNA molecule after cleavage of the 5' substrate sequence and the 3' substrate sequence by the catalytic sequences.

142. The method of any of embodiments 120-141, wherein the incubation in step (2) generates a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule.

143. The method of any of embodiments 124-142, wherein the solution comprises sodium acetate (Na2OAc), magnesium acetate (MgOAc2) or both.

144. The method of any of embodiments 124-143, wherein the solution does not comprise potassium chloride (KC1) or magnesium chloride (MgCh).

145. The method of any of embodiments 124-144, wherein the solution comprises cyclic di-guanosine monophosphate (c-di-GMP).

146. The method of embodiment 145, wherein the solution comprises c-di-GMP at a concentration of between about 0.5 mM and about 10 mM.

147. The method of embodiment 145 or 146, wherein the solution comprises c-di-GMP at a concentration of about 5 mM.

148. The method of any of claims 124-142, wherein the solution comprises distilled water (DW).

149. The method of any of claims 124-142 and 148, wherein the solution consists of distilled water (DW).

150. The method of any of claims 124-142, wherein the solution comprises Tris-EDTA (TE) buffer, optionally TE buffer pH 7.0 or TE buffer pH 8.0.

151. The method of any of claims 124-150, further comprising a denaturation step and a renaturation step.

152. The method of embodiment! 51, wherein the denaturation step is performed between about 60°C and about 85 °C, or about 65 °C and about 80 °C, or at about 65 °C, or at about 80 °C.

153. The method of embodimentl51 or 152, wherein the renaturation step comprises incubating at ambient temperature or 4 °C after the denaturation step. 154. The method of any of claims 151-153, wherein the denaturation step and the renaturation step are performed in the solution for generating the cleaved RNA molecule.

155. The method of embodimentl54, wherein the solution comprises Tris-EDTA (TE) buffer, optionally TE buffer pH 7.0 or TE buffer pH 8.0.

156. The method of any of claims 120-155, wherein among a population of the RNA molecules generated by the method, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

157. The method of any of claims 124-156, wherein among a population of the RNA molecules generated by the method, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

158. The method of any of claims 144-157, wherein the hydroxyl group at the 5' terminus and the 2',3'-cyclic phosphate at the 3' terminus of the cleaved RNA molecule is capable of being ligated in the presence of an RNA ligase to generate a circular RNA molecule.

159. The method of any of claims 129-133 and 139-158, wherein the incubating with the RNA ligase in step (3) is performed for between about 5 and about 60 minutes, about 10 and about 30 minutes, about 15 and about 25 minutes, or about 10 and about 20 minutes.

160. The method of any of claims 129-133 and 139-159, wherein the incubating with the RNA ligase in step (3) is performed for about 20 minutes.

161. The method of any of claims 129-133 and 139-160, wherein the incubating with the RNA ligase in step (3) is performed between about 30°C and about 40 °C, about 35 °C and about 39 °C, or about 36 °C and about 38 °C.

162. The method of any of claims 129-133 and 139-161, wherein the incubating with the RNA ligase in step (3) is performed at about 37°C.

163. The method of any of claims 129-133 and 139-162, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that comprises Mg²⁺.

164. The method of embodimentl63, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that comprises Tris-HCl, KC1, MgCh, and DTT.

165. The method of embodimentl63 or 164, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that comprises 50 mM Tris-HCl, 75 mM KC1, 3 mM MgCb, and 10 mM DTT. 166. The method of any of claims 129-133 and 139-162, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that does not comprise Mg²⁺.

167. The method of any of claims 129-166, wherein the RNA ligase is a tRNA splicing ligase.

168. The method of any of claims 129-167, wherein the RNA ligase is an RtcB ligase.

169. The method of any of claims 129-168, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

170. The method of any of claims 120-169, wherein the method further comprises purifying the cleaved linear RNA molecule or the circular RNA molecule.

171. The method of embodimentl70, wherein the purifying is carried out by chromatography.

172. The method of embodimentl71, wherein the chromatography comprises high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEC) or size exclusion chromatography-high performance liquid chromatography (SEC-HPLC).

173. The method of any of claims 120-172, wherein the method further comprises enriching for the circular RNA molecules.

174. The method of embodimentl73, wherein the enriching is carried out by incubation with a kinase.

175. The method of embodimentl74, wherein the kinase comprises a polynucleotide kinase (PNK).

176. The method of embodiment! 73, wherein the enriching is carried out by incubation with a phosphatase.

177. The method of any of claims 173-176, wherein the enriching is carried out by incubation with one or more ribonucleases.

178. The method of embodimentl77, wherein the one or more ribonucleases comprises an RNase R and/or a 5' phosphate-dependent exonuclease.

179. The method of any of claims 120-178, wherein among a population of the RNA molecule generated by the method, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase.

180. The method of any of claims 120-179, wherein among a population of the RNA molecule generated by the method, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

181. The method of any of claims 120-180, wherein the RNA molecule comprises a modified nucleoside.

182. The method of embodimentl81, wherein the modified nucleoside comprises a pseudouridine or a N1 -methylmethylpseudouridine.

183. An RNA molecule generated by the method of any of claims 120-182.

184. A cleaved RNA molecule generated by the method of any of claims 124-182.

185. The RNA molecule of embodimentl83 or 184, wherein the RNA molecule is a linear RNA molecule.

186. A circular RNA molecule generated by the method of any of claims 129-182.

187. A composition comprising the RNA molecule of any of claims 1-57, 183, and 185.

188. A composition comprising the cleaved RNA molecule of any of claims 7-57, 184, and 185.

189. A composition comprising the circular RNA molecule of any of claims 14-57 and 186.

190. A composition comprising the combination of any of claims 58-112.

191. The composition of any of claims 187-190, wherein the composition is a pharmaceutical composition.

192. The composition of embodimentl91, wherein the composition comprises a pharmaceutically acceptable excipient.

193. The composition of any of claims 187-192, wherein the composition comprises a lipid nanoparticle (LNP).

194. A method of vaccinating a subject, the method comprising administering the RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-112, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193.

195. A method of treating a disease or disorder in a subject, the method comprising administering the RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-112, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193.

196. The RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-112, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193 for use in vaccinating a subject, wherein the RNA molecule or the composition is administered to the subject.

197. The RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-112, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193 for use in treating a disease or disorder in a subject, wherein the RNA molecule or the composition is administered to the subject.

198. Use of the RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-112, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193 in the manufacture of medicament for vaccinating a subject, wherein the medicament is administered to the subject.

199. Use of the RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-112, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193 in the manufacture of medicament for treating a disease or disorder in a subject, wherein the medicament is administered to the subject.

VII. EXAMPLES

[0405] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Generation of constructs encoding RNA molecules with Twister-Sister and Twister ribozymes, for circular RNA (circRNA) generation

[0406] Various DNA constructs encoding RNA molecules comprising different ribozymes were designed and tested for efficiency in the generation of circular RNA (circRNA) molecules.

[0407] The two general approaches for designing the constructs included: (1) a unimolecular approach using an RNA molecule comprising a 5' Twister- Si st er ribozyme; an insert and a 3' Twister ribozyme; and (2) a bimolecular approach comprising an RNA molecule comprising a 5' Twister- Sister ribozyme; an insert and a 3' substrate sequence of a Twister, and a separate RNA molecule comprising a trans-acting Twister ribozyme. [0408] An exemplary construct for the unimol ecular approach included sequences encoding an RNA molecule (also called a “precursor RNA”) that comprises, in 5' to 3' order: a 5' Twister- Sister-1 (TS-1) ribozyme based on a microbial metagenomic DNA source, a coxsackievirus B3 internal ribosome entry site (CVB3 IRES), a firefly luciferase coding sequence (FLuc), and a 3' Twister ribozyme from N. vitripennis (FIG. 1A, Construct Ul; DNA sequence set forth in SEQ ID NO:28, generated RNA sequence set forth in SEQ ID NO:29). An exemplary construct for the bimolecular approach included sequences encoding an RNA molecule that comprises, in 5' to 3' order: a 5' Twister- Si ster-1 (TS-1) ribozyme, a coxsackievirus B3 internal ribosome entry site (CVB3 IRES), a firefly luciferase coding sequence (FLuc), and a 3' substrate sequence of the N. vitripennis type Pl Twister ribozyme (FIG. IB, Construct Bl; DNA sequence set forth in SEQ ID NO:36, generated RNA sequence set forth in SEQ ID NO:37), and separately another RNA molecule comprising a trans-acting N. vitripennis type Pl Twister ribozyme (FIG. IB, Construct B2; DNA sequence set forth in SEQ ID NO:34, generated RNA sequence set forth in SEQ ID NO:35). For Construct Bl, the substrate was designed to be cleaved only in the presence of the trans-acting Twister ribozyme in Construct B2.

[0409] A large insert sequence between the 5' and 3' ribozyme sequences, including the coxsackievirus B3 internal ribosome entry site (CVB3 IRES; DNA set forth in SEQ ID NO: 13; encoded RNA set forth in SEQ ID NO: 14; 741 nt), and the firefly luciferase coding sequence (FLuc; DNA set forth in SEQ ID NO: 15; encoded RNA set forth in SEQ ID NO: 16; 1653 nt), totaling 2394 nt, was used to test the cleavage efficiency and circularization efficiency, as generation of circRNA containing large inserts have been difficult and inefficient based on existing methods for generating circRNA.

[0410] In the unimolecular approach, the constructs were designed such that the 5' ribozyme and 3' ribozyme each self-cleaves at the specific cleavage site. In the bimolecular approach, constructs were designed such that the 5' ribozyme self-cleaves at its cleavage site, and the 3' substrate sequence of the first RNA molecule is cleaved by the trans-acting ribozyme in the separate RNA molecule (i.e., second RNA molecule). The cleavage reactions in both cases result in a cleaved RNA molecule (also called “pre-circRNA”) with a 5' cleavage product with a terminal 5' hydroxyl group and a 3' cleavage product with 2', 3 '-cyclic phosphate group, which are substrates for ligase-mediated ligation. In some aspects, the portion of the substrate sequences that remains on the RNA molecule after cleavage are called overhang sequences (i.e., 5' overhang sequence and 3' overhang sequence). The constructs were also designed such that in the cleaved RNA molecule, homology regions present near the 5' and 3' cleaved ends form a stem and loop structure, which resembles the structure of tRNA (FIGS. 1A-1B). The constructs were designed to produce a stem that comprises 21 nucleotides (nt), as well as a 6 nucleotide overhang on the 5' end and a 4 nucleotide overhang on the 3' end (FIG. 1C and FIG. ID). In some aspects, circular RNA molecules generated from the process described herein are called circular RNA (circRNA). The intermediates required to generate circRNA are designated as precursor RNA, which refers to linear RNAs which contains the ribozymes before cleavage, and pre-circRNA, which refers to linear RNAs that have been cleaved but not yet circularized.

Example 2: Assessment of cleavage efficiency of unimolecular constructs encoding RNA molecules with Twister-Sister and Twister ribozymes, for circular RNA (circRNA) generation

[0411] The cleavage efficiency of various unimolecular constructs for circRNA generation were assessed.

[0412] The efficiency of the unimolecular approach, using an RNA comprising a 5' TS-1 and 3' Twister ribozymes in forming circular RNA was tested using the exemplary Construct U1 described above in Example 1, compared to various different constructs, including a construct encoding an RNA comprising a 5' P3 Twister U2A ribozyme (Construct 3).

[0413] The DNA constructs encoding the RNA molecules for circRNA generation were synthesized and cloned using Gibson Assembly into pFlA T7 Flexi Vector (Promega). After colony screening and sequence verification, the plasmids were linearized and in vitro transcribed for 3 hours at 37°C using MEGAScript T7 Transcription Kit (Thermo Fisher), and digested with DNase following transcription. RNA was then purified using Monarch RNA Cleanup Kit (NEB), generating purified precursor RNA.

[0414] After in vitro transcription, the linear RNAs (precursor RNA) were incubated at room temperature for 1 hour in Cleavage Buffer 1 (30 mM HEPES pH 7.5, 100 mM KC1, 20 mM MgCb), and RNA was purified, generating purified pre-circRNA.

[0415] The pre-circRNA were then ligated in the presence of RtcB RNA Ligase (New England Biolabs) at approximately 10 pmol pre-circRNA, and 15 pmol of RtcB RNA Ligase, in Ligase Buffer 1 (0.1 mM GTP, 1 mM MnCh) including RNase Inhibitor Murine (New England Biolabs) to help prevent RNase-mediated degradation, for 1 .5 hours at 37 °C, and RNA was purified, generating purified circRNA.

[04161 After cleanup, the circRNA was enriched by RNase R digestion to digest all linear RNA, by incubation of 10 pg of total RNA sample, 2 pl of RNase R (Abeam ab286929) at 37 °C for 1 hour (in the presence of RNase Inhibitor, which is not known to inhibit RNase R). The RNA was purified, generating enriched circRNA. At various stages, the samples were subject to electrophoresis, and intensity of the various RNA species in the sample was determined by densitometry.

[0417] FIG. 2A shows an electrophoresis profile of various RNA intermediates and products after generation, cleavage, ligation (using RtcB) and enrichment (using RNase R), from Construct 3 (RNA comprising a 5' P3 Twister U2A ribozyme and a 3' type Pl Twister ribozyme). For Construct 3, circularization efficiency (circRNA/(circRNA+linear RNA) xlOO) was about 10%, without treatment with RNase R. The low circularization efficiency was likely due to the relatively low cleavage efficiency of 5’ P3 Twister U2A ribozyme and/or 3' type Pl Twister ribozyme, leaving fewer molecules with a 5' hydroxyl group terminus and a 3' cleavage product with 2', 3 '-cyclic phosphate group that could be ligated by RtcB.

[0418] DNA constructs encoding different ribozyme pairs for the 5' and 3' ribozymes, were assessed to determine ribozyme pairs that could efficiently cleave both the 5' and 3' termini of precursor RNAs to generate pre-circular RNAs that could be ligated by RtcB. For the 5' end, Twister- Sister 1 (TS-1) was selected as a candidate in part because the cleavage site is located near the 3' end of the ribozyme to leave minimal residual ribozyme sequences, and leave a 5' hydroxyl group at the 5' end of the pre-circRNA after cleavage. For the 3' ribozyme, several ribozymes that cleave near its 5' end, to leave as little residual ribozyme sequence as possible, and a 2', 3 '-cyclic phosphate at the 3' end of the pre-circRNA after cleavage, including a Twister ribozyme from N. vitripennis, hatchet ribozyme and a type P l Twister ribozyme Oryza sativa Osa- 1-4, were selected for testing. As shown in FIGS. 2B and 2C, cleavage efficiency of 5' TS-1 was generally at least three- to five-fold higher than that a 5' Twister U2A ribozyme. The highest cleavage efficiency was observed with Construct Ul, comprising a 5' TS-1 ribozyme and a 3' type Pl Twister ribozyme. The results support the utility and advantage of generating circRNA using a 5' Twister-Sister ribozymes and a 3' Twister ribozymes, particularly for generating circRNA with a large insert.

Example 3: Assessment of reaction conditions on circular RNA (circRNA) formation [0419] Various reaction conditions, including buffers and buffer components for were assessed for their effect in cleavage and ligation/circularization of RNA molecules to generate circular RNA (circRNA).

A. Cleavage buffers

[0420] After in vitro transcription, the majority of RNA species in the sample were indicated to be uncleaved precursor RNA or semi-cleaved RNA (with only one of the 5' end or 3' end underwent cleavage), as the direct circularization efficiency was observed to be low, possibly due to low cleavage efficiency and a large insert size and the large distance between the 5' and 3' ribozymes. By contrast, the majority of linear RNA prepared after a separate cleavage reaction were likely pre- circRNA, as the circularization efficiency was relatively high when treated with a ligase such as RtcB. In addition, chelator reagents such as EDTA contained in the T7 RNA polymerase buffer and pyrophosphates produced during the transcription potentially inhibit or hinder the action of Mg2⁺ which promotes ribozyme activity. Accordingly, buffers were selected and tested to determine whether buffer components for the separate cleavage reaction affect cleavage and circularization efficiency.

[0421] Buffers tested for the cleavage reaction include T7 RNA transcription buffers from different sources (Thermo Fisher, Promega, Roche, and MEGAScript) and Cleavage Buffer 1 (30 mM HEPES pH 7.5, 100 mM KC1, 20 mM MgCh). The in vitro transcribed RNA from Construct U1 was incubated in the different buffers. The samples were then subject to a circularization reaction by incubation with RtcB ligase, generally as described in Example 2 above, and by electrophoresis and densitometry.

[0422] FIG. 3A shows the electrophoresis profile and densitometry quantification of circularization efficiency of RNA molecules that were subject to a cleavage reaction using T7 RNA polymerase buffers from Thermo Fisher (Lane 2), Promega (Lane 3), Roche (Lane 4), and MEGAScript (Lane 6); Cleavage Buffer 1 (Lane 5); and in vitro transcribed RNA without a separate cleavage reaction as a negative control (Lane 1). Among the tested buffers, the circularization efficiency of Cleavage Buffer 1 and the MEGAScript buffer were the highest (see FIG. 3A).

[0423] In view of the results, different components of the buffer were further investigated, to assess their effects on cleavage activity and circularization. A buffer containing sodium acetate (Na2OAc) and magnesium acetate (M OAc2) instead of potassium chloride (KC1) and magnesium chloride (MgCh), Cleavage Buffer 2 (40 mM HEPES pH 7.5, 40 mM DTT, 10 mM NaOAc, 75 mM MgOAc2, and 0.2 mM spermidine) was tested. As acetic acid (CH3COOH) is a weak acid while acetate ion (CH3COO ) is a strong base and is only able to deprotonate an acid with a pKa lower than 5.0, the acetate ion could induce deprotonation of the hydroxyl group of the ribose at the catalytic site of the ribozymes and enhance cleavage and circularization efficiency. In addition, a different Cleavage Buffer 3 (100 mM HEPES pH 7.5, 10 mM MgCh, 2 mM spermidine, 40 mM DTT, and 0.1 mg/ml BSA) containing MgCh was also tested.

[0424] As shown in FIG. 3B, circularization efficiency was increased by 1.47-fold when Cleavage Buffer 2 containing sodium acetate and magnesium acetate (lane 4; 63.44%), compared to that of Cleavage Buffer 1 (lane 1; 54.1%).

[0425] The addition of cyclic di-guanosine monophosphate (c-di-GMP) as an additive in the buffer was also tested. C-di-GMP was reported to exhibit picomolar or nanomolar dissociation constant (KD) for riboswitches, which is more than three orders of magnitude higher binding affinity compared to those of guanine analogs (Lee et al. Science, 329(5993), 845-848, Sudarsan et al., Science, 321(5887), 411-413). This strong binding affinity potentially allows c-di-GMP access the catalytic sites of ribozymes to enhance cleavage activity by providing a more nucleophilic environment at the sites.

[0426] As shown in FIG. 3B, the addition of c-di-GMP to Cleavage Buffer 1 at a high concentration (5 mM) increased the circularization efficiency by 1.61-fold (lane 3; 65.5%), compared to that of Cleavage Buffer 1 (lane 1; 54.1%). As shown in FIG. 3B, circularization efficiency was increased by 1.47-fold when Cleavage Buffer 2 containing sodium acetate and magnesium acetate (lane 4; 63.44%), compared to that of Cleavage Buffer 1 (lane 1; 54.1%).

[0427] The effect of c-di-GMP on the circularization efficiency in Cleavage Buffer 1 and Cleavage Buffer 2 were also measured. Unexpectedly, the effect of c-di-GMP in increasing circularization efficiency was more evident in Cleavage Buffer 1, compared to c-di-GMP in Cleavage Buffer 2 (FIG. 3C). The results support the advantage of using c-di-GMP in the cleavage reaction in promoting the generation of circRNAs.

[0428] The effect of the presence of magnesium ion (Mg²⁺) on circRNA was also assessed. Mg²⁺ has been reported to lead to nicking of generated circRNAs, Generated and gel-purified circRNA were incubated at different temperatures (room temperature, 4°C, -20°C, and -80°C) with or without 10 mM MgCh for 16 hrs. Nicked circRNA bands represent single nicks that occur at random positions in an intact circRNA (FIG. 3D). As shown in FIG. 3D, MgCk-mediated nicking occurred at all temperatures tested.

B. Ligase Conditions

[0429] The effect of the amount of ligase present in the reaction and the length of time of the ligase reaction on circularization efficiency of RNA were also assessed. 500 ng of cleaved RNA generated from Construct U1 was used for the ligation reaction with various amounts of RtcB ligase (15 pmole/pL) in 0.5, 1, 2, 3, 4, or 5 pL of Cleavage Buffer 1 for 1 hr 30 min at 37 °C.

[0430] As shown in FIG. 3E, circularization efficiency gradually increased in a dose-dependent manner and reached the plateau at 2 uL of RtcB ligase (30 pmole). These results demonstrate that RtcB-mediated circularization is generally dose-dependent until a plateau.

[0431] The effect of the length of time of the ligase reaction on ligation efficiency was also assessed. 1.5 mg of cleaved pre-circRNA, generated from Construct U1 after in vitro transcription and cleavage reaction in the presence of c-di-GMP, was column-purified and incubated with RtcB at 37°C for 5, 10, 20, 30, 60, and 90 min at room temperature. After purification, RNA samples were subject to electrophoresis.

[0432] Unexpectedly, the highest circularization efficiency (75.5%) was shown when the incubation was RtcB was at 37 °C for 20 min (see FIG. 3F). After 20 min, the circularization efficiency gradually decreased and linear RNA gradually increased (see FIG. 3F). The results indicate that circular RNA may be linearized as the incubation time increased beyond the maximum circularization efficiency. One reason for the apparent linearization of generated circRNA, may be that Mn²⁺ ions are present in the ligase buffer, similar to Mg²⁺ as shown in Example 3A and FIG. 3D above, contributes to nicking of circRNA, and ultimately linearization of the generated circRNA. The results support the advantage of limiting the ligation reaction time for generating circRNA.

Example 4: Stable expression of gene products in cells using circRNA and intermediates

[0433] Circular RNA (circRNA) and various intermediate preparations produced using the unimolecular approach as described in Examples 1-4 above were introduced into cell lines and the expression of the encoded gene product were assessed.

[0434] HEK 293T cells were cultured and seeded onto a 12-well cell-culture treated dish one day before transfection to reach 60-80% confluence at time of transfection. Three different RNA intermediate samples (RNA preparation produced after in vitro transcription (“IVT”); RNA preparation after cleavage reaction (“CLV”), or enriched RNA prepared after ligation reaction and RNase R treatment (“RtcB+RR”), as described in FIG. 1A and Example 2) generated using an exemplary Construct Ul, were transfected using lipofectamine as follows: 3 pL of Messenger Max lipid was added into 50 pL of pre-warmed OPTI-MEM (Gibco); 1 pg of RNA was added to the OPTI-MEM and incubated at RT for 5 minutes; and the lipid/RNA mixture was added to the HEK 293T cells.

[0435] As shown in FIG. 4A, before transfection into cells, circRNA generation and enrichment by cleavage reaction followed by ligase and RNase R treatment (enrichment) substantially increased the circRNA content in the RNA sample (73%, lane 3, “RtcB+RR”).

[0436] 24 hours post-transfection, the conversion of linear RNA to circRNA was analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using primers specific for the junctional site of the circRNA. The primers for the junctional site of circRNA included: 5'- GCAAGACCATGACCGAGAAG-3 ' (forward primer; SEQ ID NO:38), and 5'- GATCAAAACGTGGCTGGTGT-3' (reverse primer; SEQ ID NO:39). Reverse transcription reactions was performed on total RNA extracted from the transfected HEK293 cells using a cDNA synthesis kit using random hexamers or gene-specific primers.

[0437] In some aspects, because RtcB is ubiquitously and endogenously expressed by mammalian cells, even linear RNA intermediates (IVT and CLV samples) when transfected into mammalian cells could result in the generation of circRNA in the cell. As shown in FIG. 4B, transfection with IVT or CLV samples also produced detectable circRNA based on qPCR of the junctional sites. However, the quantity of circRNA generated by transfection of IVT and CLV preparations was about 1.3 times lower than the amount of circRNA in the cell when the enriched circRNA preparation was transfected (FIG. 4B). The data indicate that linear RNA transcribed in vitro and cleaved (Lane 2; “CLV”) from Construct Ul can be circularized in mammalian cells.

[0438] Luciferase activity post-transfection was measured to determine the level of protein expression in cells that were transfected with the various RNA intermediates. 24, 48, 72, 96, and 120 hours post-transfection, cells were washed with PBS and lysed in 200 pL of passive lysis buffer (Promega) at 4°C for 15 minutes. 10 pL of cell lysate was transferred to a 96 well solid white plate and 90 pl of Bio-Gio substrate (Promega) was added further incubated for 5 minutes at room temperature. Relative light unit measurement was performed using a plate reader. As shown in FIG. 4C, the cleavage reaction followed by ligase and RNase R enrichment (enriched circRNA sample) substantially increased luciferase expression.

[04391 The kinetics of protein production was also measured, to assess whether the generated circRNA molecules or intermediates resulted in substantially more expression of the encoded protein over their lifetimes due to increased stability or expression than the same protein encoded by linear mRNA molecules that were not designed to generate circular RNA (in some cases, containing unmodified or modified N1 -methylpseudouridine (pU)). As shown in FIG. 4D, the half-lives of protein production of transfected linear IVT and CLV preparations were 109.2 and 121.1 hrs respectively, which was comparable to that of enriched circRNA (119.6 hrs). In comparison, linear mRNAs containing unmodified or modified uridine had half-lives of luciferase production of approximately 56.9 and 58.9 hrs, respectively (FIG. 4D). These result show that transfection of IVT and CLV preparations, intermediates of constructs designed to produce circRNA, and enriched circRNA preparation, resulted in an extended half-life of protein expression compared to expression by linear RNA expressing the same protein. The results support the utility of the exemplary constructs and RNA intermediates (e.g., IVT and CLV preparations) in successfully generating circRNA when introduced into mammalian cells, and the utility of the generated circRNA in stably expressing the delivered encoded protein for much longer time compared to the same protein expressed using a linear mRNA.

Example 5: Assessment of cleavage efficiency of bimolecular constructs encoding RNA molecules with Twister-Sister and Twister ribozymes, for circular RNA (circRNA) generation

[0440] The cleavage efficiency and ligation efficiency of various generated bimolecular constructs using a trans-acting ribozyme for circRNA generation were assessed.

[0441] For the bimolecular approach, the cleavage efficiency and generation of precursor RNA was tested using a first RNA molecule comprising a 5' TS-1, insert, and 3' substrate (exemplary Construct Bl) and a second RNA molecule comprising a trans-acting ribozyme from various classes, including a trans-acting N. vitripennis type Pl Twister ribozyme (exemplary Construct B2), pistol, hatchet, and Neurospora VS ribozymes. The 3' substrate sequence of each of the first RNA molecule included the corresponding substrate sequence of each of the trans-acting ribozyme. The trans-acting ribozymes with cleavage activity were synthesized separately from the first RNA molecules containing the substrate. The first RNA molecule containing the substrate and the second RNA molecule containing the trans-acting ribozyme were heated at 80 °C for 1 min and cooled to room temperature. 100 nM of the substrate and 5 pM of the trans-acting ribozyme were incubated together in Cleavage Buffer 1, containing MgCh, or MEGAScript T7 RNA transcription buffer for the cleavage reaction.

[0442] After the cleavage reaction to cleave the 5' TS-1 ribozyme and 3' substrate sequence mediated by the trans-acting ribozyme, the constructs were subjected to RtcB-mediated ligation. Among the tested trans-acting ribozymes, only the Construct Bl -B2 pair, with trans-acting N. vitripennis type Pl Twister ribozyme, successfully generated circRNA from the RNA molecule containing the cognate substrate (3' N. vitripennis type Pl Twister ribozyme substrate sequence). The efficiency of circularization as measured by band densitometry was 35% for cleavage in Cleavage Buffer 1, and 27% for cleavage in MEGAScript T7 RNA transcription buffer, without enrichment using RNase R (FIG. 5), showing relatively lower circularization efficiency compared to the unimolecular approach using Construct Ul. Although linear RNA produced by the transacting ribozyme showed relatively lower cleavage efficiency, the generated circular RNA was completely resistant to RNase R, which allowed for the enrichment of circRNA using RNase R (FIG. 5). The results support the utility of the described exemplary bimolecular approach, employing a 5' Twister- Sister ribozyme and a trans-acting Twister ribozyme, in successfully generating a cleaved RNA intermediate (pre-circRNA), and subsequently circRNA after ligation. The results also show that the generated circRNA can be further enriched using RNase R, indicating that the generated circRNA has similar characteristics and behavior as the circRNA generated using the exemplary unimolecular approach.

Example 6: DNA sequencing of the junctional site for circularization using the unimolecular and bimolecular approaches circular RNA (circRNA) generation

[0443] To confirm the circularization of pre-circRNA was produced using the exemplary unimolecular and bimolecular approaches described in Examples 1-5 above, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primers that target the junction site of the circularized circRNA, and the products were sequenced.

[0444] Pre-circRNA was prepared from in vitro transcripts of Construct U 1 and Construct B 1 , subject to cleavage reaction (for Construct Bl incubating together with Construct B2), and ligated in the presence or absence of RtcB ligase to generate circRNA. Reverse transcription reactions was performed on the generated circRNA molecules using a cDNA synthesis kit using gene-specific primers. The primers for amplifying the junctional site of circRNA by PCR included: 5'- GCAAGACCATGACCGAGAAG-3' (forward primer; SEQ ID NO:38), and 5'- GATCAAAACGTGGCTGGTGT-3' (reverse primer; SEQ ID NO:39); and for an internal region as a control, 5'-ACTTCTGTTACCCCGGACTG-3' (forward primer; SEQ ID NO:40), and 5'- CAAAGTAGTCGGTTCCGCTG-3' (reverse primer; SEQ ID NO:41). The PCR products were purified and sequenced to determine the sequence of the junctional site of generated circular RNA.

[0445] The expected size of PCR products amplifying the junctional sites of circRNA was detected from both the transcripts of Construct U1 and Construct B1/B2 only in the presence of RtcB but not in the absence of RtcB (FIG. 6A). These results indicate that the primers were specific for the junctional sites of circRNA generated by pre-circRNA from Construct U1 and Constructs B1/B2. The positive control PCR products specific for a non-junctional, internal site in the IRES were amplified from all transcripts regardless of RtcB ligation (see FIG. 6A). Sanger sequencing of the PCR products showed that the exact predicted sequences of the junctional site of circRNAs generated from Construct U1 and Constructs Bl /B2, confirming that both Construct U1 and Constructs B1/B2 produce circRNAs with the correct junctional site sequence (FIG. 6B). The results support the utility of the exemplary unimolecular and bimolecular approaches in efficiently generating circRNAs that are cleaved at the correct junctional site, even for RNA molecules containing large insert sequences. The results also show that both of the approaches can be used to generate identical circRNAs in terms of sequence and characteristics.

Example 7: Stable expression of gene products in cells using circRNA generated using the bimolecular approach

[0446] Circular RNA (circRNA) and various intermediates produced using the bimolecular approach as described in Examples 1, 5 and 6 above were introduced into cell lines and the expression of the encoded gene product were assessed.

[0447] HEK 293 T cells were cultured and transfected, generally as described in Example 4, with three different RNA intermediate samples generated using the Construct B1/B2 pair: Construct Bl RNA alone, which does not contain a 2', 3 '-cyclic phosphate at 3' end due to lack of treatment with the trans-acting Twister ribozyme of N. vitripennis,' Construct Bl /B2 CLV preparation, in which Constructs Bl and B2 (trans-acting Twister ribozyme of N. vitripennis) were incubated together for 1 hr for cleavage to generate a cleaved pre-circRNA; and Construct Bl /B 2 enriched circRNA preparation (“RtcB+RR”), in which Constructs Bl and B2 were incubated together for cleavage, ligated by RtcB, followed by RNase R treatment to enrich for circRNA. qRT-PCR and luciferase activity assays were performed on RNA extracted from the transfected cells, generally as described in Example 4.

[0448] Construct Bl RNA transcripts that contained only the 3' substrate sequence, which cannot be cleaved or converted into circRNA without treatment of the trans-acting Twister ribozyme of N. vitripennis of Construct B2, did not produce RT-PCR products using primers targeting the junctional site of circular RNA (FIG. 7A). The quantity of circular RNA formed in the HEK 293T cells was measured by both semi-quantitative PCR and quantitative PCR. As shown in FIG. 7B and FIG. 7C, the quantity of circRNA formed by Construct B1/B2 CLV preparation transfected into the HEK 293T cells was slightly lower than that of enriched Construct Bl /B2 RtcB+RR preparation. Linear RNAs from CLV can be converted to circRNAs in mammalian cells, whereas linear RNAs from IVT are not converted to circRNAs.

[0449] The ability of circRNA and RNA intermediates generated using the exemplary bimolecular approach to express functional luciferase in mammalian cells was assessed using a luciferase assay, generally as described in Example 4. As shown in FIG. 7D, the cleaved linear Construct B1/B2 CLV preparation showed substantially higher luciferase activity (~30X higher) than Construct Bl RNA, which is unable to circularize due to the remaining 3' substrate sequence that is not cleaved in the absence of the trans-acting ribozyme. The luciferase activity level was approximately 2 fold higher in cells transfected with enriched Construct B1/B2 RtcB+RR preparation compared to cells transfected with Construct Bl ZB2 CLV preparation (see FIG. 7D).

[0450] The results support that transfection of linear pre-circRNA preparations generated using the exemplary bimolecular approach (Construct Bl /B2 pair) results in the generation of circRNA when transfected into a mammalian cell, and leads to expression of functional luciferase in the cell. The results support the utility of the bimolecular approach in efficiently generating circRNAs, even for RNA molecules with large inserts, and leading to stable expression of the encoded protein in the cell. The results also show that the linear pre-circRNA and circRNA generated using the bimolecular approach have similar characteristics and behavior as the linear pre-circRNA and circRNA generated using the exemplary unimolecular approach.

Example 8: Generation of circRNA containing modified uridine using Twister-Sister and

Twister ribozymes [0451] The exemplary unimolecular approach using an RNA molecule that contains a 5' Twister- Sister ribozymes and a 3’ Twister ribozymes as described in Examples 1-6 above, were assessed for generating circRNA that contains modified uridine residues.

[0452] The permuted intron-exon (PIE) method of circularizing RNA utilizes fused partial exons flanked by half-intron sequences (i.e., permuted intron-exon system) that can undergo double transesterification reactions characteristic of group I catalytic introns to generate circRNAs. However, it has been reported that the PIE method cannot be used to generate a circRNA that contains pseudouridines, as the complete replacement of unmodified uridines with m h|/ abolished splicing activity, and partial replacement dramatically reduced splicing efficiency (Wesselhoeft et al., (2019), Mol Cell, 74(3), 508-520 e504).

[0453] Construct U1 described above, and a PIE method construct containing Group I intron sequences for splicing and circularization, were transcribed with a complete replacement of unmodified uridines with a N1 -methylpseudouridine. A HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) was used for in vitro transcription, replacing the UTP with an equivalent amount of Nl-methylpseudouridine-5'-triphophspate (ml\|/; Trilink), and incubating for 3 hours at 37°C.

[0454] As shown in FIG. 8, circRNA was successfully generated using Construct U1 with complete replacement of unmodified uridine with ml\|/ (lane 7, FIG. 8), with a circularization efficiency of 48%. The produced circRNA containing mli|/ was resistant to RNase R treatment, which allowed enrichment of circRNA using RNase R (lane 8, FIG. 8). In contrast, no circRNA was generated from the PIE method Group I intron-containing RNA with complete replacement with ml\|/. Furthermore, even with unmodified uridines, the circularization efficiency of Construct U1 was substantially higher than that of PIE method Group I intron-containing system (57% vs 31%, after RtcB ligation but without RNase R enrichment, see FIG. 8). The results support the advantage of the described RNA molecules containing a 5' Twister-Sister ribozyme and a 3' Twister ribozyme, in generating circRNAs that contain modified uridine residues, which were not possible using existing PIE method with Group I introns.

Example 9; Purification of circRNA and intermediates

[0455] CircRNAs and their intermediates (pre-circRNAs) generated as described above, including circRNAs containing modified uridine residues were subject to purification to remove contaminating species such as nicked circRNAs. [0456] Using the permuted intron-exon (PIE) system based on Group I introns to generate circRNAs has disadvantages in that even though circRNAs can be generated, the circRNA preparations are often contaminated with nicked circRNAs, making purification difficult. Because nicked circRNA is resistant to RNase R treatment and has the same molecular weight, complete removal of nicked circRNA is not possible using size exclusion high performance liquid chromatography (HPLC) (Wesselhoeft et al., (2018) Nat Commun, 9(1), 2629). Additionally, small amounts of nicked circRNA present have been reported to be immunogenic, inducing robust cellular immune responses and reducing the stability of circRNA preparations and protein expression (Wesselhoeft et al., (2019), Mol Cell, 74(3), 508-520 e504). There is a need to increase the purity of the circRNA preparation, for maximizing protein production and reducing innate cellular immune responses from the contaminating nicked circRNA.

[0457] As described in Example 4, pre-circRNA (cleaved linear RNA) can be directly introduced into mammalian cells for the endogenous ligase (e.g., RtcB ligase) to circularize the RNA in the cell, resulting in stable expression of the encoded proteins. To assess the potential for immunogenicity of the circRNAs and intermediates, a preparation of RNA from Construct U1 after cleavage (linear pre-circRNA) containing unmodified uridine or N1 -methylpseudouridine (m l ), were purified through HPLC after the cleavage reaction. In addition, circRNA preparations generated after ligation by RtcB treatment were also purified by HPLC.

[0458] HPLC was performed using an SRT SEC-2000 column (Sepax) and a buffer (10 mM Tris-HCl pH 6 and 1 mM EDTA), at a flow rate of 0.6 ml/min. For fractionation, peak-based volume slices (threshold intensity of 5 maU) of 0.2 ml were taken. After HPLC, a small volume was analyzed on a 2% agarose gel. The fractions were combined and concentrated using a centrifugal filter column.

[0459] As shown in FIG. 9A and FIG. 9C, highly pure preparation of linear RNAs were obtained after the cleavage reaction and purification, without the cleaved ribozyme fragments or free cleaved substrates. For the circRNA preparations after RtcB ligase incubation, resolution of the circRNA and linear RNA peaks (both for RNAs containing unmodified uridines or ml ) by HPLC was difficult, even though the molecular weight of the circRNA and the linear RNAs were different (see FIG. 9B and FIG. 9D). After HPLC, a small volume of the main fraction was analyzed on a 2% agarose gel to confirm the linear RNA with the expected size, and no difference was observed between the purified RNAs containing unmodified uridine (U) and ml\|/ (FIG. 9E). The results support that in some contexts, a highly pure RNA preparation of the circRNA intermediates of the exemplary RNA molecules as described herein, can be achieved using purification by HPLC.

Example 10; Immunogenicity of circRNA and intermediates

[0460] CircRNA preparations and their intermediates, including circRNAs containing modified uridine residues, were assessed for immunogenicity and their effect on innate immunity.

[0461] Capped and polyadenylated linear mRNAs can be recognized by stimulating toll-like receptors (TLRs), which triggers cytokine secretion, while circRNA is not recognized by TLRs. For example, TLR-3, -7, and -8 are known to recognize RNA in endosomes and initiate an inflammatory cascade; TLR-3 binds to dsRNA and stem structures in viral ssRNA; and TLR-7 and TL-R8 bind to ssRNA and nucleoside degradation products (guanosine for TLR-7 and uridine for TLR-8), with both ligands necessary for complete TLR activation. For linear capped and polyadenylated mRNA delivery, various approaches have been utilized to reduce the innate cellular immune response to the RNA, including employing nucleoside modifications such as pseudouridine ( ), N1 -methylpseudouridine (ml ), and 5-methoxyuridine (5moU), which can be reduce the activation of TLRs and RIG-I by the mRNAs.

[0462] Accordingly, the enriched circRNA preparations and purified intermediates generated as described in Examples 1-4, 8 and 9 above, were assessed for their ability to activate TLRs using a reporter cell line. HEK-Blue TLR-3, TLR-7, and TLR-8 reporter cells, in which embryonic alkaline phosphatase (SEAP) is expressed and secreted upon ligand-mediated activation of the respective TLRs, were transfected with 200 ng of HPLC-purified pre-circRNA generated from exemplary construct U1 containing unmodified uridine residues or m l using Lipofectamine MessengerMax (Invitrogen). Secreted SEAP was detected by harvesting the culture media 24 h after transfection and combined with QUANTLBlue Detection reagent (Invivogen), then incubated at 37°C overnight. Absorbance at 640 nm was measured using a plate reader. PolyFC and R848 were used as positive controls for TLR-3 and TLR-7/-8 activation, respectively. SEAP secretion was normalized to the absorbance measured in the non-treatment control group. CleanCap®Firefly Luciferase mRNA (mRNA; TriLink) was also used as a positive control.

[0463] As shown in FIGS. 10A-10C, surprisingly, purified cleaved linear RNA (pre-circRNA) from Construct Ul, containing unmodified uridine residues or ml\|/, did not elicit a TLR-mediated response in any of the reporter cells. Likewise, the mixture of enriched circRNA and cleaved linear RNA from Construct Ul, produced after incubation with RtcB and purification by HPLC, containing unmodified or ml\|/, also did not activate the TLRs tested.

[04641 These results show that cleaved linear RNA (pre-circRNA) or the mixture of enriched circRNA and linear RNA, generated after incubation with the RtcB ligase, did not elicit TLR- mediated immune response and were able to evade detection by TLRs, similar to linear mRNAs containing modified uridines such as ml\|/. In contrast, the positive control linear capped and polyadenylated mRNA encoding firefly luciferase significantly activated TLR-3 and TLR-7 reporter response (see FIGS. 10A-10B). The results support the advantage and utility of the circRNA molecules and intermediates generated as described herein, using the exemplary unimolecular and bimolecular approaches, in stably delivering and expressing gene products into cells without triggering undesired immune reactions.

Example 11; Enrichment of circRNA

[0465] CircRNA preparations generated as described above were further assessed for enrichment using polynucleotide kinase (PNK), which can render semi-cleaved RNAs to be subject to nuclease-mediated degradation, thus removing the semi-cleaved species from the RNA preparation.

[0466] Further enrichment of circRNA was performed by treatment of RNA samples after a cleavage reaction and a ligation reaction, with a T4 polynucleotide kinase (PNK), a bifunctional enzyme with 5' kinase and 3' phosphatase activities. FIG. 11A depicts a schematic showing various purification and enrichment steps that were used to purify and enrich for circRNA in the preparation. In the case of exemplary construct Ul, after the cleavage reaction, a mixture of linear RNA species including precursor RNA, pre-circRNA, and semi-cleaved RNA are purified by HPLC, which remove the cleaved, free 5' TS-1 and 3’ Twister ribozymes. Only pre-circRNA, which have both the 5' and 3' terminal ribozymes cleaved off, can be ligated to form a circRNA. Semicleaved RNAs, in which only one of the two terminal ribozymes underwent self-cleavage, either could contain a 5' hydroxyl group with the 3' Twister ribozyme intact; or a 5' Twister- Sister ribozyme intact with a 2', 3 '-cyclic phosphate at the 3' end. PNK is a bifunctional enzyme with 5'- kinase and 3 '-phosphatase activities that typically is involved in RNA and DNA repair. Particularly, the physiological substrate for the PNK phosphatase activity is an RNA with a 2’, 3 '-cyclic phosphate end. By employing the bifunctional activities of PNK, 5' hydroxyl ends of the 5' semicleaved RNA can be phosphorylated, and the 2',3'-cyclic phosphate at the 3' end of a 3' semi- cleaved RNA can be removed. After PNK treatment, a 5' phosphate-dependent exonuclease can degrade linear RNA species with 5' phosphorylated ends, which can include semi-cleaved RNA with 5' phosphorylated ends and precursor RNA; and RNase R can degrade semi-cleaved RNAs that do not have a 2', 3 '-cyclic phosphate at the 3' end, including 3' semi -cleaved RNA with the 2', 3'- cyclic phosphate removed by PNK and precursor RNA (see FIG. 11 A).

[0467] For assessing purification and enrichment, 10 pg of in vitro transcribed RNA from Construct U1 was denatured at 80 °C for 1 min then cooled down to room temperature. Then, the sample was mixed with an RNase inhibitor (New England Biolabs) and incubated in Cleavage Buffer 1 for 1 hr at RT. After cleavage, the RNA sample was cleaned up, and ligation reaction was performed by incubation with RtcB RNA Ligase, generally as described in Example 3 above. After the ligation reaction, the RNA sample was cleaned up, then incubated with the T4 polynucleotide kinase (PNK; New England Biolabs) at 37°C for 30 min, for 5' phosphorylation and 3’ hydroxylation of the uncleaved linear and semi -cleaved RNAs. After PNK treatment, the RNA sample was cleaned up, and incubated with a Terminator 5'-Phosphate dependent Exonuclease (Lucigen) at 30°C for 1 hr and/or RNase R (Lucigen) at 37°C for 15 min, for degradation of linear RNAs. The RNA sample was further cleaned up.

[0468] As shown in FIG. 11B, indeed, when RtcB ligase-treated RNAs were incubated with 5' phosphate-dependent exonuclease, circRNA were dramatically enriched, going from 53.2% to 81.9% circRNA. In addition, as treatment with PNK can remove the 2’, 3 '-cyclic phosphate end in the 3' semi -cleaved RNA (see FIG. 11 A), PNK treatment renders the 3' semi-cleaved RNA to RNase R-mediated degradation. As shown in FIG. 11B, combined treatment with PNK and RNase R resulted in dramatic enrichment of circRNA, going from 53.2% to 84.8% circRNA. When RtcB- ligase-treated RNAs were serially incubated with PNK, 5' phosphate-dependent exonuclease, and RNase R, circRNA in the sample was further enriched, up to 95.2% (FIG. 11B). The overall yield of circRNA was reduced, however, potentially due to loss from multiple incubation and cleanup procedures. The results support the advantage of using PNK, 5' phosphate-dependent exonuclease, and/or RNase R, to enrich for circRNA in an RNA preparation.

Example 12: Assessment of cleavage efficiency of 3' end Twister ribozyme in the generation of circRNA using extended end sequences

[0469] The exemplary unimolecular approach using an RNA molecule that contains a 5' Twister- Sister ribozymes and a 3’ Twister ribozyme as described in Examples 1-4 above, was assessed for the cleavage efficiency of 3' Twister ribozyme in the generation of circRNA.

[0470] For the exemplary Construct Ul, the length of the 3' ribozyme fragment after cleavage (portion of the precursor RNA that is cleaved away from the pre-circRNA after ribozyme cleavage) is relatively short (78 bp), making it difficult to detect on an electrophoresis gel. To further visualize and assess the cleavage efficiency of a 3’ Twister ribozyme, Construct Ul as described in Examples 1-4 were modified to extend the 3' end sequence (see FIG. 13A) as set forth in Table 1 below:

Table 1.

[0471] Cleavage and circularization reactions were performed generally as described in Examples 1-4. The expected size of cleaved 3’ ribozyme fragments with extended sequences were observed from each construct, in addition to 5' ribozyme fragments (FIG. 13B). Surprisingly, cleaved linear RNA of identical sizes (2468 bp) were produced from all constructs, indicating that 3' Twister ribozyme was efficiently cleaved by all constructs, including constructs with long extended 3' sequences. Linear RNA with uncleaved 3' end would have resulted in a larger band size than 2468 bp, which were not detected. Furthermore, the results show that the constructs do not lead to substantial incomplete cleavage of the 3' Twister ribozyme. The results support the advantage and utility of a 3' ribozyme comprising a catalytic sequence and a 3' substrate sequence of a Twister ribozyme in the effective cleavage for a circular RNA preparation, including using constructs with long extended 3' end sequences.

Example 13: Assessment of additional reaction conditions on improving circularization efficiency

[0472] Various reaction conditions, including buffers and buffer components were assessed for their effect in cleavage and ligation/circularization of RNA molecules to generate circular RNA (circRNA).

[0473] In some cases, cleaved ribozyme fragments could potentially interfere with the formation of the stem and loop structure, which is recognized by the RtcB ligase, by interacting with the homology region of the cleaved pre-circRNA, preventing efficient circularization of the RNA. Positive ions, such as Na⁺ and Mg²⁺ in the cleavage buffer, have the potential to partially induce renaturation between the homology region of the cleaved ribozyme fragment and the cleaved pre-circRNA. Additional cleavage buffers were tested for their effect on cleavage reaction and circularization efficiency.

A. Sodium and Magnesium Ions for Cleavage

[0474] To assess the effect of Na⁺ and Mg²⁺ in the cleavage buffer on cleavage and circularization efficiency, distilled water (DW) was added to the exemplary Construct U1 RNA samples after in vitro transcription (IVT), as opposed to proceeding to the cleavage reaction, to minimize the renaturation ("No cleavage (DW)” in FIG. 14A). The RNA was then subjected to a denaturation/renaturation reaction. Briefly, the IVT -RNA was denatured at 65°C for 2 minutes. The sample was then cooled at room temperature for 3 minutes. RNA was then purified using Monarch RNA Cleanup Kit (NEB), generating purified precursor RNA.

[0475] After the denaturation/renaturation, the RNA sample was ligated, generally as described in Example 2, and circularization efficiency was assessed compared to a cleavage buffer-exposed control pre-circRNA (“Cleavage” in FIG. 14A). FIG. 14A shows the electrophoresis profile of the no cleavage (DW) and cleavage conditions. Surprisingly, circularization efficiency was substantially improved by the no cleavage (DW) condition compared to incubation with a cleavage buffer. Additionally, the extra bands species (dotted arrows; potential concatemer band (larger size band above circular RNA band) and extra linear RNA band migrating slightly below the circRNA band), frequently observed in the cleavage condition sample, were completely absent in the sample of the no cleavage (DW) condition (FIG. 14A).

B. Manganese Ions for Ligation

[0476] The effect of the concentration of manganese during the ligation reaction by RtcB on circRNA was also assessed. Manganese has been reported to be important for efficient ligation of the 2’,3'-cyclic phosphate and the 5'-OH termini by RtcB.

[0477] Exemplary Construct U4 was cleaved and ligated for circRNA generation by RtcB in a minimum buffer (50 mM Tris, pH 7.6, 1 mM GTP) containing increasing concentrations of MnCh, as compared to the control RtcB reaction buffer with 0.25 mM MnCh (FIG. 14B). As shown in FIG. 14B, the circularization efficiency of Construct U4 decreased as manganese concentration increased in the minimum buffer as compared to the RtcB reaction buffer. Additionally, when 0.25 mM manganese was used in the RtcB reaction buffer, circularization efficiency was higher than minimum buffer with same concentration of manganese (84.4 % vs 77.2%).

C. pH

[0478] The effect of the pH on circRNA in the ligation reaction was also assessed. RNA is most stable at a pH of about 4-5, and is unstable at an alkaline pH. The RtcB reaction buffer is reported to be a weak alkaline solution (pH=8.3). To assess whether circularization efficiency would improve in lower pH conditions, cleaved Construct U4 was ligated in RtcB reaction buffer at various lower pH (7.4, 7.0, and 6.5, as compared to the control pH of 8.0). As shown in FIG. 14C, circularization efficiency was increased at pH 7.4 or pH 7.0 conditions, compared with that of pH 8.0.

[0479] The effect of the individual components in the RtcB reaction buffer (assessed at pH=7.6) on the circularization efficiency of Construct U4 was also investigated. Although the individual components did not have substantial effect on the circulation efficiency, each component increased the circulation efficiency by more than 10% at pH 7.6 compared to pH 8.3 in the RtcB reaction buffer (FIG. 14D).

D. Denaturation/Renaturation

[0480] To assess whether the properly formed stem and loop structure plays a role in optimizing RtcB ligase activity, exemplary Construct U4 was subject to denaturation/renaturation using Tris- EDTA (TE) buffer at pH 7.0 or pH 8.0. After denaturation and renaturation in TE buffer, the pre- circRNA was then ligated in the RtcB-mediated circularization reaction in the RtcB reaction buffer (50 mM Tris (pH 7.6), 75 mM KC1, 3mM MgCh, 10 mM DTT, 0.25 mM MnCh, 0.1 mM GTP, as described in FIG. 14D). As shown in FIG. 14E, this combination of denaturation/renaturation and ligation conditions increased the circularization efficiency to more than 90%.

[0481] To confirm the reproducibility of the circularization efficiency, Constructs Ul, U2, U3, and U4 as described above were assessed under denaturation and renaturation in TE buffer and ligation in RtcB reaction buffer as described above. As shown in FIG. 14F, the circularization efficiency of all constructs was over 90%, indicating that the denaturation and renaturation in TE buffer and ligation in RtcB reaction buffer reproducibly led to efficient circularization of all tested exemplary Constructs Ul, U2, U3, and U4. The results support the advantage and utility of a denaturation/renaturation reaction step, followed by ligation in a RtcB reaction buffer for efficiently generating circRNA. Example 14; Reducing heat-induced circular RNA cleavage during gel running

[0482] To analyze circular RNA after the RtcB-mediated circularization reaction, reaction samples were loaded onto pre-cast 2% E-Gel EX agarose gels (Invitrogen) using E-gel iBase (Invitrogen). E-Gel EX generates heat during this process, and this heat could cut circular RNA to produce linear RNA while the gel was running, reducing circularization efficiency.

[0483] To test whether E-Gel EX generated heat could cut circular RNA, we loaded the same circular RNA onto the gel and ran the gel at room temperature (RT) or 4°C (FIG 15A and 15B). Linear RNA without RtcB treatment was used as a control. As shown in FIGS. 15A and 15B, a linear RNA band was observed when running the gel with circular RNA under RT conditions, whereas no linear RNA band was observed when running the gel at 4°C.

[0484] To determine the stability of circular RNA under heat, we exposed circular RNA to denaturation conditions at 65°C for 3 min and then ran a gel run with two controls, circular RNA and linear RNA at 4°C. As shown in FIG. 15C, most circular RNA exposed to denaturation conditions was observed to be in a linear state, while circular RNA that was not exposed to the denaturation condition remained circular.

[0485] Even when running the gel at 4°C, we observed that circular RNA was cleaved when the running time was relatively long (about 10 minutes) (FIG. 16A). To determine if the heat generated while running the gel at 4°C can cleave circular RNA, we reduced the gel running time to 3 minutes at 4°C and then incubated the gel on ice for about 10 minutes to cool it down, repeating the process about 3 times, and finally running the gel at 4°C for 3 minutes (FIG.16B). After this process, we were able to prevent the cleavage of circular RNA during gel running at 4°C. These results clearly show that circular RNA can be cleaved even by the heat generated during running gels at 4°C for relatively long periods.

[0486] Based on this method of repeating the conditions of running the gel at 4°C and cooling it on ice, we succeeded in circulating RNAs of various lengths, including EGFP (SEQ ID NO: 66, 720 bp), human erythropoietin (hEPO, SEQ ID NO: 68, 582 bp), firefly luciferase (FLuc, SEQ ID NO: 16, 1653 bp), and Cas9 (SEQ ID NO: 69, 3270 bp), into RtcB with 100% efficiency. (FIG. 17). Agarose gel running

[0487] To check the circularization and purity of circular RNA, 250 ng of circularized RNA was separated on precast 2% E-Gel EX agarose gel (Invitrogen) on the E-gel iBase (Invitrogen) at room temperature (RT) or 4°C with two modified methods. Briefly, 2 % E-Gel EX agarose gel was cooled in the ice before loading and running. The RNA sample was loaded with 2X RNA Loading Dye (NEB) and running gel using E-Gel 1-2% program for 3 min at 4°C, then cool down the gel on ice for 10 min. Repeat this step 3 times which makes 12 min total running time. Alternatively, the RNA sample was loaded with 2X RNA Loading Dye (NEB) and running gel using E-Gel 1-2% program for 2 min, then cooled down the gel on ice for 3 min. Repeat this step as 5 times which makes 10 min total running time at RT. ssRNA ladder (NEB) was used as standard-size marker, in vitro transcribed RNA without circularization was used as a linear RNA control, which was mixed with 2X RNA Loading Dye and denatured at 65° C for 2 min and immediately transferred on ice 2 min before loading into the E-Gel. The bands image was visualized using the iBright CL150 (Invitrogen) imaging system and was quantified using Image Lab software (Bio-Rad).

Example 15: Engineering IRES of circRNA to improve protein expression

[0488] The internal ribosome entry site (IRES) determines the protein expression level of genes located in circular RNA by interacting with important translational factors, including eIF4F and eIF4A. We first constructed LVT14 expressing FLuc based on the IRES of the coxsackievirus B3 (CVB3, FIG.18A). The IRES RNA sequences tested included IRES sequences from Coxackie Bl virus (CVB1, SEQ ID NO: 71), encephalomyocarditis virus (EMCV, SEQ ID NO: 73), Epstein- Barr nuclear antigen 1 (EBNA1, SEQ ID NO: 75), enterovirus serotype EV-B107 (SEQ ID NO: 77), enterovirus serotype EV-D94 (SEQ ID NO: 79), Echovirus El 1 (EchoVl 1, SEQ ID NO: 81), Coronavirus disease 19 (Covidl9, SEQ ID NO: 83), coxsackievirus A20 (CVA20, SEQ ID NO: 85), poliovirus serotype 3 (PV3, SEQ ID NO: 87), Simian V4 (SEQ ID NO: 89), Human Rhinovirus Al (HRV-A1, SEQ ID NO: 91), Hepatitus C virus (HCV, SEQ ID NO: 93), Human Rhinovirus A21 (HRV-A21, SEQ ID NO: 95), Human Rhinovirus B17 (HRV-B17, SEQ ID NO: 97), Human Rhinovirus (HRV-A100, SEQ ID NO: 99), Human Rhinovirus B37 (HRV-B37, SEQ ID NO: 101), Human Rhinovirus B92 (HRV-B92, SEQ ID NO: 103), Human Rhinovirus B3 (HRV-B3, SEQ ID NO: 105), and Human Rhinovirus C54 (HRV-C54, SEQ ID NO: 107). Nineteen IRES derived from different viruses showed greater activity than CVB3 IRES except EMCV, EBNA1 EchoVl 1, Covidl9 and CVA20 (Chen et al., 2023). To test whether the 19 viral IRES introduced can increase protein expression compared to the CVB3 IRES, we compared their functions by measuring the activity of FLuc expressed by each IRES. In terms of FLuc activity, these IRES did not induce significantly higher protein expression than the CVB3 IRES. Some of these, for instance an IRES from EV-B107, HRV-B3, HRV-A1 and HRV-B92 had much stronger activity in their construct expressed in HEK293 cells as around 20-fold higher compared to the one from CVB3. Nevertheless, two IRES, EV-B107 and HRV-B3, were found to be approximately 1.5-fold higher than CVB3 IRES in FLuc activity. (FIG. 18B).

[0489] The trans-elements to recruit factors that regulate mRNA translation such as RNA binding proteins, poly(A)-binding protein (PABP), poly(C)-binding protein (PCBP) as well as translation initiation factors eIF4G, affects CVB3 IRES activity for downstream gene expression. (Chen et al., 2023). The LVT14 platform containing CVB3 IRES/FLuc was used to test how PABP- binding RNA binding elements or eIF4G-binding aptamers affect IRES function in circular RNAs. We designed several constructs based on LVT-14 with a combination of binding sites for these two translational factors, which include LVT-18 (SEQ ID NO: 109), LVT-20 (SEQ ID NO: 111), LVT- 22 (SEQ ID NO: 113), and LVT14/eIF4G (SEQ ID NO: 115) (FIG. 19A). We found that all the constructs we modified exhibited higher FLuc activity than LVT-14 under the control of coxsackievirus wild-type IRES. The LVT-18, which includes 5’ PABP binding site and eIF4G binding site in the middle of CVB3 IRES showed 2-fold higher than LVT-14 (FIG. 19B). LVT14/eIF4G, in which only e!F4G is located between IRES and FLuc, also showed similar levels of FLuc activity to LVT18, with about 2-fold higher FLuc activity compared to LVT14. LVT-20, which moved the eIF4G binding site of LVT18 between the CVB3 IRES and FLuc, showed slightly higher levels than LVT18 and about 2.5-fold higher FLuc activity than LVT14. Interestingly, LVT- 22, which only contains a PABP binding site at the 5' end of the CVB3 IRES, also exhibited slightly higher FLuc activity than LVT18 and approximately 2.5-fold higher FLuc activity when compared to LVT14. This result suggests that the elements for PABP binding alone, without the eIF4G aptamer, are sufficient to enhance CVB3 IRES activity on the LVT platform.

Cloning and mutagenesis

[0490] To generate a modified construct, an open reading frame of hEPO, various IRES fragments were synthesized from TelesisBio and cloned into a linearized LVT-14 backbone DNA by Gibson assembly. To modify or add a regulatory element for translation, Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used according to the manufacturer’s instructions. For testing various IRESes, the information of sequence was from Chen R et al., 2022.

Circular RNA production, purification, and analysis

[0491] Circular RNA precursors were synthesized by in-vitro transcription (IVT) from a linearized plasmid DNA template using a MEGAscript T7 Kit (Invitrogen, AMB 1334-5) according to the manufacturer's instructions. For circularization, the IVT RNA was treated with 3 pM RtcB ligase along with a buffer containing 50 mM Tris-HCl, 75 mM KC1, 3 mM MgC12, 10 mM DTT, 100 p.M GTP, 100 pM MnC12, and 40U RNase inhibitor for 15 minutes at 37°C. Subsequently, the RNA was column-purified using a Monarch RNA Cleanup kit.

[0492] To purify circular RNA using high-performance liquid chromatography (HPLC), 1-2 mg of RNA was passed through a 21.2 x 300 mm size-exclusion column with a particle size of 5 pm and a pore size of 2000 A (Sepax Technologies; part number: 215980-21230) on an Agilent 1260 Series HPLC (Agilent). RNA was run in RNase-free TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) at a flow rate of 3.0 mL/minute. The RNA was detected by UV absorbance at 260 nm but was collected without UV detection.

[0493] Fractions containing circular RNA were selected by separating the RNA on precast 2% E-gel EX agarose gels (Invitrogen) on the E-gel iBase (Invitrogen) using the E-gel EX l%-2% program. Subsequently, fractions containing circular RNA were pooled and concentrated using the Amicon® Ultra-15 Centrifugal Filter Unit (Millipore Sigma, UFC900308). The purity of circular RNA was determined by separating the RNA on precast 2% E-gel EX agarose gel and the RNA was stored at -80°C until use.

Example 16: Resistance of circular RNA to detection by TLRs, RIG-L and MDA5

[0494] The innate immune response is known to be triggered by RNA through interaction with pattern recognition receptors (PRRs) such as TLR3 (Agonist: dsRNA), TLR7/8 (Agonist: ssRNA), RIG-I (Agonist: 5’ triphosphate on short dsRNAs), and MDA5 (Agonist: long double-stranded RNA). To investigate whether our circular RNA can activate the innate immune response by stimulating these PRRs, HEK-TLR reporter cell lines overexpressing human TLR3, 7, 8, or RIG1 were treated with HPLC-purified linear RNA, circular RNA, or mRNA (5MoU) for 24 hours. We then measured SEAP, whose expression is induced by NF-KB activation, or lucia-luciferase activity, whose expression is induced by IRF3/7 activation, to measure TLR-dependent NF-KB activation or RIG1 -dependent IRF3/7 activation in each of the corresponding cell lines, respectively.

[0495] When the reporter cell lines were treated with highly pure linear and circular RNA, SEAP secretion in HEK-TLR3, -TLR7, or -TLR8 was not detected upon treatment with linear RNA or circular RNA, whereas each PRR ligand, such as polyFC (TLR3 agonist), R848 (TLR7/8 agonist), substantially induced SEAP in the cells expressing the corresponding receptors (FIGS. 20A to 20D) [0496] There are conflicting results on the immunogenicity that circular RNAs can cause. Previously, Daniel G. Anderson's group reported that highly pure circular RNA does not induce immunogenicity because it does not stimulate RIG-I (Wesselhoeft et al., 2019). Howard Y. Chang's group reported that foreign circular RNA, such as synthetic circular RNA lacking adenine methylation, induces immunogenicity by stimulating RIG-I. However, his group did not provide information on the purity of the circular RNA they used in their paper. Therefore, to determine whether the immunogenicity induced by circular RNA was due to contamination of impurities during circular RNA purification, we investigated immunogenicity using highly pure circular RNA (FIG. 20A).

[0497] To investigate whether RIG-I is stimulated by circular RNA, we treated the RIG-I reporter cell line with high-purity linear and circular RNA. As shown in FIG. 20E, no luciferase activity of HEK-RIG1 was detected upon linear RNA or circular RNA treatment, whereas each PRR ligand, such as polyLC (TLR3 agonist) and 3p-hpRNA (RIG1 agonist), significantly induced luciferase secretion in cells expressing the corresponding receptor. To further investigate whether RIG-I or MDA5 recognizing double-stranded RNA is a crucial factor in the induction of immune responses by circular RNA, RIG-I or MDA5 deficient A549 reporter cell lines were treated with highly pure linear and circular RNA. These cell lines express Lucia-luciferase reporter protein to monitor IRF3/7 activation in the absence of RIG-I or MDA5, allowing us to examine the immunogenicity induced by the interaction between circular RNA and these two receptors (FIG 20F and 20G). Interestingly, neither linear RNA nor circular RNA induced Lucia-luciferase expression in RIG-I or MDA5 deficient A549 reporter cell lines, suggesting that pure circular RNA does not interact with RIG-I or MDA5 and consequently does not induce immunogenicity.

[0498] Finally, to investigate whether circular RNA can induce the expression of pro- inflammatory cytokines, A549 WT cells were treated with the highly pure linear and circular RNA, and the expression of IL-6, IFN- , and RANTES was examined from the supernatants using ELISA (FIG. 21). As expected, neither linear RNA nor circular RNA induced the expression of inflammatory cytokines, but the positive controls, 3p-hpRNA and Poly IC, did. In the case of linear RNA, the 5'OH and 3' cyclic phosphates of linear RNA formed as a result of ribozyme-mediated cleavage are present. These two moieties are recognized and ligated by the endogenous RtcB enzyme and converted to circular RNA in the cells, which is a possible mechanism by which RIG-I activation can be avoided. [0499] In conclusion, highly pure circular RNA produced by the present technology does not induce immunogenicity as it avoids recognition by TLRs, RIG-I, and MDA5. Considering that immunogenicity limits protein expression and induces side effects in vivo, the use of highly pure circular RNA is a factor in circular RNA-based drug development.

Preparation of Lipid nanoparticle (LNP)-RNA complexes

[0500] l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), N-palmitoyl-sphingosine-1- { succinyl [methoxy(poly ethylene glycol)2000]} (PEG2000Ceramide) and 1,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). SM102 was purchased from MedKoo Biosciences, Inc. (Morrisville, NC). 1,2-Dimyristoyl-sn- glycerol, methoxypolyethylene Glycol (DMG-PEG 2,000) was purchased from NOF EUROPE GmbH (Frankfurt am Main, Germany). STP1244, SMARTCap® FLuc mRNA (mly) and hEPO (ml\|/) were obtained from STPharm (Seoul, South Korea). CleanCap® hEPO mRNA (5MoU) was obtained from TriLink Biotechnologies (San Diego, CA, USA).

[0501] The lipid nanoparticle (LNP) formulation process was prepared by mixing the ethanol and aqueous phases at a 1:3 volumetric ratio using the NanoAssemblr Platform (Precision Nanosystems Inc., Vancouver, BC, Canada) in a staggered herringbone micromixer. The ethanol phase was prepared by dissolving SM102, DSPC, cholesterol, and DMG-PEG 2,000 (SM102-LNP) or STP1244, DOPE, cholesterol, and PEG2000Ceramide (STP1244-LNP) in a molar ratio of 50: 10:38.5: 1.5 or 36.5: 15:47: 1.5, respectively. The aqueous phase consisted of mRNA or circRNA dissolved in a 25 mM sodium acetate buffer (pH 5). The LNPs were dialyzed against PBS using Slide-A-Lyzer G2 Dialysis Cassettes, 10,000 MWCO (Thermo Fisher) overnight at room temperature. To determine the concentration of RNA encapsulated in the LNPs, the Quant-iT RiboGreen assay (Thermo Fisher) was employed following the manufacturer’s protocol. The efficiency of RNA encapsulation into LNPs was assessed by comparing measurements in the absence and presence of 1% (v/v) Triton X-100. The nanoparticle size, poly dispersity (PDI), and z- potential were analyzed using dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK).

Immunogenicity assay

[0502] HEK-Blue human TLR3, TLR7, TLR8, HEK-Lucia™ RIG-I cells, A549-Dual™ KO- RIG-I cells, A549-Dual™ K0-MDA5 cells, and A549 cells (50,000 cells/200 pL/well in a 96-well plate) were treated with 362 fmol of RNA encapsulated with LNP or R848, polyFC, and 3p- hpRNA, which were encapsulated with Lipofectamine MessengerMax (Thermo Fisher Scientific) for 24 hours. To determine SEAP secretion in HEK-Blue TLR 3, 7, 8 cells upon RNA-LNP treatment, culture supernatant was harvested 24 hours after transfection and incubated with HEK- Blue detection reagent (Invivogen) for 3 hours at 37°C. The absorbance at 640 nm was measured using GloMax® Explorer Multimode Microplate Reader (Promega). For determining Lucia- Luciferase activities in HEK-Lucia RIGl, A549-Dual™ KO-RIG-I Cells, and A549-Dual™ KO- MDA5 Cells in the treatment with RNA-LNP, the culture supernatant was harvested at 24 hours, and incubated with QUANTI-Luc™ 4 substrate reagent (InvivoGen) for 3 minutes. Luciferase activity was measured using GloMax® Explorer Multimode Microplate Reader (Promega). To investigate the production of IL-6, IFN-0, and RANTES in A549 cells upon RNA-LNP treatment, culture supernatants were harvested at 24 hours and subjected to ELISA according to the manufacturer's protocol (Biolegend, San Diego, CA). 293T and A549 cells were purchased from ATCC. HEK-Blue human TLR3, TLR7, TLR8, HEK-Lucia™ RIG-I Cells, A549-Dual™ KO-RIG- I Cells, A549-Dual™ K0-MDA5 Cells, and R848, polyLC, and 3p-hpRNA were obtained from InvivoGen.

Example 17; Circular RNA shows significantly higher protein expression and duration both ex vivo and in vivo compared to mRNA

[0503] According to previous studies, circular RNA is known to be more stable, resulting in higher protein expression in cells compared to mRNA (Wesselhoeft et al., 2018). To investigate whether this phenomenon is also observed in our circular RNA, we conducted experiments comparing protein expression by treating 293T cells with circular RNA or mRNA encoding firefly Luciferase (FFLuc) for 7 days.

[0504] As shown in FIG. 22, the cumulated firefly luciferase activity in the 293T cells treated with circular RNA over 7 days was approximately 45 times higher FLuc activity than that induced by linear RNA. Additionally, the half-life of FLuc activity by linear RNA was observed to be 54 hours, while circular RNA exhibited a half-life of approximately 158 hours, indicating it is about three times longer.

[0505] Finally, to determine if this phenomenon also occurs in vivo, we administered mRNA and circular RNA intramuscularly to the hind limbs of Balb/c mice and then assessed protein expression levels using in vivo bioluminescence imaging experiments (FIG. 23). Similar to the trend of the experimental results observed in ex vivo, the in vivo FLuc activity induced by the circular RNA was 7.6-fold higher than that induced by the mRNA. In addition, the FLuc activity by mRNA sharply decreased after 24 hours while FLuc activity by circular RNA was observed until 9 days.

[0506] In conclusion, our study demonstrates that circular RNA exhibits superior protein expression levels and duration both in cells and in vivo compared to mRNA. We used LVT18-based circular RNA in the ex vivo and in vivo experiments described above. Compared to LVT14, LVT18 showed higher FLuc activity both ex vivo and in vivo, suggesting that IRES engineering is an important factor in determining the level of protein expression by circular RNAs in vivo as well as ex vivo.

Ex vivo protein expression of FLuc

[0507] 293 T cells (10,000 cells/5 mL/well in a 6-well plate) were treated with 4.2 pmol of FLuc mRNA (ml\|/)- or FFLuc circular RNA-encapsulated with STP1244-LNP for 7 days. Every day the cells were harvested and lysed with Passive Lysis Buffer (Promega) and incubated with Dual-Glo luciferin reagent (Promega) for 3 min on a microplate shaker. Then, bioluminescence was measured with a GloMax® Explorer Multimode Microplate Reader (Promega).

In vivo Bioluminescence Imaging

[0508] In vivo Bioluminescence Imaging was conducted by Explora BioLabs, Inc. (San Diego, CA). Briefly, eight-week-old female Balb/C mice (n = 5 for each group) were intramuscularly injected with 13.78 pmol/50 pL of FLuc mRNA (ml\|/)- or FFLuc circular RNA-encapsulated with STP1244-LNP. In vivo Bioluminescence imaging (BLI) for firefly luciferase was performed daily for up to 9 days. At each time point, the animals were subcutaneously injected with 0.1 mL of 30 mg/mL XenoLight D-Luciferin Potassium Salt (PerkinElmer 122799) and imaged on the ventral side 15 minutes later (exposure time=auto, binning=medium, and f/stop=l). Total Flux (photon/ second) was measured using Living Image software.

Statistical Analysis

[0509] One-way analysis of variance (ANOVA) post hoc tests was used to determine the significance of differences between experimental and relevant control values within each experiment. All analyses were performed using the Graph Pad Prism 8.0 statistical software. Significant differences were reported as *p < 0.05, **p < 0.01, and ***p < 0.001.

References

[0510] Chen, R., Wang, S. K., Belk, J. A., Amaya, L., Li, Z., Cardenas, A., Abe, B. T., Chen, C. K., Wender, P. A., & Chang, H. Y. (2023). Engineering circular RNA for enhanced protein production. Nat Biotechnol, 41(2), 262-272. https://doi.org/10.1038/s41587-022-01393-0

[05111 Wesselhoeft, R. A., Kowalski, P. S., & Anderson, D. G. (2018). Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun, 9(1), 2629. https://doi.org/10.1038/s41467-018-05096-6

[0512] Wesselhoeft, R. A., Kowalski, P. S., Parker-Hale, F. C., Huang, Y., Bisaria, N., & Anderson, D. G. (2019). RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo. Mol Cell, 74(3), 508-520 e504. https://doi.Org/10.1016/j.molcel.2019.02.015

[0513] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Sequences

Claims

2. The RNA molecule of claim 1, wherein the 5' substrate sequence comprises a 5' overhang sequence, and the 3' substrate sequence comprises a 3' overhang sequence

3. The RNA molecule of claim 1 or 2, wherein the 5' ribozyme and the 3' ribozyme together are capable of cleaving the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence; the insert sequence; and the 3' overhang sequence.

4. The RNA molecule of any of claims 1-3, further comprising a 5' homology region and 3' homology region.

5. The RNA molecule of claim 4, wherein the 5' homology region is located 3 ' of the 5' ribozyme.

6. The RNA molecule of claim 4 or 5, wherein the 3' homology region is located 3' of the insert sequence.

8. The RNA molecule of any of claims 4-7, wherein at least a portion of the 5' homology region and at least a portion of 3' homology region are complementary and are capable of forming a stem structure.

9. The RNA molecule of any of claims 3-8, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus.

10. The RNA molecule of claim 9, wherein the hydroxyl group at the 5' terminus and the 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase to generate a circular RNA molecule.

11. The RNA molecule of claim 10, wherein the RNA ligase is a tRNA splicing ligase.

12. The RNA molecule of claim 10 or 11, wherein the RNA ligase is an RtcB ligase.

13. The RNA molecule of any of claims 10-12, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

15. The RNA molecule of any of claims 1-14, wherein the Twister- Sister ribozyme comprises a Twister- Si ster-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme.

16. The RNA molecule of any of claims 1-15, wherein the Twister- Sister ribozyme comprises a TS-1 ribozyme.

17. The RNA molecule of any of claims 1-16, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6.

18. The RNA molecule of any of claims 1-17, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6.

19. The RNA molecule of any of claims 1-18, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:4.

20. The RNA molecule of any of claims 1-19, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

21. The RNA molecule of any of claims 1-20, wherein the Twister ribozyme comprises a type Pl Twister ribozyme.

22. The RNA molecule of any of claims 1-21, wherein the Twister ribozyme comprises a Nasonia vitripennis Type Pl Twister ribozyme.

23. The RNA molecule of any of claims 1-22, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:21.

24. The RNA molecule of any of claims 1-23, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

25. The RNA molecule of any of claims 1-24, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO 23.

26. The RNA molecule of any of claims 1-25, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23.

27. The RNA molecule of any of claims 1-26, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NOTO.

28. The RNA molecule of any of claims 1-27, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10.

29. The RNA molecule of any of claims 1-28, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:19.

30. The RNA molecule of any of claims 1-29, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19.

31. The RNA molecule of any of claims 1-30, wherein the insert sequence comprises a translation initiation element.

32. The RNA molecule of claim 31, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element.

33. The RNA molecule of claim 31 or 32, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:14.

34. The RNA molecule of any of claims 31-33, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

35. The RNA molecule of any of claims 1-34, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules.

36. The RNA molecule of claim 35, wherein the one or more exogenous molecule is selected from among a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecule comprises a vaccine antigen.

38. The RNA molecule of claim 37, wherein the vaccine antigen comprises a viral vaccine antigen.

39. The RNA molecule of claim 38, wherein the vaccine antigen comprises a cancer antigen, optionally a cancer neoantigen.

40. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecule comprises a sequence-specific nuclease.

41. The RNA molecule of claim 40, wherein the sequence-specific nuclease is a Cas nuclease.

42. The RNA molecule of claim 41, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas 12 nuclease, or a Cas 13 nuclease.

43. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

44. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecule comprises an immunomodulatory polypeptide.

45. The RNA molecule of claim 44, wherein the immunomodulatory polypeptide comprises a cytokine.

46. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecule comprises a transcription factor.

47. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecule comprises a reporter molecule.

48. The RNA molecule of claim 47, wherein the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP).

49. The RNA molecule of claim 48, wherein the reporter molecule comprises a firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16.

50. The RNA molecule of any of claims 1-49, wherein the insert sequence is at least about 500 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

51. The RNA molecule of any of claims 1-50, wherein among a population of the RNA molecules, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

52. The RNA molecule of any of claims 3-13 and 15-51, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

53. The RNA molecule of any of claims 1-52, wherein among a population of the RNA molecules, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase.

54. The RNA molecule of any of claims 10-53, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

55. The RNA molecule of any of claims 1-54, wherein the RNA molecule comprises a modified nucleoside.

56. The RNA molecule of claim 55, wherein the modified nucleoside comprises a pseudouridine or a N1 -methylmethylpseudouridine.

57. The RNA molecule of any of claims 1-56, wherein the RNA molecule leads to reduced activation of one or more toll-like receptors (TLRs) or evades detection by one or more TLRs when incubated with a cell comprising the TLR.

59. The combination of claim 58, wherein the 5' substrate sequence comprises a 5' overhang sequence.

60. The combination of claim 58 or 59, wherein the 5' ribozyme of the first RNA molecule and the trans-acting ribozyme of the second RNA molecule together are capable of cleaving the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence; the insert sequence; and the 3' overhang sequence.

61. The combination of claim 60, wherein the cleaved RNA molecule further comprises a 5' homology region and 3' homology region.

62. The combination of claim 61, wherein the 5' homology region is located 3' of the 5' ribozyme.

63. The combination of claim 61 or 62, wherein the 3' homology region is located 3' of the insert sequence.

64. The combination of any of claims 61-63, wherein at least a portion of the 5' homology region and at least a portion of 3' homology region are complementary and are capable of forming a stem structure.

65. The combination of any of claims 60-64, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus.

66. The combination of claim 65, wherein the hydroxyl group at the 5' terminus and the 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule are capable of being ligated together in the presence of an RNA ligase to generate a circular RNA molecule.

67. The combination of claim 66, wherein the RNA ligase is a tRNA splicing ligase.

68. The combination of claim 66 or 67, wherein the RNA ligase is an RtcB ligase.

69. The combination of any of claims 66-68, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

70. The combination of any of claims 58-69, wherein the Twister- Sister ribozyme comprises a Twister- Si ster-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme.

71. The combination of any of claims 58-70, wherein the Twister- Sister ribozyme comprises a TS-1 ribozyme.

72. The combination of any of claims 58-71, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:6.

73. The combination of any of claims 58-72, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6.

74. The combination of any of claims 58-73, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:4.

75. The combination of any of claims 58-74, wherein the catalytic sequence of a Twister- Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

76. The combination of any of claims 58-75, wherein the Twister ribozyme comprises a type Pl Twister ribozyme.

77. The combination of any of claims 58-76, wherein the Twister ribozyme comprises a Nasonia vitripennis Type Pl Twister ribozyme.

78. The combination of any of claims 58-77, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:21.

79. The combination of any of claims 58-78, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

80. The combination of any of claims 58-79, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 23.

81. The combination of any of claims 58-80, wherein the catalytic sequence of a Twister ribozyme comprises the sequence set forth in SEQ ID NO:23.

82. The combination of any of claims 58-81, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 10.

83. The combination of any of claims 58-82, wherein the 5' homology region comprises the sequence set forth in SEQ ID NO: 10.

84. The combination of any of claims 58-83, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 19.

85. The combination of any of claims 58-84, wherein the 3' homology region comprises the sequence set forth in SEQ ID NO: 19.

86. The combination of any of claims 58-85, wherein the insert sequence comprises a translation initiation element.

87. The combination of claim 86, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element.

88. The combination of claim 86 or 87, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:14.

89. The combination of any of claims 86-88, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

90. The combination of any of claims 58-89, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules.

91. The combination of claim 90, wherein the one or more exogenous molecule is selected from among a vaccine antigen, a cancer antigen, optionally a cancer neoantigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

92. The combination of claim 90 or 91, wherein the one or more exogenous molecule comprises a vaccine antigen.

93. The combination of claim 92, wherein the vaccine antigen comprises a viral vaccine antigen.

94. The combination of claim 92, wherein the vaccine antigen comprises a cancer antigen, optionally a cancer neoantigen.

95. The combination of claim 90 or 91, wherein the one or more exogenous molecule comprises a sequence-specific nuclease.

96. The combination of claim 95, wherein the sequence-specific nuclease is a Cas nuclease.

97. The combination of claim 96, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas 12 nuclease, or a Cas 13 nuclease.

98. The combination of claim 90 or 91, wherein the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

99. The combination of claim 90 or 91, wherein the one or more exogenous molecule comprises an immunomodulatory polypeptide.

100. The combination of claim 99, wherein the immunomodulatory polypeptide comprises a cytokine.

101. The combination of claim 90 or 91, wherein the one or more exogenous molecule comprises a transcription factor.

102. The combination of claim 90 or 91, wherein the one or more exogenous molecule comprises a reporter molecule.

103. The combination of claim 102, wherein the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP).

104. The combination of claim 103, wherein the reporter molecule comprises a firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16.

105. The combination of any of claims 58-104, wherein the insert sequence is at least about 500 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length.

106. The combination of any of claims 58-105, wherein among a population of the RNA molecules, cleavage of the 5' substrate sequence and/or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

107. The combination of any of claims 60-106, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

108. The combination of any of claims 58-107, wherein among a population of the RNA molecules, ligation of the 5' terminus and the 3' terminus occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population, in the presence of an RNA ligase.

109. The combination of any of claims 66-108, wherein among a population of the RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

110. The combination of any of claims 58-109, wherein the RNA molecule comprises a modified nucleoside.

111. The combination of claim 110, wherein the modified nucleoside comprises a pseudouridine or a N1 -methylmethylpseudouridine.

112. The combination of any of claims 58-111, wherein the RNA molecule leads to reduced activation of one or more toll-like receptors (TLRs) or evades detection by one or more TLRs when incubated with a cell comprising the TLR.

113. A deoxyribonucleic acid (DNA) molecule encoding the RNA molecule of any of claims 1-57, the first RNA molecule of the combination of any of claims 58-112, the second RNA molecule of the combination of any of claims 58-112, or the first RNA molecule and the second RNA molecule of the combination of any of claims 58-112.

114. A system for generating a circular RNA molecule comprising the RNA molecule of any of claims 1-57, or the combination of any of claims 58-112.

115. A system for generating a circular RNA molecule comprising the DNA molecule of claim 113, and a reagent for in vitro transcription.

116. The system of claim 114 or 115, further comprising an RNA ligase.

117. The system of claim 116, wherein the RNA ligase is a tRNA splicing ligase.

118. The system of claim 116 or 117, wherein the RNA ligase is an RtcB ligase.

119. The system of any of claims 116-118, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

120. A method for generating a ribonucleic acid (RNA) molecule, the method comprising producing the RNA molecule of any of claims 1-57.

122. A method for generating a combination of ribonucleic acid (RNA) molecules, the method comprising producing the combination of any of claims 58-112.

123. A method for generating a combination of ribonucleic acid (RNA) molecules, the method comprising producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme.

124. The method of any of claims 120-123, further comprising incubating the RNA molecule in a solution, thereby generating a cleaved RNA molecule.

125. A method for generating a cleaved RNA molecule, the method comprising:

(1) producing the RNA molecule of any of claims 1-57; and

126. A method for generating a cleaved RNA molecule, the method comprising:

127. A method for generating a cleaved RNA molecule, the method comprising:

(1) producing the combination of any of claims 58-112; and

128. A method for generating a cleaved RNA molecule, the method comprising:

Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme; and

129. The method of any of claims 124-128, further comprising incubating the RNA molecule with an RNA ligase, thereby generating a circular RNA molecule.

130. A method for generating a circular RNA molecule, the method comprising:

(1) producing the RNA molecule of any of claims 1-57;

131. A method for generating a circular RNA molecule, the method comprising:

132. A method for generating a circular RNA molecule, the method comprising:

(1) producing the combination of any of claims 58-112;

133. A method for generating a circular RNA molecule, the method comprising:

Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme; (2) incubating the first RNA molecule and the second RNA molecule in a solution, thereby generating a cleaved RNA molecule; and

134. A method for generating a circular RNA molecule, the method comprising:

(1) producing the RNA molecule of any of claims 1-57;

135. A method for generating a circular RNA molecule, the method comprising:

136. A method for generating a circular RNA molecule, the method comprising:

(1) producing the combination of any of claims 58-112;

137. A method for generating a circular RNA molecule, the method comprising:

(1) producing a combination of RNA molecules comprising: a first RNA molecule comprising, in 5' to 3' order: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister- Sister ribozyme; an insert sequence; and a 3' substrate sequence of a Twister ribozyme comprising a 3' overhang sequence; and a second RNA molecule comprising a trans-acting ribozyme comprising a catalytic sequence of a Twister ribozyme;

138. The method of any of claims 134-137, wherein the circular RNA is generated by an RNA ligase endogenously present in the subject.

139. The method of any of claims 120-138, wherein the RNA molecule or the first RNA molecule or the second RNA molecule of the combination is produced by in vitro transcription.

140. The method of any of claims 120-139, wherein the RNA molecule or the first RNA molecule or the second RNA molecule of the combination is produced by RNA synthesis.

141. The method of any of claims 120-140, wherein the incubation in step (2) generates a cleaved RNA molecule after cleavage of the 5' substrate sequence and the 3' substrate sequence by the catalytic sequences.

142. The method of any of claims 120-141, wherein the incubation in step (2) generates a hydroxyl group at the 5' terminus, and a 2', 3 '-cyclic phosphate at the 3' terminus of the cleaved RNA molecule.

143. The method of any of claims 124-142, wherein the solution comprises sodium acetate (Na20Ac), magnesium acetate (MgOAc2) or both.

144. The method of any of claims 124-143, wherein the solution does not comprise potassium chloride (KC1) or magnesium chloride (MgCk).

145. The method of any of claims 124-144, wherein the solution comprises cyclic diguanosine monophosphate (c-di-GMP).

146. The method of claim 145, wherein the solution comprises c-di-GMP at a concentration of between about 0.5 mM and about 10 mM.

147. The method of claim 145 or 146, wherein the solution comprises c-di-GMP at a concentration of about 5 mM.

152. The method of claim 151, wherein the denaturation step is performed between about 60°C and about 85 °C, or about 65 °C and about 80 °C, or at about 65 °C, or at about 80 °C.

153. The method of claim 151 or 152, wherein the renaturation step comprises incubating at ambient temperature or 4 °C after the denaturation step.

154. The method of any of claims 151-153, wherein the denaturation step and the renaturation step are performed in the solution for generating the cleaved RNA molecule.

155. The method of claim 154, wherein the solution comprises Tris-EDTA (TE) buffer, optionally TE buffer pH 7.0 or TE buffer pH 8.0.

161. The method of any of claims 129-133 and 139-160, wherein the incubating with the

RNA ligase in step (3) is performed between about 30°C and about 40 °C, about 35 °C and about 39 C, or about 36 °C and about 38 °C.

164. The method of claim 163, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that comprises Tris-HCl, KC1, MgCh, and DTT.

165. The method of claim 163 or 164, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that comprises 50 mM Tris-HCl, 75 mM KC1, 3 mM MgCk, and 10 mM DTT.

166. The method of any of claims 129-133 and 139-162, wherein the incubating with the RNA ligase in step (3) is performed in a buffer that does not comprise Mg²⁺.

169. The method of any of claims 129-168, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence that has at least at or about 90%, 95%, 96%>, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO:43.

171. The method of claim 170, wherein the purifying is carried out by chromatography.

172. The method of claim 171, wherein the chromatography comprises high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEC) or size exclusion chromatography-high performance liquid chromatography (SEC-HPLC).

174. The method of claim 173, wherein the enriching is carried out by incubation with a kinase.

175. The method of claim 174, wherein the kinase comprises a polynucleotide kinase (PNK).

176. The method of claim 173, wherein the enriching is carried out by incubation with a phosphatase.

178. The method of claim 177, wherein the one or more ribonucleases comprises an RNase R and/or a 5' phosphate-dependent exonuclease.

182. The method of claim 181, wherein the modified nucleoside comprises a pseudouridine or a N1 -methylmethylpseudouridine.

183. An RNA molecule generated by the method of any of claims 120-182.

184. A cleaved RNA molecule generated by the method of any of claims 124-182.

185. The RNA molecule of claim 183 or 184, wherein the RNA molecule is a linear RNA molecule.

186. A circular RNA molecule generated by the method of any of claims 129-182.

190. A composition comprising the combination of any of claims 58-112.

192. The composition of claim 191, wherein the composition comprises a pharmaceutically acceptable excipient.

199. Use of the RNA molecule of any of claims 1-57 and 183-186, the combination of any of claims 58-1 12, an RNA molecule generated by the method of any of claims 120-182, or the composition of any of claims 187-193 in the manufacture of medicament for treating a disease or disorder in a subject, wherein the medicament is administered to the subject.

200. The RNA molecule of any one of claims 1-57, the combination of any one of claims 58-112, the system of claims 114-119, or the method of any one of claim 120-182, wherein the RNA molecule comprises an internal ribosome entry site (IRES).

201. The RNA molecule, the combination, the system, or the method of claim 200, wherein the IRES comprises an IRES from Coxackie B3 virus, coxackie Bl virus, encephalomyocarditis virus, Epstein-Barr nuclear antigen 1, enterovirus serotype EV-B107, enterovirus serotype EV-D94, Echovirus El l, Coronavirus disease 19, coxsackievirus A20, poliovirus serotype 3, Simian V4, Human Rhinovirus Al, Hepatitus C virus, Human Rhinovirus A21, Human Rhinovirus Bl 7, Human Rhinovirus, Human Rhinovirus B37, Human Rhinovirus B92, Human Rhinovirus B3, or Human Rhinovirus C54.

202. The RNA molecule, the combination, the system, or the method of claims 200 or 201, wherein the IRES comprises at least at or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 14, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, or 107.

203. The RNA molecule, the combination, the system, or the method of any one of claims 200-202, wherein the RNA molecule comprises a poly(A)-binding protein (PABP) binding site.

204. The RNA molecule, the combination, the system, or the method of any one of claims 200-203, wherein the RNA molecule comprises a poly(C)-binding protein (PCBP) binding site.

205. The RNA molecule, the combination, the system, or the method of any one of claims 200-204, wherein the RNA molecule comprises an eIF4G binding site.

206. The RNA molecule, the combination, the system, or the method of any one of claims 200-205, wherein the RNA molecule comprises LVT-18 (SEQ ID NO: 109), LVT-20 (SEQ ID NO: 111), LVT-22 (SEQ ID NO: 113), or LVT14/eIF4G (SEQ ID NO: 115).

207. The RNA molecule, the combination, the system, or the method of any one of claims 200-206, wherein the RNA molecule has reduced immunogenicity.

208. The RNA molecule, the combination, the system, or the method of any one of claims 200-207, wherein the reduced immunogenicity comprises reduced detection by a Toll-like Receptor (TLR).

209. The RNA molecule, the combination, the system, or the method of any one of claims 200-208, wherein the reduced immunogenicity comprises reduced detection by a retinoic acidinducible gene I (RIG-I).

210. The RNA molecule, the combination, the system, or the method of any one of claims 200-209, wherein the reduced immunogenicity comprises reduced detection by a melanoma differentiation-associated protein 5 (MDA5).

211. The RNA molecule, the combination, the system, or the method of any one of claims 207-210, wherein the RNA molecule has reduced immunogenicity compared to an RNA molecule without the IRES sequence.

212. The RNA molecule, the combination, the system, or the method of any one of claims 207-210, wherein the RNA molecule has reduced immunogenicity compared to an RNA molecule without the PABP, PCBP, or eIF4G binding sites.

213. The RNA molecule, the combination, the system, or the method of any one of claims 200-212, wherein the RNA molecule is a circular RNA and shows higher protein expression compared to a non-circular mRNA counterpart.

214. The RNA molecule, the combination, the system, or the method of claim 213, wherein the RNA molecule shows higher protein expression in vivo.

215. The RNA molecule, the combination, the system, or the method of claim 213 or 214, wherein the RNA molecule shows higher protein expression ex vivo.