Circular RNA molecules

Abstract

Provided herein are compositions and methods comprising a circular RNA that expresses an RNA-encoded polypeptide. Also provided herein are reaction mixtures i) comprising a DNA-dependent RNA polymerase, a plurality of nucleotide triphosphates, and a transcription template encoding an RNA and ii) an RNA and a ligase to produce a circular DNA.

Description

CIRCULAR RNA MOLECULESCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 610,356, filed on December 14, 2023, the disclosure of which is hereby incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The Sequence Listing titled 207653-606601_SL.xml, which was created on December 7, 2024 and is 62,054 bytes in size, is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0003] The present disclosure relates generally to circular nucleic acids and more specifically to circular RNA molecules with modified nucleosides, and methods for producing the same.BACKGROUND

[0004] Nucleoside-modified mRNA have been used in the development of vaccines (e.g., as a vaccine against the COVID-19 pandemic). Exemplary nucleoside modifications include pseudouridine (T), N1 -methylpseudouridine (ml'P), and 5 -methy oxyuridine (5moU).However, for non-immunogenic purposes, the expression period of mRNA is often too short to drive adequate protein expression (e.g., in some applications of Cas9 or chimeric antigen receptors). Circular RNA (circRNA) produced by back-splicing of precursor mRNAs have been used in efforts to increase stability compared to mRNAs, taking advantage of the closed loop structure without 5’-end cap and 3’-end poly A tails. However, existing exogenous circRNAs can trigger an undesirable immune response to the RNA itself. To overcome these disadvantages of circRNAs, we discovered a method to minimize foreign sequences for circularization.SUMMARY

[0005] In view of the foregoing, there is a need for improved RNA-based compositions for expression of desired polypeptides. Compositions and methods disclosed herein address this need, and provide other advantages as well. In some particular embodiments, methods for the production of circRNA are provided that (a) reduce the presence of foreign sequences in theresulting circRNA (such as residual foreign sequences typically associated with ribozyme- related production methods), and / or (b) incorporate modified nucleotides. In accordance with some embodiments, use of modified nucleotides and / or avoiding incorporation of certain foreign sequences reduces immunogenenicity of circRNA, thereby improving the protein expression therefrom.

[0006] In one aspect, the present disclosure provides a composition comprising a circular RNA. In some embodiments, (i) the circular RNA comprises an internal ribosomal entry site (IRES) and modified nucleotides; and (ii) the IRES is a tobacco etch virus (TEV) IRES, a human β-globin (Hbb-bl) IRES, a heat shock protein 70 (Hsp70) IRES, or an apoptotic peptidase activating factor- 1 (Apaf-1) IRES.

[0007] In some embodiments, the IRES comprises the modified nucleotides. In some embodiments, the circular RNA comprises the modified nucleotides outside of the IRES. In some embodiments, the circular RNA comprises the modified nucleotides inside and outside of the IRES. In some embodiments, all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the circular RNA are modified nucleotides. In some embodiments, the modified nucleotides comprise one or more of 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5- methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5 -hydroxyuridine, 5 -methyluridine, 5,6- dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'- deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5 -formyluridine, 5- methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1- methylpseudouridine (melψ), 2'-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7- deazaguanosine, 8-oxoguanosine, or 6-O-methylguanosine. In some embodiments, the modified nucleotides comprise N1-methylpseudouridine (melψ). In some embodiments, the modified nucleotides comprise 5-methoxyuridine (5moU).

[0008] In some embodiments, the IRES comprises any one of SEQ ID NO: 13-17. In some embodiments, the circular RNA encodes a polypeptide. In some embodiments, the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length. In some embodiments, the polypeptide comprises a chimeric antigen receptor (CAR). In some embodiments, the circularRNA is effective to induce translation of the polypeptide in a host cell. In some embodiments, the host cell is a human cell.

[0009] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a lipid formulation. In some embodiments, the lipid formulation is selected from a lipid nanoparticle, a lipoplex, a liposome, a polymer-based carrier, an exosome, a lamellar body, a micelle, and an emulsion. In some embodiments, the lipid formulation is a lipid nanoparticle. In some embodiments, the lipid nanoparticle has a size of less than about 200 nm, 180 nm, 130 nm, or 80 nm. In some embodiments, the lipid formulation comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid has a structure of Formula I:or a pharmaceutically acceptable salt or solvate thereof, wherein R5and R6are each independently selected from the group consisting of a linear or branched C1-C31alkyl, C2-C31alkenyl or C2-C31alkynyl and cholesteryl; L5and L6are each independently selected from the group consisting of a linear C1-C20alkyl and C2-C20alkenyl; X5is -C(O)O-, whereby -C(O)O- R6is formed or -OC(O)- whereby -OC(O)-R6is formed; X6is -C(O)O- whereby -C(O)O-R5is formed or -OC(O)- whereby -OC(O)-R5is formed; X7is S or O; L7is absent or lower alkyl; R4is a linear or branched C1-C6alkyl; and R7and R8are each independently selected from the group consisting of a hydrogen and a linear or branched C1-C6 alkyl. In some embodiments, the ionizable cationic lipid is selected from Table 1.

[0010] In some embodiments, the lipid formulation comprises a helper lipid. In some embodiments, (a) the helper lipid is a phospholipid, or (b) the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In someembodiments, the lipid formulation comprises cholesterol. In some embodiments, the lipid formulation comprises a polyethylene glycol (PEG)-lipid conjugate.

[0011] In some embodiments, the composition comprising the circular RNA is provided for use as a medicament.

[0012] In one aspect, the present disclosure provides for use of a composition comprising a circular RNA disclosed herein in the manufacture of a medicament.

[0013] In one aspect, the present disclosure provides a method of expressing a polypeptide in a cell in vitro. In some embodiments, the method comprises contacting the cell with a composition comprising a circular RNA according to any of the various aspects or embodiments disclosed herein.

[0014] In one aspect, the present disclosure provides a method of expressing a polypeptide in a subject. In some embodiments, the method comprises administering to the subject a composition comprising a circular RNA according to any of the various aspects or embodiments disclosed herein.

[0015] In one aspect, the present disclosure provides a reaction mixture comprising: (a) a DNA-dependent RNA polymerase; (b) a plurality of nucleotide triphosphates, wherein the plurality of nucleotide triphosphates comprise modified nucleotides; and (c) a transcription template encoding an RNA, wherein (i) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA produces a circular RNA, (ii) the circular RNA comprises an IRES, and (iii) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES. In some embodiments, the IRES comprises any one of SEQ ID NO: 13-17. In some embodiments, the circular RNA encodes a polypeptide. In some embodiments, the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length. In some embodiments, the polypeptide comprises a chimeric antigen receptor (CAR).

[0016] In some embodiments, all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides in the reaction mixture are modified nucleotides. In some embodiments, the modified nucleotides comprise one or more of 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5- methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5 -hydroxyuridine, 5 -methyluridine, 5,6- dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'- deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5 -formyluridine, 5-methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5 -fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1- methylpseudouridine (melψ), 2'-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7- deazaguanosine, 8-oxoguanosine, or 6-O-methylguanosine. In some embodiments, the modified nucleotides comprise N1-methylpseudouridine (melψ). In some embodiments, the modified nucleotides comprise 5-methoxyuridine (5moU).

[0017] In some embodiments, the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5 ’ to 3 ’ direction, and is located within 5 nucleotides of the first internal sequence. In some embodiments, the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length. In some embodiments, at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, there are no intervening nucleotides between the first internal sequence and the second internal sequence.

[0018] In one aspect, the present disclosure provides a reaction mixture comprising an RNA and a ligase. In some embodiments, (i) the RNA comprises modified nucleotides; (ii) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA with the ligase produces a circular RNA; (iii) the circular RNA comprises an IRES; and (iv) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES. In some embodiments, the IRES comprises any one of SEQ ID NO: 13-17. In some embodiments, the circular RNA encodes a polypeptide. In some embodiments, the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length. In some embodiments, the polypeptide comprises a chimeric antigen receptor (CAR).

[0019] In some embodiments, the IRES comprises the modified nucleotides. In some embodiments, the circular RNA comprises the modified nucleotides outside of the IRES. In some embodiments, all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the RNA are modified nucleotides. In some embodiments, the modified nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-0-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5 -methoxyuridine (5moU), 5- propynyluridine, 5-bromouridine, 5-iodouridine, 5 -fluorouridine, pseudouridine, 2'-O-methyl- pseudouridine, N1-hydroxypseudouridine, N1-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine. In some embodiments, the modified nucleotides comprise N1- methylpseudouridine (melψ). In some embodiments, the modified nucleotides comprise 5- methoxyuridine (5moU).

[0020] In some embodiments, the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5 ’ to 3 ’ direction, and is located within 5 nucleotides of the first internal sequence. In some embodiments, the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length. In some embodiments, at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, there are no intervening nucleotides between the first internal sequence and the second internal sequence.

[0021] In one aspect, the present disclosure provides a method of generating a circular RNA. In some embodiments, the method comprises joining a 5’ nucleotide of an RNA to a 3’ nucleotide of the RNA with a ligase, thereby generating the circular RNA. In some embodiments, (i) the RNA comprises modified nucleotides; (ii) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA with the ligase produces a circular RNA; (iii) the circular RNA comprises an IRES; and (iv) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES. In some embodiments, the IRES comprises any one of SEQ ID NO: 13- 17. In some embodiments, the circular RNA encodes a polypeptide. In some embodiments, the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length. In some embodiments, the polypeptide comprises a chimeric antigen receptor (CAR).

[0022] In some embodiments, the IRES comprises the modified nucleotides. In some embodiments, the circular RNA comprises the modified nucleotides outside of the IRES. Insome embodiments, all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the circular RNA are modified nucleotides. In some embodiments, the modified nucleotides comprise one or more of 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5- methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5 -hydroxyuridine, 5 -methyluridine, 5,6- dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'- deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5 -formyluridine, 5- methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5 -fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1- methylpseudouridine (melψ), 2'-O-methyl-N1-methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3- methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7- deazaguanosine, 8-oxoguanosine, or 6-O-methylguanosine. In some embodiments, the modified nucleotides comprise N1-methylpseudouridine (melψ). In some embodiments, the modified nucleotides comprise 5-methoxyuridine (5moU).

[0023] In some embodiments, the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5 ’ to 3 ’ direction, and is located within 5 nucleotides of the first internal sequence. In some embodiments, the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length. In some embodiments, at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, there are no intervening nucleotides between the first internal sequence and the second internal sequence.

[0024] In some embodiments, the method further comprises transcribing the RNA from a transcription template with a DNA-dependent RNA polymerase in the presence of a plurality of nucleotide triphosphates, wherein the plurality of nucleotide triphosphates comprise the modified nucleotides.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIGS. 1A-1B show circularization of RNA. (FIG. 1A) Circularization using ribozyme and ligase methods. (FIG. IB) Circularization of RNA with modified nucleosides.

[0026] FIGS. 2A-2C show circularization using T4 RNA ligase without splint oligonucleotides. (FIG. 2A) Predicted 2D of RNA with self-complementarity, and expanded view of a portion showing nick structure for circularization (portions of the illustrated sequence are, in 5’ to 3’ order, represented by SEQ ID NOS: 28-30). (FIG. 2B) Agarose gel electrophoresis of RNA with unmodified nucleosides or the indicated modified nucleosides after circularization. The arrowhead indicates circRNA. (FIG. 2C) NLuc expression from circRNA.

[0027] FIGS. 3A-3E show circularization and expression of CAR-GFP RNA. (FIG. 3A) Predicted 2D RNA structure (left), with nick indicated (arrowhead, structure at right; portions of the illustrated sequence are, in 5’ to 3’ order, represented by SEQ ID NOS: 31-32). (FIG. 3B) Agarose gel electrophoresis of RNA with unmodified and modified nucleosides before and after circularization. (FIG. 3C) Expression of GFP from the indicated CAR-GFP RNA. (FIG. 3D) PKR mRNA levels after transfection of the indicated CAR-GFP RNA. (FIG. 3E) RIG-I mRNA after transfection of the indicated CAR-GFP RNA.

[0028] FIGS. 4A-4B show the function of IRES sequences in circular RNA. (FIG. 4A) Agarose gel electrophoresis of constructs with the indicated IRES. (FIG. 4B) Luciferase Assay after transfection of RNAse R-treated circRNA constructs.

[0029] FIGS. 5A-5C show translation activity of circular RNA with unmodified or modified nucleosides and encoding FLuc. (FIG. 5A) Agarose gel electrophoresis. (FIG. 5B) Luciferase Assay after transfection of RNAse R-treated circRNA constructs. (FIG. 5C) PKR mRNA and RIG-I mRNA levels after transfection with the indicated control or FLuc-encoding circular RNA.

[0030] FIGS. 6A-6C show translation activity of circular RNA with unmodified or modified nucleosides and encoding NLuc. (FIG. 6A) Agarose gel electrophoresis. (FIG. 6B) Luciferase Assay after transfection of RNAse R-treated circRNA constructs. (FIG. 6C) PKR mRNA and RIG-I mRNA levels after transfection with the indicated control or NLuc-encoding circular RNA.

[0031] FIGS. 7A-7L show predicted 2D circRNA structures. (FIG. 7A) circNLuc - prepared with ribozyme. (FIG. 7B) circNLuc - prepared with ligase. (FIG. 7C) circNLuc -prepared with ribozyme + ligase. (FIG. 7D) circFLuc-TEV. (FIG. 7E) circFLuc-Hbb-bl. (FIG. 7F) circFLuc-Hbb-bl-113. (FIG. 7G) circFLuc-Hsp70. (FIG. 7H) circFLuc-EV71. (FIG. 71) circFLuc-Apaf-1. (FIG. 7 J) circFLuc-CVB3. (FIG. 7K) circNLuc-TEV. (FIG. 7L) circCD19-CAR-GFP.DETAILED DESCRIPTION

[0032] The present disclosure provides, in various aspects, compositions comprising circular RNA, and reaction mixtures and methods for producing the same. In some particular embodiments, methods for the production of circRNA are provided that (a) reduce the presence of foreign sequences in the resulting circRNA (such as residual foreign sequences typically associated with ribozyme-related production methods), and / or (b) incorporate modified nucleotides.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0034] Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, that have been made throughout this disclosure are hereby incorporated herein in their entirety for all purposes.Definitions

[0035] As used herein, the term “nucleic acid” refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analogues. A DNA or RNA molecule can be double-stranded or single-stranded and can be of any size. Exemplary nucleic acids include, but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell- free DNA (cfDNA), mitochondrial DNA, chloroplast DNA, viral DNA, mRNA, tRNA, rRNA, long non-coding RNA, siRNA, micro RNA (miRNA or miR), hnRNA, and viral RNA. Exemplary nucleic analogues include peptide nucleic acid, morpholino- and locked nucleic acid, glycol nucleic acid, and threose nucleic acid. As used herein, the term “nucleic acid molecule” is meant to include fragments of nucleic acid molecules as well as any full- length or non-fragmented nucleic acid molecule, for example. As used herein, the terms “nucleic acid” and “nucleic acid molecule” can be used interchangeably, unless context clearly indicates otherwise.

[0036] As used herein, the term “polynucleotide” refers to a nucleic acid sequence that includes at least two nucleotide monomers. The term “polynucleotide” can refer to polymersof DNA, RNA, nucleic acid analogues, or combinations of these. A “polynucleotide” can be double-stranded or single-stranded and can be of any size. A polynucleotide can be a separate nucleic acid molecule or be a part of a nucleic acid molecule. Accordingly, the term “polynucleotide” can refer to a nucleic acid molecule or to a region of a nucleic acid molecule. Various polynucleotides may be referred to herein by a sequence, or a percent identity thereto. An RNA sequence may be referred to herein with reference to the sequence of the RNA itself, or with reference to a DNA sequence encoding the same. Likewise, a DNA sequence may be referred to by the sequence of the DNA itself, or with reference to an RNA it encodes. It will be appreciated that T present in DNA is substituted with U in RNA, and vice versa. Sequences presented herein as RNA sequences may be encoded by a corresponding DNA sequence, in which U is replaced with T. Similarly, sequences presented herein as DNA sequences may be converted to the corresponding RNA sequence encoded thereby by replacing T with U. In general, the RNA sequence “encoded” by a DNA sequence refers herein to an RNA with the same 5’ to 3’ orientation and order of bases (except for U replacing T), and not the reverse complement. Both the DNA and RNA versions of a given sequence are contemplated herein, unless context clearly indicates otherwise. In cases where a given embodiment refers to an RNA polynucleotide by referencing a SEQ ID NO including T nucleotides, it will be understood that the T nucleotides are U nucleotides (or modified versions thereof) in the RNA, unless context clearly indicates otherwise. Likewise, in cases where a given embodiment refers to a DNA polynucleotide by referencing a SEQ ID NO including U nucleotides, it will be understood that the U nucleotides are T nucleotides (or modified versions thereof) in the DNA, unless context clearly indicates otherwise. In some cases, a DNA sequence may include elements not found in an RNA encoded thereby (e.g., a promoter sequence). In some cases, an RNA sequence may include elements not found in a DNA construct encoding the RNA (e.g., a poly-A tail when added post-transcriptionally).

[0037] As used herein, the term “protein” refers to any polymeric chain of amino acids. The terms “peptide” and “polypeptide” can be used interchangeably with the term protein, unless context clearly indicates otherwise, and can also refer to a polymeric chain of amino acids. The term “protein” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A protein may be monomeric or polymeric. The term “protein” encompasses fragments and variants (including fragments of variants) thereof, unless otherwise contradicted by context.

[0038] In general, “sequence identity” or “sequence homology,” which can be used interchangeably, refer to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby or the amino acid sequence of a polypeptide, and comparing these sequences to a second nucleotide or amino acid sequence. As used herein, the term “percent (%) sequence identity” or “percent (%) identity,” also including “percent homology,” refers to the percentage of amino acid residues or nucleotides in a sequence that are identical with the amino acid residues or nucleotides in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Thus, two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity,” also referred to as “percent homology.” The percent identity to a reference sequence (e.g., nucleic acid or amino acid sequences), which may be a sequence within a longer molecule (e.g., polynucleotide or polypeptide), may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a reference sequence and a claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exactmatch indicates 100% identity over the length of the reference sequence. Additional programs and methods for comparing sequences and / or assessing sequence identity include the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at ebi.ac.uk / Tools / psa / emboss needle / , optionally with default settings), the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at ebi.ac.uk / Tools / psa / emboss water / , optionally with default settings), the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Drive, Madison, Wis.). In some embodiments, reference to percent sequence identity refers to sequence identity as measured using BLAST (Basic Local Alignment Search Tool). In some embodiments, ClustalW is used for multiple sequence alignment. Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[0039] As used herein, the term “drug” or “medicament,” means a pharmaceutical formulation or composition as described herein.

[0040] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the composition” includes one or more compositions, and / or components of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0041] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +20%, or ±10%, or ±5%, or even ±1% from the specified value, as such variations are appropriate for the disclosed compositions or to perform the disclosed methods.

[0042] The term “expression” refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA or other RNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.”

[0043] As used herein, “operably linked,” “operable linkage,” “operatively linked,” or grammatical equivalents thereof refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, an internal ribosomal entry site (IRES), etc.,wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which can comprise promoter and / or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. Similarly, an IRES in an RNA is operably linked to a polypeptide- encoding portion of the RNA if the IRES facilitates entry of a ribosome for translation of the desired polypeptide. There may be intervening residues between the regulatory element and coding region so long as the desired functional relationship is maintained.

[0044] As used herein, the term “circular RNA” refers to a class of RNA molecules that distinguishes itself from linear RNA by adopting a covalently sealed, continuous structure. Unlike linear RNA, which typically features distinct 3' and 5' ends, circular RNA achieves its circular configuration by connecting these ends. The term “circular” does not imply or require any particular three-dimensional shape, only the absence of free 5’ and 3’ ends. On the contrary, various regions of self-complementarity may result in the circular RNA forming complex secondary and tertiary structures (see, e.g., FIGS. 7A-7L). In some embodiments, the circular configuration imparts notable advantages, such as resistance to degradation by exonucleases, rendering it potentially more stable within cellular environments compared to otherwise corresponding linear RNA molecules.

[0045] As used herein, the term “chimeric antigen receptor (CAR)” refers to a genetically engineered receptor comprising an extracellular target-binding domain fused to an intracellular signaling domain, and which is effective to trigger an immune response in the presence of the target. The engineered receptor can be introduced into immune cells, such as T cells or NK cells for the activation thereof in response to cells comprising the target. Nonlimiting examples of CARs are provided herein. A variety of additional suitable CARs are available.Compositions

[0046] In one aspect, the present disclosure provides a composition comprising a circular RNA. In some embodiments, (i) the circular RNA comprises an internal ribosomal entry site (IRES) and modified nucleotides; and (ii) the IRES is a tobacco etch virus (TEV) IRES, a human P-globin (Hbb-bl) IRES, a heat shock protein 70 (Hsp70) IRES, or an apoptotic peptidase activating factor- 1 (Apaf-1) IRES.

[0047] In general, an internal ribosomal entry site (IRES) refers to an RNA regulatory sequence that governs cap-independent translation initiation. Ribosomes can be recruited directly to an IRES in an RNA for the translation of a polypeptide encoded therein. In this way, an IRES may act as a landing pad for ribosomes, allowing them to attach to an RNA (e.g., a circular RNA) at the IRES region and start protein synthesis. A variety of IRES sequences are known, and are often named for the biological context in which they were originally identified. For example, an IRES of a tobacco etch virus (i.e., a “TEV IRES”) is an IRES sequence from TEV. Likewise, a human P-globin (Hbb-bl) IRES is an IRES sequence from an Hbb-bl gene. The source of an IRES does not limit the biological context in which it may be employed. For example, a TEV IRES may be used for the expression of a circular RNA in a human cell.

[0048] In some embodiments, the IRES is a tobacco etch virus (TEV) IRES. In some embodiments, the TEV IRES comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the IRES comprises a sequence with at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the IRES comprises a sequence with at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 13.

[0049] In some embodiments, the IRES is a human P-globin (Hbb-bl) IRES. In some embodiments, the Hbb-bl IRES comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the IRES comprises a sequence with at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the IRES comprises a sequence with at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 14. In some embodiments, the I-Ibb-bl IRES comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the IRES comprises a sequence with at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the IRES comprises a sequence with at. least 95% sequence identity to SEQ ID NO: 15. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 15 (also referred to herein as an “Hbb-bl -113 IRES”).

[0050] In some embodiments, the IRES is a heat shock protein 70 (Hsp70) IRES. In some embodiments, the Hsp70 IRES comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the IRES comprises a sequence with at least 90% sequence identity' to SEQ ID NO: 16. In someembodiments, the IRES comprises a sequence with at toast 95% sequence identity to SEQ ID NO: 16. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 16.

[0051] In some embodiments, the IRES is an apoptotic peptidase activating factor- 1 (Apaf- 1) IRES. In some embodiments, the Apaf-1 IRES comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the IRES comprises a sequence with at ieast 90% sequence identity to SEQ ID NO: 17. In some embodiments, the IRES comprises a sequence with at least 95% sequence identity to SEQ ID NO: 17. In some embodiments, the IRES comprises the sequence of SEQ ID NO: 17.

[0052] In some embodiments, the circular RNA comprises modified nucleotides. Modified nucleotides may be any of a variety of modified nucleotides, may comprise two or more types of modified nucleotides, and may be modified at any of a variety of positions within the circular RNA. In some embodiments, the IRES comprises modified nucleotides. In some embodiments, the circular RNA comprises the modified nucleotides inside and / or outside of the IRES. In some embodiments, all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the circular RNA are modified nucleotides. Examples of nucleic acid monomers include non-natural, modified, and chemically-modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the base portion or the sugar portion. In nature, most polynucleotides comprise nucleotides that are “unmodified” or “natural” nucleotides, which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). These bases are typically fixed to a ribose or deoxy ribose at the 1’ position. The use of RNA polynucleotides comprising chemically modified nucleotides have been shown to improve RNA expression, expression rates, half-life and / or expressed protein concentrations. RNA polynucleotides comprising chemically modified nucleotides have also been useful in optimizing protein localization thereby avoiding deleterious bio-responses such as immune responses and / or degradation pathways.

[0053] Examples of modified or chemically-modified nucleotides include 5- hydroxycytidines, 5-alkylcytidines, 5-hydroxyalkylcytidines, 5-carboxycytidines, 5- formylcytidines, 5-alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N4- alkylcytidines, N4-aminocyti dines, N4-acetylcytidines, andN4,N4-dialkylcytidines. Examples of modified or chemically-modified nucleotides include 5 -hydroxy cytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5 -bromocytidine, 5 -iodocytidine, 2-thiocytidine; N4-methylcytidine, N4- aminocytidine, N4-acetylcytidine, and N4,N4-dimethylcytidine.

[0054] Examples of modified or chemically-modified nucleotides include 5- hydroxyuridines, 5-alkyluridines, 5-hydroxyalkyluridines, 5-carboxyuridines, 5- carboxyalkylesteruridines, 5-formyluridines, 5-alkoxyuridines, 5-alkynyluridines, 5- halouridines, 2-thiouridines, and 6-alkyluridines. Examples of modified or chemically- modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as “5MeOU”), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5- iodouridine, 2-thiouridine, and 6-methyluridine. Examples of modified or chemically-modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2- thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2’-O-methyluridine, l-methyl-3- (3-amino-3-carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5- carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2- thiouridine, 5-(isopentenylaminomethyl)uridine, 2’-O-methylpseudouridine, 2-thio-2’O- methyluridine, and 3,2’-O-dimethyluridine.

[0055] Examples of modified or chemically-modified nucleotides include N6- methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 8-azaadenosine, 7-deazaadenosine,8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N6-methyladenosine, N6- i sopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O- dimethyl-adenosine, N6,N6,2'-O-trimethyl-adenosine, l,2'-O-dimethyl-adenosine, 2'-O- ribosyladenosine, 2-amino-N6-methyl-purine, 1 -thio-adenosine, 2'-F-ara-adenosine, 2'-F- adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0056] Examples of modified or chemically-modified nucleotides include Nl- alkylguanosines, N2-alkylguanosines, thienoguanosines, 7-deazaguanosines, 8- oxoguanosines, 8-bromoguanosines, O6-alkylguanosines, xanthosines, inosines, and Nl- alkylinosines.

[0057] Examples of modified or chemically-modified nucleotides include Nl- methylguanosine, N2-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-methylguanosine, xanthosine, inosine, and Nl-methylinosine.

[0058] Examples of modified or chemically-modified nucleotides include pseudouridines. Examples of pseudouridines include Nl-alkylpseudouridines, Nl-cycloalkylpseudouridines,N1 -hydroxypseudouridines, Nl-hydroxyalkylpseudouri dines, Nl-phenylpseudouri dines, Nl- phenylalkylpseudouri dines, Nl-aminoalkylpseudouri dines, N3 -alkylpseudouridines, N6- alkylpseudouridines, N6-alkoxypseudouridines, N6-hydroxypseudouridines, N6- hydroxyalkylpseudouridines, N6-morpholinopseudouridines, N6-phenylpseudouridines, andN6-halopseudouridines. Examples of pseudouridines include Nl-alkyl-N6- alkylpseudouridines, Nl-alkyl-N6-alkoxypseudouridines, Nl-alkyl-N6- hydroxypseudouridines, Nl-alkyl-N6-hydroxy alkylpseudouridines, Nl-alkyl-N6- morpholinopseudouridines, Nl-alkyl-N6-phenylpseudouridines, and Nl-alkyl-N6- halopseudouridines. In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted, or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents. Examples of pseudouridines include Nl-methylpseudouridine (also referred to herein as “N1MPU”), Nl-ethylpseudouridine, Nl-propylpseudouridine, Nl-cyclopropylpseudouridine, Nl-phenylpseudouridine, Nl-aminomethylpseudouridine, N3 -methylpseudouridine, Nl- hydroxypseudouridine, and N1 -hydroxymethylpseudouridine.

[0059] Examples of nucleic acid monomers include modified and chemically-modified nucleotides, including any such nucleotides known in the art. In some embodiments, the modified nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5- methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5 -methoxyuridine (5moU), 5- propynyluridine, 5-bromouridine, 5-iodouridine, 5 -fluorouridine, pseudouridine, 2'-O-methyl- pseudouridine, N1-hydroxypseudouridine, N1-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine. In some embodiments, the modified nucleotides comprise N1-methylpseudouridine (melψ). In some embodiments, the modified nucleotides comprise 5- methoxyuridine (5moU).

[0060] In some embodiments, the circular RNA encodes one or more polypeptides. Any of a variety of polypeptides my be selected for expression from a circular RNA according to the present disclosure. Examples of polypeptides include, but are not limited to, an enzyme (e.g. a luciferase), a receptor (e.g., a chimeric antigen receptor (CAR)), a fluorescent protein (e.g., green fluorescent protein (GFP), and an antibody (or antigen-binding fragment thereof). In some embodiments, the polypeptide comprises a CAR. In some embodiments, the polypeptide is at least 100, 150, 200, 300, 400, 500, 750, 1000, 1250, 1500, or more amino acids in length. In some embodiments, the polypeptide is at least 200 amino acids in length. In some embodiments, the polypeptide is at least 500 amino acids in length. In some embodiments, the polypeptide is at least 1000 in length. In some embodiments, the polypeptide is at least 2000 amino acids in length.

[0061] In some embodiments, the polypeptide is a luciferase, such as a firefly luciferase. In some embodiments, the luciferase comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24 or 25. In some embodiments, the luciferase comprises a sequence with at least 90% sequence identity to SEQ ID NO: 24 or 25. In some embodiments, the luciferase comprises a sequence with at least 95% sequence identity to SEQ ID NO: 24 or 25. In some embodiments, the luciferase comprises the sequence of SE Q ID NO: 24. In some embodiments, the luciferase comprises the sequence of SFQ ID NO: 25. In some embodiments, the luciferase is encoded by a polynucleotide with at least 70%, 80%, 85%, 90%, 95%o, 96%), 97%, 98%), or 99% sequence identity to SEQ ID NO: 20 or 21. In some embodiments, the luciferase is encoded by a polynucleotide with at least 90% sequence identity to SEQ ID NO: 20 or 21. In some embodiments, the luciferase is encoded by a polynucleotide with at least 95%) sequence identity to SEQ ID NO: 20 or 21. In some embodiments, the luciferase is encoded by a polynucleotide having the sequence of SEQ ID NO: 20. In some embodiments, the luciferase is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 21.

[0062] In some embodiments, the polypeptide is a GFP. In some embodiments, the GFP comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 27. In some embodiments, the GFP comprises a sequence with at least 90%) sequence identity to SEQ ID NO; 27. In some embodiments, the GFP comprises a sequence with at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, theGFP comprises the sequence of SEQ ID NO: 27. In some embodiments, the GFP is encoded by a polynucleotide with at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23. In some embodiments, the GFP is encoded by a polynucleotide with at least 90% sequence identity to SEQ ID NO: 23. In some embodiments, the GFP is encoded by a polynucleotide with at least 95% sequence identity to SEQ ID NO: 23. In some embodiments, the GFP is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 23.

[0063] In some embodiments, the polypeptide is a CAR. In some embodiments, the CAR comprises a binding domain that binds to CD19, and is referred to herein as a “CD19-CAR ” In some embodiments, the CAR comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 26. In some embodiments, the CAR comprises a sequence with at least 90% sequence identity to SEQ ID NO: 26. In some embodiments, the CAR comprises a sequence with at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 26. In some embodiments, the CAR is encoded by a polynucleotide with at least with at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22. In some embodiments, the CAR is encoded by a polynucleotide with at least 90% sequence identity to SEQ ID NO: 22. In some embodiments, the CAR is encoded by a polynucleotide with at least 95% sequence identity to SEQ ID NO: 2'2. In some embodiments, the CAR is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 22.

[0064] In some embodiment, the circular RNA is effective to induce translation of the polypeptide in a host cell. A variety of suitable host cells is available to those skilled in the art. Examples of host cell include, but are not limited to, a human cell, a mammalian cell, a yeast cell, and a bacterial cell. In some embodiments, the circular RNA is prepared for delivery to a cell in vitro. In some embodiments, the circular RNA is prepared for delivery to a cell in vivo, such as by being formulated for administration to a subject. In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells include cells of any of human, hamster, mouse, monkey, rat, pig, cow, or rabbit. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a human cell. Examples of human cells include, but are not limited to, an immune cell (e.g., a T cell or an NK cell), a brain cell, a muscle cell, a nervous cell, or a cell line such as HEK-293, HeLa, Jurkat, SH-SY5Y, A549, MCF-7, PC-3, U-87, and HspG2.Formulations

[0065] In some embodiments, compositions disclosed herein (e.g., compositions comprising a circular RNA in accordance with any of the various aspects and embodiments herein) further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises a lipid formulation. In some embodiments, the lipid formulation comprises a transfection reagent, a lipoplex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a lamellar body, a micelle, or an emulsion. In some embodiments, the lipid formulation comprises a liposome (e.g., a cationic liposome). In some embodiments, the lipid formulation comprises a lipid nanoparticle. In some embodiments, the lipid formulation comprises one or more cationic lipids (e.g., an ionizable cationic lipid). In some embodiments, the lipid formulation comprises an anionic lipid, a zwitterionic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, a phospholipid, a glycolipid, or a combination thereof. In some embodiments, the lipid formulation comprises a helper lipid, cholesterol, a polyethylene glycol (PEG)-lipid conjugate, or any combination of one or more of these.Liposomes

[0066] Conventional liposomes are vesicles that consist of at least one bilayer and an internal aqueous compartment. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally present as spherical vesicles and can range in size from 20 nm to a few microns. Liposomal formulations can be prepared as a colloidal dispersion or they can be lyophilized to reduce stability risks and to improve the shelf-life for liposome-based drugs. Methods of preparing liposomal compositions are known in the art and would be within the skill of an ordinary artisan.

[0067] Liposomes that have only one bilayer are referred to as being unilamellar, and those having more than one bilayer are referred to as multilamellar. The most common types of liposomes are small unilamellar vesicles (SUV), large unilamellar vesicle (LUV), and multilamellar vesicles (MLV). In contrast to liposomes, lysosomes, micelles, and reversed micelles are composed of monolayers of lipids. Generally, a liposome is thought of as having a single interior compartment, however some formulations can be multivesicular liposomes (MVL), which consist of numerous discontinuous internal aqueous compartments separated by several nonconcentric lipid bilayers.

[0068] Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes, and can be prepared from both natural and synthetic phospholipids (Int J Nanomedicine. 2014; 9: 1833-1843). In their use as drug delivery vehicles, because a liposome has an aqueous solution core surrounded by a hydrophobic membrane, hydrophilic solutes dissolved in the core cannot readily pass through the bilayer, and hydrophobic compounds will associate with the bilayer. Thus, a liposome can be loaded with hydrophobic and / or hydrophilic molecules. When a liposome is used to carry a nucleic acid such as RNA, the nucleic acid will be contained within the liposomal compartment in an aqueous phase.

[0069] In some embodiments, the lipid formulation comprises a cationic liposome, a nanoliposome, a proteoliposome, a unilamellar liposome, a multilamellar liposome, a ceramide-containing nanoliposome, or a multivesicular liposome.Cationic Liposomes

[0070] Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic portion. In addition to the general characteristics profiled above for liposomes, the positively charged moieties of cationic lipids used in cationic liposomes provide several advantages and some unique structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic and thus will direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moiety will associate with aqueous media and more importantly with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being researched for use in gene therapy due to their favorability towards negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large- scale production required for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed herein below.Lipid Nanoparticles

[0071] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that can include a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell aredirectly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0072] For lipid nanoparticle nucleic acid delivery systems in accordance with some embodiments, the lipid shell is formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below or about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid’s negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0073] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine® reagent, have found considerable utility for in vitro transfection. However, these first- generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (Imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation. In some embodiments, nucleic acid molecules provided herein and lipids or lipid formulations provided herein form a lipid nanoparticle (LNP).

[0074] In some embodiments, polynucleotides (e.g., circular RNAs disclosed herein) are incorporated into a lipid formulation (e.g., a lipid-based delivery vehicle). In some embodiments, the polynucleotide is encapsulated within the lipid formulation or lipidnanoparticle. In some embodiments, the polynucleotide is complexed to the lipid formulation or lipid nanoparticle.

[0075] In the context of the present disclosure, a lipid-based delivery vehicle typically serves to transport a desired RNA to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid- based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing an RNA or mRNA of the disclosure. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and a polynucleotide of the disclosure. In some embodiments, the lipid-based delivery vehicle (e.g., lipid nanoparticle, lipid monolayer or bilayer, or other lipid formulation) further comprises a neutral lipid or a polymer. In some embodiments, the lipid formulation comprises a liquid medium. In some embodiments, the formulation further encapsulates a nucleic acid. In some embodiments, the lipid formulation further comprises a nucleic acid and a neutral lipid or a polymer. In some embodiments, the lipid formulation encapsulates the nucleic acid.

[0076] In some embodiments of the nucleic acid-lipid formulations, the polynucleotide may be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, a lipid formulation comprising a polynucleotide is fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, the polynucleotide in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37°C for at least 20, 30, 45, or 60 minutes. In some embodiments, the polynucleotide in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37°C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In some embodiments, the polynucleotide is complexed with the lipid portion of the formulation. One of the benefits of the formulations of the present disclosure is that the nucleic acid-lipid compositions are substantially non-toxic to animals such as humans and other mammals.

[0077] In the context of nucleic acids, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent- mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it tointeract with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (10 - I) / I0, where / and 10 refers to the fluorescence intensities before and after the addition of detergent.

[0078] In some embodiments, the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-lipid nanoparticles.

[0079] In some embodiments, the lipid formulations comprise RNA that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about95%, from about 60% to about 95%, from about 70% to about 95%, from about 80% to about95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof or range therein) of the particles have the RNA encapsulated therein. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0080] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0081] In some embodiments, a nucleic acid-lipid composition disclosed herein comprises a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-lipid nanoparticles.

[0082] In some embodiments, the lipid formulations have a total lipidmucleic acid molecule weight ratio (mass / mass ratio) of from about 1 : 1 to about 100: 1, from about 1 : 1 to about 50: 1, from about 2: 1 to about 45: 1, from about 3: 1 to about 40: 1, from about 5: 1 to about 45: 1, or from about 10: 1 to about 40:1, or from about 15: 1 to about 40: 1, or from about 20:1 to about 40: 1; or from about 25: 1 to about 45: 1; or from about 30: 1 to about 45: 1; or from about 32: 1 to about 42: 1; or from about 34: 1 to about 42: 1. In some embodiments, the total lipid: nucleic acid molecule weight ratio (mass / mass ratio) is from about 50: 1 to about 10: 1. The ratio may be any value or subvalue within the recited ranges, including endpoints.

[0083] In some embodiments, the lipid nanoparticles have a size (e.g., a mean diameter) of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm. The diameter may be any value or sub-value within the recited ranges, including endpoints. In some embodiments, the lipid nanoparticle has a size of less than about 200 nm, less than about 150 nm, less than about 100 nm, or about 55 nm to about 90 nm. In some embodiments, the lipid nanoparticle has a size of about 55 nm to about 90 nm. In some embodiments, a lipid nanoparticle has a size of less than about 200 nm, 180 nm, 130 nm, or 80 nm. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, generally are resistant in aqueous solution to degradation with a nuclease.

[0084] In some embodiments, a lipid formulation is a cationic liposome or a lipid nanoparticle (LNP) comprising: (a) an RNA of the present disclosure, (b) a cationic lipid, (c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and optionally (e) a sterol.

[0085] In some embodiments, pharmaceutically acceptable carriers comprise substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional nontoxic pharmaceuticallyacceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.Cationic Lipids

[0086] In some embodiments, the pharmaceutically acceptable carrier comprises a lipid formulation comprising one or more cationic lipids. In some embodiments, the one of more cationic lipids comprises one or more of 5-carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3 -Dimethylammonium -Propane (DODAP), l,2-Dioleoyl-3- Trimethylammonium-Propane (DOTAP), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), l,2-dioleyloxy-N,N-dimethyl-3 -aminopropane (DODMA), 1,2-dilinoleyloxy- N,N-dimethyl-3 -aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethyl-3- aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest- 5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy l-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di oleyloxybenzylamine (DMOBA), 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3 -dimethylaminopropane (DLinCDAP), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), or 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2-DMA). In some embodiments, the one or more cationic lipids comprises an ionizable cationic lipid.

[0087] In some embodiments, the ionizable cationic lipid has a structure of Formula I:or a pharmaceutically acceptable salt or solvate thereof, wherein R5and R6are each independently selected from the group consisting of a linear or branched C1-C31alkyl, C2-C31alkenyl or C2-C31alkynyl and cholesteryl; L5and L6are each independently selected from the group consisting of a linear C1-C20alkyl and C2-C20alkenyl; X5is -C(O)O-, whereby -C(O)O- R6is formed or -OC(O)- whereby -OC(O)-R6is formed; X6is -C(O)O- whereby -C(O)O-R5is formed or -OC(O)- whereby -OC(O)-R5is formed; X7is S or O; L7is absent or lower alkyl; R4is a linear or branched C1-Ce alkyl; and R7and R8are each independently selected from the group consisting of a hydrogen and a linear or branched C1-C6alkyl.

[0088] In some embodiments, X7 is S. In some embodiments, X5 is -C(O)O-, whereby - C(O)O-R6 is formed and X6 is -C(O)O- whereby -C(O)O-R5 is formed. In some embodiments, R7 and R8 are each independently selected from the group consisting of methyl, ethyl and isopropyl. In some embodiments, L5 and L6 are each independently a C1-C1 0alkyl. In some embodiments, L5 is C1-C3alkyl, and L6 is C1-C5alkyl. In some embodiments, L6 is C1-C2alkyl. In some embodiments, L5 and L6 are each a linear C7 alkyl. In some embodiments, L5 and L6 are each a linear C9 alkyl. In some embodiments, R5 and R6 are each independently an alkenyl. In some embodiments, R6 is alkenyl. In some embodiments, R6 is C2 -C9 alkenyl. In some embodiments, the alkenyl comprises a single double bond. In some embodiments, R5 and R6 are each alkyl. In some embodiments, R5 is a branched alkyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C9 alkyl, C9 alkenyl and C9 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a Cl 1 alkyl, Cl 1 alkenyl and Cl 1 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C7 alkyl, C7 alkenyl and C7 alkynyl. In some embodiments, R5 is -CH((CH2)pCH3)2 or -CH((CH2)pCH3)((CH2)p-lCH3), wherein p is 4-8. In some embodiments, p is 5 and L5 is a C1 -C3 alkyl. In some embodiments, p is 6 and L5 is a C3 alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5 is a C1-C3 alkyl. In some embodiments, R5 consists of -CH((CH2)pCH3)((CH2)p-lCH3), wherein p is 7 or 8. In some embodiments, R4 is ethylene or propylene. In some embodiments, R4 is n-propylene or isobutylene. In some embodiments, L7 is absent, R4 is ethylene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is n-propylene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is ethylene, X7 is S and R7 and R8 are each ethyl. In some embodiments, X7 is S, X5 is -C(O)O-, whereby -C(O)O-R6 is formed, X6 is -C(O)O- whereby-C(O)O-R5 is formed, L5 and L6 are each independently a linear C3-C7 alkyl, L7 is absent, R5 is -CH((CH2)pCH3)2, and R6 is C7-C12 alkenyl. In some further embodiments, p is 6 and R6 is C9 alkenyl.

[0089] In some embodiments, the ionizable cationic lipid is selected from Table 1.Table 1. Exemplary Ionizable Cationic Lipids

[0090] In some embodiments, the composition disclosed herein comprises the lipid formulation comprising a helper lipid. In some embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoylphosphatidyl glycerol (DPPG), dipalmitoyl phosphatidylcholine (DPPC), dioleoylphosphatidyl phosphatidylcholine (DOPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the lipid formulation comprises cholesterol.

[0091] In some embodiments, the lipid formulation comprises a polyethylene glycol (PEG)- lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG (e.g., PEG2000- DMG). In some embodiments, the lipid formulation comprises about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 4 mol% to about 16 mol% DSPC, about 30 mol% to about 47 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG. In some embodiments, the composition has a total lipidmucleic acid molecule weight ratio of about 50: 1 to about 10: 1.

[0092] In some embodiments, the helper lipid comprises from about 2 mol% to about 20 mol%, from about 3 mol% to about 18 mol%, from about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, from about 6 mol% to about 12 mol%, from about 5 mol% to about10 mol%, from about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0093] The lipid portion, or the cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 35 mol%, or about 28 mol% to about 35 mol%; or about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, or about 37 mol% of the total lipid present in the lipid formulation.

[0094] In some embodiments, the lipid portion of the lipid formulation is about 35 mol% to about 42 mol% cholesterol.

[0095] In some embodiments, the phospholipid component in the mixture may comprise from about 2 mol% to about 20 mol%, from about 3 mol% to about 18 mol%, from about 4 mol % to about 16 mol %, about 5 mol % to about 14 mol %, from about 6 mol % to about 12 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0096] In some embodiments, the lipid portion of the lipid formulation comprises about, but is not necessarily limited to, 40 mol% to about 60 mol% of the ionizable cationic lipid, about 4 mol% to about 16 mol% DSPC, about 30 mol% to about 47 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG.

[0097] In some embodiments, the lipid portion of the lipid formulation may comprise, but is not necessarily limited to, about 42 mol% to about 58 mol% of the ionizable cationic lipid, about 6 mol% to about 14 mol% DSPC, about 32 mol% to about 44 mol% cholesterol, and about 1 mol% to about 2 mol% PEG2000-DMG.

[0098] In some embodiments, the lipid portion of the lipid formulation may comprise, but is not necessarily limited to, about 45 mol% to about 55 mol% of the ionizable cationic lipid,about 8 mol% to about 12 mol% DSPC, about 35 mol% to about 42 mol% cholesterol, and about 1.25 mol% to about 1.75 mol% PEG2000-DMG.

[0099] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ± 5 mol%.

[0100] A lipid formulation that includes a cationic lipid compound or ionizable cationic lipid compound may be on a molar basis about 30-70% cationic lipid compound, about 25-40 % cholesterol, about 2-15% helper lipid, and about 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percent is of the total lipid present in the formulation. In some embodiments, the composition is about 40-65% cationic lipid compound, about 25- 35% cholesterol, about 3-9% helper lipid, and about 0.5-3% of a PEG-lipid, wherein the percent is of the total lipid present in the formulation.

[0101] The formulation may be a lipid particle formulation, for example containing 8-30% nucleic acid compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4- 25% helper lipid, 2- 25% cholesterol, 10- 35% cholesterol -PEG, and 5% cholesterol-amine; or 2-30% cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol -PEG, and 1- 20% cholesterol-amine; or up to 90% cationic lipid and 2-10% helper lipids, or even 100% cationic lipid.Use o f the composition

[0102] In one aspect, the present disclosure provides for use of a composition comprising a circular RNA disclosed herein in the manufacture of a medicament. In some embodiments, the composition comprising the circular RNA is for use as a medicament. Any of a variety of polypeptides can be encoded by circular RNA provided herein, including enzymes, receptors, antigens, structural proteins, signal transduction proteins, fusion proteins, reporters, and others. In some embodiments, the polypeptide is phenylalanine hydroxylase (PAH), ornithine transcarbamylase (OTC), cystic fibrosis transmembrane regulator (CFTR), an infectious disease antigen, or a cancer antigen. In some embodiments, the medicament comprises the circular RNA disclosed herein encoding one or more polypeptide. In some embodiments, the medicament comprises the circular RNA encoding a polypeptide in accordance with any of the various aspects and embodiments herein. The particular polypeptide selected, its formulation, and mode of delivery will depend on the particular indication selected. For example, CD19-CAR-T cells find use in the treatment of cancer, such as leukemia (see, e.g.,US20210347851A1). A circular RNA encoding a CD19-CAR may be transfected into cells in culture, such as T cells, which may then be administered directly to a subject, or cultured first to prepare the therapeutic composition comprising the cells or progeny thereof. Alternatively, a composition comprising the circular RNA may be administered to a subject to deliver the circular RNA into target cells (e.g., T cells).Methods of use

[0103] In one aspect, the present disclosure provides a method of expressing a polypeptide in a cell in vitro. In some embodiments, the present disclosure provides a method of expressing a polypeptide in a cell in vivo. In some embodiments, the method comprises contacting the cell with a composition comprising a circular RNA according to any of the various aspects and embodiments disclosed herein.

[0104] In one aspect, the present disclosure provides a method of expressing a polypeptide in a subject. In some embodiments, the method comprises administering to the subject a composition comprising a circular RNA according to any of the various aspects and embodiments disclosed herein. Any of a variety of polypeptides can be expressed by the methods provided herein, including enzymes, receptors, antigens, structural proteins, signal transduction proteins, fusion proteins, reporters, and others. In some embodiments, the polypeptide is phenylalanine hydroxylase (PAH), ornithine transcarbamylase (OTC), cystic fibrosis transmembrane regulator (CFTR), an infectious disease antigen, or a cancer antigen.

[0105] As used herein, the term “subject” refers to any individual or patient on which the methods disclosed herein are performed. The term “subject” can be used interchangeably with the term “individual” or “patient.” The subject can be a human, although the subject may be an animal, as will be appreciated by those in the art. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject. As used herein, the term “effective amount” or “therapeutically effective amount” refers to that amount of a circular RNA molecule, composition, or pharmaceutical composition described herein that is sufficient to effect the intended application, including but not limited to inducing an immune response and / or disease treatment, as defined herein. The therapeutically effective amount may vary depending upon the intended application (e.g., inducing an immune response, treatment, application in vivo), or the subject or patient and disease condition being treated, e.g., theweight and age of the subject, the species, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in a target cell. The specific dose will vary depending on the particular circular RNA molecule, composition, or pharmaceutical composition chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.Reaction Mixtures

[0106] In one aspect, the present disclosure provides reaction mixtures for preparing circular RNAs or compositions comprising the same, in accordance with any of the various aspects and embodiments herein. In some embodiments, the reaction mixture comprises (a) a DNA- dependent RNA polymerase; (b) a plurality of nucleotide triphosphates, wherein the plurality of nucleotide triphosphates comprise modified nucleotides; and (c) a transcription template encoding an RNA, wherein (i) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA produces a circular RNA, (ii) the circular RNA comprises an IRES, and (iii) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES. In some embodiments, incubating the reaction mixture produces a linear RNA comprising two ends, that if joined, form the circular RNA. In some embodiments, the IRES is not formed until the 5’ nucleotide and 3’ nucleotide are joined. The circular RNA formed by joining the 5’ nucleotide and the 3’ nucleotide (e.g., by ligation) can be any circular RNA described herein, including with regard to any of the various aspects and embodiments (e.g., with regard to compositions described above).

[0107] In one aspect, the present disclosure provides a reaction mixture comprising an RNA and a ligase. In some embodiments, (i) the RNA comprises modified nucleotides; (ii) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA with the ligase produces a circular RNA; (iii) the circular RNA comprises an IRES; and (iv) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES. In some embodiments, the RNA is a linear RNA produced in a reaction mixture comprising a DNA-dependent RNA polymerase, such as in the preceding aspect. An RNA produced from an in vitro transcription reaction may be isolated prior to incubating in a separate reaction mixture comprising the ligase. The circular RNA can be any circular RNA described herein, including with regard to any of the various aspects and embodiments (e.g., with regard to compositions described above).

[0108] A DNA-dependent RNA polymerase is an enzyme that catalyzes the synthesis of an RNA transcript from a complementary DNA template. A variety of suitable RNA polymerases are available, examples of which include but are not limited to, phage RNA polymerases such as T7 RNA polymerase, T3 RNA polymerase, Kl l RNA polymerase, SP6 RNA polymerase, and variants thereof. In some embodiments, the reaction mixture is an in vitro transcription (IVT) reaction mixture. In an illustrative IVT method, a target polynucleotide sequence encoding an RNA molecule is first selected for incorporation into a vector which will be amplified to produce a DNA template. Optionally, the template polynucleotide sequence and / or any flanking sequences may be codon optimized. After production, the RNA molecule of the disclosure may undergo purification and clean-up processes. In some embodiments, the reaction mixture comprises a transcription buffer, the nucleotide triphosphates (NTPs), an RNase inhibitor, and the RNA polymerase. The NTPs may be selected from, but are not limited to, those described herein, including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.

[0109] For the transcription of an RNA to be circularized, the present disclosure also provides expression vectors comprising a polynucleotide sequence encoding the RNA that is preferably operably linked to at least one regulatory sequence. Regulatory sequences are art- recognized and are selected to direct expression of the encoded polypeptide. Accordingly, the term regulatory sequence in the context of a DNA template includes promoters, enhancers, and other expression control elements. In some embodiments, DNA molecules provided herein comprise a promoter. As used herein, the term “promoter” refers to a regulatory sequence that initiates transcription. A promoter can be operably linked to one or more polynucleotides of DNA molecules provided herein, with the one or more polynucleotides of DNA molecules encoding one or more polynucleotides of RNA molecules provided herein. Any suitable promoter for in vitro transcription can be included in DNA molecules provided herein, such as a T7 promoter, a T3 promoter, an SP6 promoter, and others. In some embodiments, DNA molecules provided herein comprise a T7 promoter.

[0110] In some embodiments, the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the firstinternal sequence in the 5 ’ to 3 ’ direction, and is located within 5 nucleotides of the first internal sequence. An illustrative example of such a structure is indicated by the arrow (open triangle) in FIG. 2 A, where the “G” below the arrow is the 5’ terminal nucleotide of the RNA, and the “C” above the arrow is the 3’ terminal nucleotide. As illustrated, the first internal sequence is a “CC” dinucleotide paired with the “GG” dinucleotide below the arrow, and the second internal sequence is the 9-nucleotide sequence of bases complementary to corresponding bases ending with the 3’ terminus. In some embodiments, the first end sequence and the second end sequence are each at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides in length. In some embodiments, the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length. In some embodiments, at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length. In some embodiments, one or both of the first end sequence and the second end sequence is at least 5 nucleotides in length. In some embodiments, one or both of the first end sequence and the second end sequence is at least 9 nucleotides in length. In some embodiments, the first end sequence and the second end sequence have a combined total length of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more nucleotides in length. In some embodiments, the first end sequence and the second end sequence have a combined total length of at least 5 nucleotides. In some embodiments, the first end sequence and the second end sequence have a combined total length of at least 10 nucleotides. In some embodiments, the first internal sequence and the second internal sequence have proximal ends that are separated by fewer than 5, 4, 3, 2, or 1 nucleotides. In some embodiments, there are no intervening nucleotides between the first internal sequence and the second internal sequence (i.e., the first nucleotide after the end of the first internal sequence is the beginning of the second internal sequence).

[0111] In some embodiments, the reaction mixture comprising a DNA-dependent RNA polymerase comprises nucleotide triphosphates (NTPs) comprising modified nucleotides. The NTPs may comprise unmodified (natural) nucleotides as well. In some embodiments, the NTPs comprise two or more different modified nucleotides. Combinations of any of these are contemplated herein. For example, NTPs may comprise unmodified A, U, G, and C, combined with modified U NTPs. As a further example, NTPs may comprise unmodified A, G, and C, with all U NTPs comprising modified nucleotides (same or different modification). In yet another example, two or more of A, U, G, and C are modified among the NTPs (e.g., some or all of U and some or all of C are modified, and none of the A and G NTPs are modified). In some embodiments, all of the NTPs comprise modified nucleotides. In some embodiments,the modified nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5- methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5 -methoxyuridine (5moU), 5- propynyluridine, 5-bromouridine, 5-iodouridine, 5 -fluorouridine, pseudouridine, 2'-O-methyl- pseudouridine, ISf-hydroxypseudouridine, ISf-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine. In some embodiments, the modified nucleotides comprise N1- methylpseudouridine (melψ). In some embodiments, the modified nucleotides comprise 5- methoxyuridine (5moU). In some embodiments, modified nucleotides incorporated into the linear RNA formed by the DNA-dependent RNA polymerase are retained in the circular RNA formed by joining the 5’ and 3’ ends thereof, thereby forming a circular RNA comprising modified nucleotides.

[0112] In some embodiments, the reaction mixture comprises an RNA and a ligase. The ligase catalyzes formation of phosphodiester bonds between 5 '-phosphate and 3 '-hydroxyl termini of a nucleic acid. In some embodiments, a 5’ nucleotide of the RNA joins to a 3’ nucleotide of the RNA with the ligase to produce a circular RNA. A variety of suitable ligases are available. Examples of ligases include, but not limited to, T4 RNA ligase (e.g., T4 RNA ligase 1, T4 RNA ligase 2, T3 RNA ligase, Rte A ligase, RtcB Ligase, and CIRCLIGASE. In some embodiments, the ligase is T4 RNA ligase. In some embodiments, the ligase is a T4 RNA ligase.Methods

[0113] In one aspect, the present disclosure provides a method of generating a circular RNA. In some embodiments, the method comprises joining a 5’ nucleotide of an RNA to a 3’ nucleotide of the RNA with a ligase, thereby generating the circular RNA. In some embodiments, (i) the RNA comprises modified nucleotides, (ii) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA with the ligase produces a circular RNA, (iii) the circular RNA comprises an IRES, and (iv) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES. Examples of particular RNAs, circular RNAs, IRES’s, modifiednucleotides, and ligases for use in and / or produced by the method are described herein, such as with regard to various aspects and embodiments of compositions and reaction mixtures described herein.

[0114] The circular RNA formed by joining the 5’ nucleotide and the 3’ nucleotide can be any circular RNA described herein, including with regard to any of the various aspects and embodiments (e.g., with regard to compositions described above). In some embodiments, the method is performed using one or more of the reaction mixtures described herein (e.g., with regard to reaction mixtures described above). In some embodiments, the method further comprises transcribing the RNA from a transcription template with a DNA-dependent RNA polymerase in the presence of a plurality of nucleotide triphosphates, wherein the plurality of nucleotide triphosphates comprise the modified nucleotides. In some embodiments, the RNA produced by the polymerase is purified before subjecting to ligation in a separate reaction.

[0115] In some embodiments, the circular RNA disclosed herein encodes a polypeptide in accordance with any of the various aspects and embodiments herein. In some embodiments, the RNA comprises modified nucleotides in accordance with any of the various aspects and embodiments herein, which may be located inside and / or outside the IRES. In some embodiments, all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides in the reaction mixture are modified nucleotides.

[0116] In some embodiments, the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5 ’ to 3 ’ direction, and is located within 5 nucleotides of the first internal sequence in accordance with any of the various aspects and embodiments herein. Exemplary arrangements of these sequence elements are described herein, such as with regard to various aspects and embodiments of the reaction mixtures herein.Ranges

[0117] Throughout this disclosure, various aspects can be presented in range format. It should be understood that any description in range format is merely for convenience and brevity and not meant to be limiting. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example 1, 2, 2.1, 2.2, 2.5, 3, 4, 4.75, 4.8, 4.85, 4.95, 5, 5.5, 5.75, 5.9, 5.00, and 6. This applies to a range of any breadth.MATERIALS AND METHODS FOR EXAMPLES 1-5

[0118] Production of circular RNA (circRNA)

[0119] circRNA precursors were synthesized via IVT from the linearized circRNA plasmid templates (Table 2). After IVT, the RNA products were treated with DNase I for 10 min to digest the DNA templates. Then, the RNA was column purified with the Monarch RNA Cleanup Kit (New England Biolabs, #T2040L). RNA precursors were ligated by T4 RNA ligase 2 (NEB, #M0239) for 2 h at 37 °C. The reactions were treated with RNase R at 37° C for 15-30 min to remove linear RNA. RNAs were resolved by agarose gel (Thermo, G402022) electrophoresis.Table 2. In Vitro Transcription Reactions* Inorganic Pyrophosphatase (iPPase)

[0120] The predicted 2D structures of circular RNAs generated during this study are shown in FIGS. 7A-7L.

[0121] Transfection and Luciferase Assay

[0122] For each sample, two wells of a 96-well plate were seeded with 8,000 cells / well in 100μl of DMEM. 24 hours after seeding, RNA samples were prepared for transfection. Nano- luciferase (NLuc; codon-optimized deep-sea shrimp luciferase) constructs were diluted to 50ng / pl, followed by combining Iμl of diluted nano-luciferase RNA at 50ng / μl, 3μL OptiMem™, and 5μL of MessengerMax™ mix. MessengerMax™ mix was prepared by adding 2 μl of MessengerMax™ to 4.8μl of OptiMem™, followed by incubation at room temperature for 5 minutes before addition to the transfection mix. Transfection mixtures (total volume of 10μl) were allowed to incubate for 15 minutes before adding to the wells for transfection. Media was collected and replaced every 24 hours until final timepoint collection. 50μl of collected media per duplicate sample were transferred to a white clear bottom 96-well plate, followed by preparation of Nano-Luc® Luciferase reaction solution (Promega, N1130) by combining Nano-Gio Substrate with Nano-Gio® Luciferase Assay Buffer and addition of 50μl of the prepared reaction solution to the wells of collected sample media. Luminescence was read immediately using the plate reader (Cytation™3, BioTek® Instruments, Inc). For Firefly Luciferase assays (Promega, E1501), IX Passive Lysis Buffer was prepared by dilution of 5X stock, and assay reagent was reconstituted with the provided substrate buffer. Media was removed from sample wells, followed by washing with 100μL PBS and addition of 40 μL of IX Passive Lysis buffer. Samples were allowed to shake in lysis buffer at 750rpm for 30 minutes to 1 hour, after which 30 pl of lysate was transferred to a white clear bottom 96-well plate. Upon addition of 80μL of reconstituted reagent, luminescence was read immediately with the plate reader Auto-gain function.

[0123] TaqMan Assay

[0124] To quantify mRNA expression, Taqman assays were performed using a TaqMan RNA-to Ct 1-Step Kit (Thermo, 4392938), PKR Probe (Thermo, EIF2A), and RIG1 Probe (Thermo, DDX58). Media was removed from the sample wells and wells were washed with 100μL PBS. 40 μL of Cell-to-Ct Lysis buffer was added, followed by shaking at 750 rpm for 30 minutes to 1 hour. For one reaction (10μl total reaction volume), 2pl of lysate, O.25μL Cells-to-Ct Enzyme mix, 5μL Cells-to Ct RT mix, 0.5μL of FAM probe, 0.5μL of VIC probe, and 1.75μL of water were added.EXAMPLE 1

[0125] This example describes circularization of RNA using ligase and ribozyme methods.

[0126] Circular RNA (circRNA) synthesized by permuted self-splicing thymidylate synthase (td) introns (Ribozyme) increases cellular innate immune responses because of the presence of extra immunostimulatory fragments (e.g., El, E2, and spacer). By contrast, circRNA produced by T4 RNA ligase that lacks these immunostimulatory sequences shows lower immunogenicity (Liu et al., Molecular Cell 82, 420-434, 2022). To determine the effect of these exogenous selfsplicing sequences on protein expression, Nano Luciferase (NLuc) expression in Hepa 1-6 cells was compared for circRNAs with or without the exogenous self-splicing sequence and made using either the Ribozyme or T4 RNA ligase ligation method (FIG. 1 A). The circRNA made with ligase without the exogenous sequences showed greater expression than the circRNA made with ribozyme. Furthermore, the circRNA that contains the exogenous self-splicing sequence but was ligated with T4 ligase shows reduced expression comparable to the ribozyme circRNA. Regardless of the circularization method, NLuc expression was reduced when extra fragments were present.

[0127] Without being limited by theory, RNA modifications can be important post- transcriptional epigenetic regulators of gene expression, including modifications such as Nl- methylpseudouridine (melψ) and 5-methoxyuridine (5moU). Nucleoside modifications can reduce immunogenicity and improve translation (Alameh & Weissman, 2022). To test whether chemically modified circRNAs containing ribozyme fragments can reduce immunogenicity, the ribozyme method was used to prepare melψ or 5moU modified circRNAs. RNase R selectively degrades linear RNA. Thus, resistance to RNase R can be used as a criterion for identifying circRNAs. RNAs produced without nucleoside modification showed resistance to RNase R, consistent with the presence of circRNA (FIG. IB). By contrast, RNA prepared inthe presence of melψ or 5moU were degraded after treatment of RNase R (FIG. IB), indicating that meh| / or 5moU modified RNA were not circularized via ribozymes in vitro, consistent with other studies using the ribozyme method (Wesselhoeft et al., Molecular Cell 74, 508-520, 2019).

[0128] These results show that RNA circularization does not occur in the presence of modified nucleosides for RNA prepared by the ribozyme method.EXAMPLE 2

[0129] This example describes preparation of chemically modified circRNA.

[0130] The T4 RNA ligase-based method was utilized to produce chemically modified circRNAs from a template of SEQ ID NO:2 (including a T7 RNA polymerase promoter sequence). The 5’ and 3’ ends of SEQ ID NO:2 are partially complimentary, forming a complete coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) upon circularization for protein expression (Qu et al., Cell 185, 1728-1744, 2022). Without being limited by theory, this complimentary sequence within the circRNA generates a two-dimensional (2D) nick structure that includes the 5’ and 3’ ends and serves as a catalytic substrate for T4 RNA ligase without the use of splint oligonucleotides (FIG. 2A). The location of pairs of complementarity sequences near the 5’ and 3’ ends are also indicated below for each of SEQ ID Nos: 2-12 by pairs of bold or underlined sequences. Unmodified (UTP), melψ-modified, or 5moU-modified RNAs were synthesized by in vitro transcription (IVT) using the DNA construct encoding the RNA with self-complementarity and circularized with T4 RNA ligase to prepare circRNA (FIG. 2B). Agarose gel electrophoresis shows that new bands appeared after adding ligase that correspond to circRNA (indicated by the arrowhead). Modification of RNA with melψ or 5moU did not affect circularization. To test the secretory expression of NLuc produced by circRNAs, purified circRNAs were transfected into HepG2 cells and NLuc activity was measured (FIG. 2C). Unmodified circRNA expressed the NLuc protein, but no NLuc activity was detected with melψ or 5moU modified circRNA.

[0131] These results show that modified nucleosides in circRNA prevented protein expression driven via a split CVB3 IRES.EXAMPLE 3

[0132] This example describes circularization and expression of larger circRNAs with and without modified nucleosides.

[0133] To investigate the circularization and expression of a chemically modified circRNA larger than circRNAs encoding Nluc described above (open reading frame (ORF) of 603 nt), the coding sequence was replaced with CAR-GFP (ORF of 3201 nt) between the split CVB3 sequences (FIG. 3 A). Linear RNAs were generated by IVT using either UTP or melψ and circularized with T4 RNA ligase (FIG. 3B). As shown in FIG. 3B (arrowheads), circularization was possible even for this large RNA size with complex secondary structure. In vitro synthesized circRNAs were introduced into Jurkat cells. Flow cytometry was carried out to measure the expression of GFP (FIG. 3C). GFP transcripts were only induced in cells transfected with unmodified circRNA, as seen by GFP expression. RIG-I and PKR have been characterized as recognizing dsRNA and activating the innate immune response (Mu & Hur, 2021). To test the immunogenicity of exogenous circRNAs, RNA levels of RIG-I and PKR were measured (FIG. 3D and FIG. 3E). Linear RNA, modified or unmodified, activated the early innate immune response, as seen by increased PKR and RIG-I mRNA levels (FIG. 3D and FIG. 3E, respectively). Unmodified circ RNA also resulted in PKR and RIG-I mRNA induction relative to Control. However, chemically modified circRNAs did not activate the early innate immune response (FIGS. 3D and 3E).

[0134] These results show that circular RNA with chemical modifications does not induce the expression of dsRNA sensors that function in innate immunity, even with an ORF of more than 3,000 nucleotides.EXAMPLE 4

[0135] This example describes identification of functional IRES sequences with nucleoside modifications.

[0136] The mechanism by which nucleoside modifications suppress circRNA expression is not well understood. Without being limited by theory, structural changes of the circRNA IRES induced by modified nucleosides may contribute to suppression of expression (Wesselhoeft et al., Molecular Cell 74, 508-520, 2019). Due to the inability of circRNA to be capped, IRES structures within the 5' untranslated region (UTR) are utilized to directly or indirectly recruit ribosomes to drive RNA translation (Yang et al., J. Mol. Cell. Biol. 11, 911-918, 2019; Yang et al., Molecular Therapy 29, 1683-1702, 2021). Without being limited by theory, IRES structure may be important because the conformation of the IRES structure within the 5' untranslated region (UTR) recruits ribosomes via interactions with RNA-binding proteins known as IRES trans-acting factors, for example. To express the chemically modifiedcircRNA, finding an active IRES that retains its function with nucleoside modification is important. circFLuc was generated containing various IRESs using RNA ligase with either UTP or melψ (FIG. 4A). Each circRNA construct was transfected into Hepa 1-6 cells to test whether the IRES increased expression from circFLuc with melψ modification as compared to expression from circFLuc without nucleoside modification (FIG. 4B). Among the construct pairs in which melψ modification produced significantly increased expression over the unmodified construct, melψ-modified circFLuc with TEV produced the most robust translation, with greater expression also seen for at least melψ-modified circFLuc with Hbb- bl, Hbb-bl-113, Hsp70, and Apaf-1 IRES sequences as compared to circRNA without nucleoside modifications.

[0137] These results show that several IRES sequences were identified that can increase expression of nucleoside-modified circRNA.EXAMPLE 5

[0138] This example describes the effect of melψ or 5moU nucleoside modification on IRES-mediated luciferase expression.

[0139] circFLuc (FIG. 5A) and circNLuc (FIG. 6A) containing TEV with UTP, melψ, or 5moU were generated and transfected into HepG2 cells (FIGS. 5B and 6B). Compared to UTP, melψ increased circRNA translation, but 5moU did not change the level of translation (FIGS. 5B and 6B). Furthermore, melψ or 5moU reduced PKR and RIG-I upregulation in HepG2 cells (Figure 5C, 6C).SEQUENCES

[0140] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising a circular RNA, wherein:(i) the circular RNA comprises an internal ribosomal entry site (IRES) and modified nucleotides; and(ii) the IRES is a tobacco etch virus (TEV) IRES, a human β-globin (Hbb-bl) IRES, a heat shock protein 70 (Hsp70) IRES, or an apoptotic peptidase activating factor-1 (Apaf-1) IRES.

2. The composition of claim 1, wherein the IRES comprises the modified nucleotides.

3. The composition of claim 1 or 2, wherein the circular RNA comprises the modified nucleotides outside of the IRES.

4. The composition of any one of claims 1-3, wherein all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the circular RNA are modified nucleotides.

5. The composition of any one of claims 1-4, wherein the modified nucleotides comprise one or more of 5 -hydroxy cytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5- carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5- hydroxyuridine, 5 -methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O- methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'- deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5 -formyluridine, 5-methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine.

6. The composition of claim 5, wherein the modified nucleotides comprise N1- methylpseudouridine (melψ).

7. The composition of claim 5, wherein the modified nucleotides comprise 5- methoxyuridine (5moU).

8. The composition of any one of claims 1-7, wherein the IRES comprises any one of SEQ ID NO: 13-17.

9. The composition of any one of claims 1-8, wherein the circular RNA encodes a polypeptide.

10. The composition of claim 9, wherein the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length.

11. The composition of claim 9, wherein the polypeptide comprises a chimeric antigen receptor (CAR).

12. The composition of any one of claims 9-11, wherein the circular RNA is effective to induce translation of the polypeptide in a host cell.

13. The composition of claim 12, wherein the host cell is a human cell.

14. The composition of any one of claims 1-13, wherein the composition further comprises a pharmaceutically acceptable carrier.

15. The composition of claim 14, wherein the pharmaceutically acceptable carrier is a lipid formulation.

16. The composition of claim 15, wherein the lipid formulation is selected from a lipid nanoparticle, a lipoplex, a liposome, a polymer-based carrier, an exosome, a lamellar body, a micelle, and an emulsion.

17. The composition of claim 16, wherein the lipid formulation is a lipid nanoparticle.

18. The composition of claim 17, wherein the lipid nanoparticle has a size of less than about 200 nm, 180 nm, 130 nm, or 80 nm.

19. The composition of any one of claims 15-18, wherein the lipid formulation comprises an ionizable cationic lipid.

20. The composition of claim 19, wherein the ionizable cationic lipid has a structure of Formula I:or a pharmaceutically acceptable salt or solvate thereof, wherein R5and R6are each independently selected from the group consisting of a linear or branched C1-C31alkyl, C2-C31alkenyl or C2-C31alkynyl and cholesteryl; L5and L6are each independently selected from the group consisting of a linear C1-C20alkyl and C2-C20alkenyl; X5is -C(O)O-, whereby -C(O)O- R6is formed or -OC(O)- whereby -OC(O)-R6is formed; X6is -C(O)O- whereby -C(O)O-R5is formed or -OC(O)- whereby -OC(O)-R5is formed; X7is S or O; L7is absent or lower alkyl; R4is a linear or branched C1-Ce alkyl; and R7and R8are each independently selected from the group consisting of a hydrogen and a linear or branched C1-Ce alkyl.

21. The composition of claim 19, wherein the ionizable cationic lipid is selected from Table 1.

22. The composition of any one of claims 15-21, wherein the lipid formulation comprises a helper lipid.

23. The composition of claim 22, wherein (a) the helper lipid is a phospholipid, or (b) the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC).

24. The composition of any one of claims 15-23, wherein the lipid formulation comprises cholesterol.

25. The composition of any one of claims 15-24, wherein the lipid formulation comprises a polyethylene glycol (PEG)-lipid conjugate.

26. The composition according to any one of claims 1-25 for use as a medicament.

27. Use of the composition of any one of claims 1-25 in the manufacture of a medicament.

28. A method of expressing a polypeptide in a cell in vitro, the method comprising contacting the cell with a composition of any one of claims 9-25.

29. A method of expressing a polypeptide in a subject, the method comprising administering to the subject a composition of any one of claims 9-25.

30. A reaction mixture comprising:(a) a DNA-dependent RNA polymerase;(b) a plurality of nucleotide triphosphates, wherein the plurality of nucleotide triphosphates comprise modified nucleotides; and(c) a transcription template encoding an RNA, wherein (i) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA produces a circular RNA, (ii) the circular RNA comprises an IRES, and (iii) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES.

31. The reaction mixture of claim 30, wherein the circular RNA encodes a polypeptide.

32. The reaction mixture of claim 30 or 31, wherein all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides in the reaction mixture are modified nucleotides.

33. The reaction mixture of any one of claims 30-32, wherein the modified nucleotides comprise one or more of 5 -hydroxy cytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5- carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5- hydroxyuridine, 5 -methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'- deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5 -formyluridine, 5-methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine.

34. The reaction mixture of claim 33, wherein the modified nucleotides comprise N1- methylpseudouridine (melψ).

35. The reaction mixture of claim 33, wherein the modified nucleotides comprise 5- methoxyuridine (5moU).

36. The reaction mixture of any one of claims 30-35, wherein the IRES comprises any one of SEQ ID NO: 13-17.

37. The reaction mixture of any one of claims 31-36, wherein the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length.

38. The reaction mixture of any one of claims 31-36, wherein the polypeptide comprises a chimeric antigen receptor (CAR).

39. The reaction mixture of any one of claims 30-38, wherein the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5’ to 3’ direction, and is located within 5 nucleotides of the first internal sequence.

40. The reaction mixture of claim 39, wherein the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length.

41. The reaction mixture of claim 39 or 40, wherein at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length.

42. The reaction mixture of any one of claims 39-41, wherein there are no intervening nucleotides between the first internal sequence and the second internal sequence.

43. A reaction mixture comprising an RNA and a ligase, wherein:(i) the RNA comprises modified nucleotides;(ii) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA with the ligase produces a circular RNA;(iii) the circular RNA comprises an IRES; and(iv) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES.

44. The reaction mixture of claim 43, wherein the circular RNA encodes a polypeptide.

45. The reaction mixture of claim 43 or 44, wherein the IRES comprises the modified nucleotides.

46. The reaction mixture of any one of claims 43-45, wherein the circular RNA comprises the modified nucleotides outside of the IRES.

47. The reaction mixture of any one of claims 43-46, wherein all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the RNA are modified nucleotides.

48. The reaction mixture of any one of claims 43-47, wherein the modified nucleotides comprise one or more of 5 -hydroxy cytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5- carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5- hydroxyuridine, 5 -methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O- methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'- deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5 -formyluridine, 5-methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-O- methylguanosine.

49. The reaction mixture of claim 48, wherein the modified nucleotides comprise N1- methylpseudouridine (melψ).

50. The reaction mixture of claim 48, wherein the modified nucleotides comprise 5- methoxyuridine (5moU).

51. The reaction mixture of any one of claims 43-50, wherein the IRES comprises any one of SEQ ID NO: 13-17.

52. The reaction mixture of any one of claims 44-51, wherein the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length.

53. The reaction mixture of any one of claims 44-51, wherein the polypeptide comprises a chimeric antigen receptor (CAR).

54. The reaction mixture of any one of claims 43-53, wherein the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5’ to 3’ direction, and is located within 5 nucleotides of the first internal sequence.

55. The reaction mixture of claim 54, wherein the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length.

56. The reaction mixture of claim 54 or 55, wherein at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length.

57. The reaction mixture of any one of claims 54 -56, wherein there are no intervening nucleotides between the first internal sequence and the second internal sequence.

58. A method of generating a circular RNA, the method comprising joining a 5’ nucleotide of an RNA to a 3’ nucleotide of the RNA with a ligase, thereby generating thecircular RNA, wherein:(i) the RNA comprises modified nucleotides;(ii) joining a 5’ nucleotide of the RNA to a 3’ nucleotide of the RNA with the ligase produces a circular RNA;(iii) the circular RNA comprises an IRES; and(iv) the IRES is a TEV IRES, an Hbb-bl IRES, an Hsp70 IRES, or an Apaf-1 IRES.

59. The method of claim 58, wherein the circular RNA encodes a polypeptide.

60. The method of claim 58 or 59, wherein the IRES comprises the modified nucleotides.

61. The method of any one of claims 58-60, wherein the circular RNA comprises the modified nucleotides outside of the IRES.

62. The method of any one of claims 58-61, wherein all uracil nucleotides, all cytosine nucleotides, all guanine nucleotides, and / or all adenine nucleotides of the circular RNA are modified nucleotides.

63. The method of any one of claims 58-62, wherein the modified nucleotides comprise one or more of 5 -hydroxy cytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5- carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5- hydroxyuridine, 5 -methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O- methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'- deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5 -formyluridine, 5-methoxyuridine (5moU), 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N1-hydroxypseudouridine, N1-methylpseudouridine (melψ), 2'-O-methyl-N1- methylpseudouridine, N1-ethylpseudouridine, N1-hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine.

64. The method of claim 63, wherein the modified nucleotides comprise N1- methylpseudouridine (melψ).

65. The method of claim 63, wherein the modified nucleotides comprise 5- methoxyuridine (5moU).

66. The method of any one of claims 58-65, wherein the IRES comprises any one of SEQ ID NO: 13-17.

67. The method of any one of claims 59-66, wherein the polypeptide is at least 100, 200, 500, 750, 1000, or more amino acids in length.

68. The method of any one of claims 59-66, wherein the polypeptide comprises a chimeric antigen receptor (CAR).

69. The method of any one of claims 58-68, wherein the RNA comprises: (i) a first end sequence comprising a 5’ terminal nucleotide; (ii) a second end sequence comprising a 3’ terminal nucleotide; (iii) a first internal sequence that is complementary to the first end sequence; and (iv) a second internal sequence that is complementary to the second end sequence, located downstream of the first internal sequence in the 5’ to 3’ direction, and is located within 5 nucleotides of the first internal sequence.

70. The method of claim 69, wherein the first end sequence and the second end sequence are each at least 2, 3, 4, or 5 nucleotides in length.

71. The method of claim 69 or 70, wherein at least one of the first end sequence or the second end sequence is at least 5, 6, 7, 8, or 9 nucleotides in length.

72. The method of any one of claims 69-71, wherein there are no intervening nucleotides between the first internal sequence and the second internal sequence.

73. The method of any one of claims 58-72, further comprising transcribing the RNA from a transcription template with a DNA-dependent RNA polymerase in the presence of a plurality of nucleotide triphosphates, wherein the plurality of nucleotide triphosphates comprise the modified nucleotides.

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