WO2025259931A1 - Compositions and methods for rna circularization - Google Patents

Abstract

The present invention provides nucleic acids and methods for making circular RNAs (circRNAs), and circular RNAs, compositions and methods of use thereof. The present invention uses selected self-splicing intron sequences (e.g., intronic sequences derived from self-splicing Group I or Group II introns) to produce circular RNAs. These self-splicing intron sequences described herein mediate efficient circularization of a linear RNA sequence.

Description

COMPOSITIONS AND METHODS FOR RNA CIRCULARIZATION

CROSS REFERENCE TO RELATED APPLICATIONS

[01] The present application claims priority to, and the benefit of U.S. Provisional Application Number 63/660,303, filed on June 14, 2024, the contents of which are incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[02] The present application is being filed with an electronically filed Sequence Listing in XML format. The sequence listing file entitled ORB-011WO1 SL. XML was created on June 11, 2025, and is 32,768 bytes in size; the information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

[03] Unlike canonical linear RNAs, circular RNAs form covalently closed, continuous stable loops. Therefore, circular RNAs are resistant to exonuclease digestion, making them more stable as compared to linear RNA. The circular form makes the RNA molecule more stable and results in the circular RNA having increased protein production capabilities and increased efficacy as therapeutics. The circular structure may also prolong product shelf life compared to current mRNA therapeutics (e.g., mRNA vaccines) and relieve stringent storage and shipping conditions.

[04] There are numerous challenges for efficiently circularizing RNA, including long protein-coding RNA sequences. Thus, there is a need for more efficient RNA circularization techniques.

SUMMARY

[05] The present invention provides nucleic acids and methods for making circular RNAs (circRNAs), and circular RNAs, compositions and methods of use thereof. The present invention utilizes self-splicing intron sequences (e.g., intronic sequences derived from selfsplicing Group I or Group II introns) to produce circular RNAs. The self-splicing intron sequences described herein mediate efficient self-splicing to circularize an RNA sequence flanked by these intronic sequences. In some embodiments, the self-splicing intronic sequences are derived from Group I or Group II introns. As non-limiting examples, the Group I introns disclosed herein include intron sequences derived from Twort-ORF142 intron, Oscillator ia-splendida-trnL intron, and Anabaena-spiroides-trnL intron.

[06] In some aspects, provided herein is a nucleic acid molecule for making a circular RNA, comprising, from 5’ to 3’ end, (i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, (ii) a nucleic acid sequence of interest, and (iii) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same Group I or Group II intron as the upstream intron sequence, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are each derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[07] In some embodiments of the nucleic acid molecule, the 3’ intron splicing fragment and the 5’ intron splicing fragment retain a catalytic core of the Group I or Group II intron.

[08] In some embodiments of the nucleic acid molecule, the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from variants or truncations of any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[09] In some embodiments, the nucleic acid molecule does not include a 5’ or 3’ homology arm.

[010] In some embodiments, the nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence of interest and/or a 3’ untranslated region (UTR) sequence between the sequence of interest and the downstream intron sequence.

[OH] In some embodiments, the 5’ spacer sequence comprises an inner homology element.

[012] In some embodiments, the 5’ spacer sequence does not comprise an inner homology element.

[013] In some embodiments, the nucleic acid molecule does not include the 5’ spacer sequence.

[014] In some embodiments, the nucleic acid molecule further comprises an internal ribosome entry site (IRES) between the 5’ spacer sequence and the sequence of interest.

[015] In some embodiments, the nucleic acid molecule comprises a 3’ UTR between the sequence of interest and the downstream intron sequence.

[016] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’ and 5’ intron splicing fragments of SEQ ID NO: 7. [017] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 119 to 282 of SEQ ID NO. 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-118 of SEQ ID NO: 7.

[018] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 140 to 282 of SEQ ID NO. 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7.

[019] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1 to 146 of SEQ ID NO: 7.

[020] In some embodiments, the upstream intron sequence comprises a sequence at least 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2.

[021] In some embodiments, the upstream intron sequence comprises the sequence of SEQ ID NO: 1, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 2.

[022] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’ and 5’ splicing fragments of SEQ ID NO: 8.

[023] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 112-231 of SEQ ID NO: 8, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-111 of SEQ ID NO: 8.

[024] In some embodiments, the upstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4.

[025] In some embodiments, the upstream intron sequence comprises the sequence of SEQ ID NO: 3, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 4.

[026] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’ and 5’ splicing fragments of SEQ ID NO: 9.

[027] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 127-264 of SEQ ID NO: 9, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-126 of SEQ ID NO: 9.

[028] In some embodiments, the upstream intron sequence comprises a sequence at least 85%, 90%, 95%, 99%, or 99% identical to SEQ ID NO: 5, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

[029] In some embodiments, the upstream intron sequence comprises the sequence of SEQ ID NO: 5, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 6.

[030] In some embodiments, the nucleic acid molecule is a DNA vector.

[031] In some embodiments, the nucleic acid molecule is a linear RNA.

[032] In some embodiments, the linear RNA is unmodified.

[033] In some embodiments, the linear RNA is modified.

[034] In some embodiments, the linear RNA comprises one or more modified nucleotides N 1 -methylpseudouridine.

[035] In some embodiments, the linear RNA comprises one or more modified nucleotides 5-methoxyuridine.

[036] In some embodiments, the linear RNA comprises one or more modified nucleotides m5C.

[037] In some embodiments, the sequence of interest is a non-coding sequence.

[038] In some embodiments, the sequence of interest is a coding sequence.

[039] In some embodiments, the sequence of interest encodes a protein.

[040] In some embodiments, the protein is a chimeric antigen receptor (CAR), a T cell receptor, a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.

[041] In some embodiments, the protein is an antigen derived from a pathogen, or specific to a tumor.

[042] In some embodiments, the CAR comprises an antigen binding domain, a hinge, a transmembrane domain, and one or more intracellular signaling domains.

[043] In some embodiments, wherein self-splicing of the upstream and downstream Group I or Group II intron sequences makes a circular RNA.

[044] In some embodiments, provided herein is a circular RNA that is made from the nucleic acid molecule of the present disclosure.

[045] In some embodiments, provided herein is a composition comprising the circular RNA of the present disclosure.

[046] In some embodiments, provided herein is a composition comprising the circular

RNA of the present disclosure formulated in a delivery vehicle.

[047] In some embodiments, the delivery vehicle is a lipid nanoparticle.

[048] In some embodiments, the lipid nanoparticle is conjugated to a targeting moiety. [049] In some embodiments, provided herein is a method for making a circular RNA using the nucleic acid molecule of the present disclosure.

[050] In some aspects, provided herein is a method for making a circular RNA comprising circularizing a nucleic acid molecule comprising, from 5’ to 3’ end, (i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, (ii) a nucleic acid sequence of interest, and (iii) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same Group I or Group II intron as the upstream intron sequence, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and wherein transcribing the nucleic acid molecule to a linear precursor RNA and circularizing the linear precursor RNA into the circular RNA occurs in one reaction.

[051] In some embodiments, the nucleic acid molecule does not include a 5’ or 3’ homology arm.

[052] In some embodiments, the nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence of interest and/or a 3’ untranslated region (UTR) sequence between the sequence of interest and the downstream intron sequence.

[053] In some embodiments, the 5’ spacer sequence comprises an inner homology element.

[054] In some embodiments, the 5’ spacer sequence does not comprise an inner homology element.

[055] In some embodiments, the nucleic acid molecule does not include the 5’ spacer sequence.

[056] In some embodiments, the nucleic acid molecule further comprises an internal ribosome entry site (IRES) between the 5’ spacer sequence and the RNA sequence.

[057] In some embodiments, the nucleic acid molecule is a DNA vector.

[058] In some embodiments, the nucleic acid molecule is a linear RNA.

[059] In some embodiments, the method comprises incubating the nucleic acid molecule at a temperature at which RNA circularization occurs.

[060] In some embodiments, the temperature is about 30°C to 60°C.

[061] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’and 5’ intron splicing fragments of SEQ ID NO: 7.

[062] In some embodiments, provided herein is a method of the present disclosure, wherein the nucleic acid comprises: (i) the upstream intron sequence comprising a sequence corresponding to positions 119 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1-118 of SEQ ID NO: 7; (ii) the upstream intron sequence comprising a sequence corresponding to positions 140 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7; or (iii) the upstream intron sequence comprising a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 146 of SEQ ID NO: 7.

[063] In some embodiments, the nucleic acid comprises the upstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 1, and the downstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO. 2.

[064] In some embodiments, the circularization occurs at 35°C to 42°C.

[065] In some embodiments of the method, the nucleic acid comprises the upstream and downstream intron sequences each corresponding to the 3 ’and 5’ splicing fragments of SEQ ID NO. 8.

[066] In some embodiments of the method, the nucleic acid comprises the upstream intron sequence comprising a sequence corresponding to positions 112-231 of SEQ ID NO: 8, and the downstream intron sequence comprising a sequence corresponding to positions 1-111 of SEQ ID NO: 8.

[067] In some embodiments of the method, the nucleic acid comprises the upstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 3, and the downstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 4.

[068] In some embodiments, the circularization occurs at a temperature between 35°C to 45°C.

[069] In some embodiments, the nucleic acid comprises the upstream and downstream intron sequences each corresponding to the 3’ and 5’ splicing fragments of SEQ ID NO. 9. [070] In some embodiments, the nucleic acid comprises the upstream intron sequence comprising a sequence corresponding to positions 127-264 of SEQ ID NO: 9, and the downstream intron sequence comprising a sequence corresponding to positions 1-126 of SEQ ID NO: 9.

[071] In some embodiments, the nucleic acid comprises the upstream intron sequence comprising a sequence at least 85%, 90%, 95%, 99%, or 99% identical to SEQ ID NO: 5, and the downstream intron sequence comprising a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

[072] In some embodiments of the method, wherein the circularization occurs at 35°C to 42°C.

[073] In some embodiments, provided herein is a method wherein the RNA of interest encodes a chimeric antigen receptor (CAR), a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.

[074] In some embodiments of the method, the RNA of interest encodes an antigen.

[075] In some embodiments, the linear RNA is unmodified.

[076] In some embodiments, the linear RNA is modified.

[077] In some embodiments, the linear RNA comprises one or more modified nucleotides N1 -methylpseudouridine and/or 5-methoxyuridine.

[078] In some embodiments, the method makes intact circular RNA.

[079] In some embodiments, provided herein is a circular RNA made by the method of the present disclosure.

[080] In some embodiments, provided herein is a method for expressing a protein of interest in a subject comprising delivering to the subject the circular RNA of the present disclosure.

[081] In some aspects, provided herein is a method of expressing a protein of interest in a subject comprising delivering to the subject a circular RNA transcribed from a nucleic acid, wherein said nucleic acid comprises the following elements: (i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, (ii) 5’ spacer, (iii) an internal ribosome entry sequence (IRES), (iv) a sequence that encodes the protein of interest, (v) a 3 ’ UTR sequence, (vi) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same intron as the upstream intron sequence, and wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and wherein the nucleic acid does not include a 5’ or 3’ homology arm.

[082] In some embodiments, the circular RNA is formulated in a delivery vehicle.

[083] In some embodiments, the delivery vehicle is a lipid nanoparticle.

[084] In some embodiments, the lipid nanoparticle is conjugated to a targeting moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[085] FIG. 1 is a representative diagram of a nucleic acid construct for making a circular RNA. It demonstrates the upstream intron fragment (“US intron”) derived, e.g., from a Group I or Group self-splicing intron and the downstream intron fragment (“DS intron”) derived, e.g., from the same Group I or Group II self-splicing intron flanking an RNA sequence (e.g., an open reading frame (ORF) or nucleic acid coding sequence). In some embodiments, as shown in FIG. 1, the RNA transcript further comprises a 5’ spacer, an Internal Ribosome Entry Site (IRES), and a 3’ UTR. Splicing of the US and DS intron fragments generates a circular RNA sequence.

[086] FIG. 2A - FIG. 2D illustrates four different embodiments of constructs for making circular RNAs. FIG. 2A shows a RNA transcript construct comprising inner homology elements (z.e., IHE) and 5’ and 3’ homology arms (z.e., outer homology arms)(OHE/IHE). FIG. 2B shows a RNA transcript construct including inner homology elements (z.e., IHE) only, without outer homology arms (-/IHE). FIG. 2C shows a RNA transcript construct that lacks both inner homology elements, and 5’ and 3’ homology arms (-/-). FIG. 2D shows a RNA transcript construct including 5’ and 3’ homology arms only, without inner homology elements (OHE/-).

[087] FIG. 3 is the sequence and secondary structure of Twort-ORF142 intron and exemplary permuted positions (also known as “splitting position” or “cut sites”). Three representative different splitting positions or cut sites (VI, V2, and V3) are shown. Splitting or cutting at these positions or sites generates a pair of upstream intron sequence and a downstream intron sequence.

[088] FIG. 4A shows the Twort-ORF142 intron comprising SEQ ID NO: 7 and an exemplary pair of the upstream intron sequence and the downstream intron sequence from SEQ ID NO: 7. FIG. 4B shows the Oscillator ia-splendida-trnL intron comprising SEQ ID NO: 8 and an exemplary pair of the upstream intron sequence and the downstream intron sequence from SEQ ID NO: 8. FIG. 4C shows the Anabaena-spiroides-trnL intron comprising SEQ ID NO: 9 and an exemplary pair of the upstream intron sequence and the downstream intron sequence from SEQ ID NO: 9.

[089] FIG. 5A - FIG. 5C shows representative gel images demonstrating that circular RNAs were successfully generated using intron sequences from Twort-ORF142 intron (FIG. 5A), Oscillator ia-splendida-trnL intron (FIG. 5B), and Anabaena-spiroides-trnL intron (FIG. 5C), respectively. A construct using the reference T4-td intron sequences (Chen et al., Nature Biotech., 2023, 41 : 262-272) was used as a control. By direct co-transcriptional circularization, all three intron fragments generated circular RNAs, more efficiently or comparably to the reference T4-td intron sequences. Similarly, after post-refolding ribozyme activation, all three intron fragments generated circular RNAs more efficiently, or comparably to the T4-td intron sequences. As shown in the gel images, circular RNAs generated using Twort-ORF142 intron, Oscillator ia-splendida-trnL intron, and Anabaena- spiroides-trnL intron sequences have less nicked circular RNAs compared to the reference T4-td intron sequences (the bands on top of “circle” in FIG. 5A - FIG. 5C).

[090] FIG. 6A shows RNA circularization using the upstream and downstream intron sequences derived from the Twort-ORF142 intron. The “zzz vitro transcription” gel images on the right shows RNA circularization using three different pairs of the upstream and downstream intron sequences split at positions VI, V2, or V3 (Twort-vl, Twort-v2 and Twort-v3, as shown in FIG. 3; the upstream and downstream intron sequences generated after each split are listed in Table 1 herein) by direct co-transcriptional circularization (z.e., during in vitro transcription (IVT)). The “ribozyme refolding” gel images on the left show RNA circularization using the Twort-vl, Twort-v2 and Twort-v3 via post-refolding ribozyme activation after co-transcriptional circularization.

[091] FIG. 6B demonstrates RNA circularization efficiency using the three different pairs of the Twort-ORF142 upstream and downstream intron sequences Twort-vl, Twort-v2 and Twort-v3) by direct co-transcriptional circularization (z.e., IVT). The “% spliced IVT” indicates the percentage of circular RNAs over total RNAs (including circular RNAs and linear RNAs).

[092] FIG. 6C demonstrates RNA circularization efficiency using the three different pairs of the Twort-ORF142 upstream and downstream intron sequences (Twort-vl, Twort-v2 and Twort-v3) via post-refolding ribozyme activation. The “% spliced refolded” indicates the percentage of circular RNAs over the whole RNAs (including circular RNAs and linear RNAs).

[093] FIG. 7A - FIG. 7D shows representative gel images of RNA circularization using the upstream and downstream intron sequences from T4 bacteriophage (FIG. 7A), Twort- ORF142 intron (Twort-v2) (FIG. 7B), Oscillatoria-splendida-trnL intron (FIG. 7C), and Anabaena-spiroides-trnL intron (FIG. 7D) at different temperatures, via post-refolding ribozyme activation.

[094] FIG. 8A and FIG. 8B show RNA circularization efficiency using the upstream and downstream intron sequences from Twort-ORF142 intron (Twort-v2), or Oscillatoria- splendida-trnL intron. Four constructs “1. IHE/OHE”, “2. IHE/-”, “3. -/OHE”, and “4. -/-”, each including Twort-ORF142 intron, or Oscillator ia-splendida-trnL intron “US” and “DS” sequences, were tested and compared for circularization efficiency. FIG. 8A shows representative gel images of RNA circularization using these four constructs after direct co- transcriptional circularization, and post-refolding ribozyme activation, respectively. As shown in FIG. 8B, the construct that only includes inner homology element (IHE) sequences without 5’ and 3’ homology arms (z.e., “-/IHE” construct (used interchangeably herein with “IHE/-”) construct design as shown in FIG. 2B) has higher percentage of RNA circularization as compared to the constructs including 5’ and 3’ homology arms (e.g., “IHE/OHE” and “-/OHE ”).

DEFINITIONS

[095] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[096] About'. As used herein, the term “about” is understood as within a range of normal tolerance in the art, for example, within two standard deviations of the mean. About can be understood as within 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[097] Associated'. As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” needs not be strictly through direct covalent chemical bonding. It may also be through ionic or hydrogen bonding or a hybridization-based connectivity sufficiently stable such that the “associated” entities remain physically associated.

[098] Circular RNA : As used herein, the term “circular RNA” or “circRNA” refers to an RNA that forms a circular structure through covalent or non-covalent bonds. The terms “circRNA” or “circular polyribonucleotide” or “circular RNA” are used interchangeably herein. In some embodiments, circRNAs are covalently closed, single stranded RNA molecules. A circular RNA can be produced by back-splicing of a linear precursor RNA, by chemical ligation and/or enzymatic ligation. Circular RNAs (circRNAs) can be endogenous or synthetic. Synthetically created and exogenously delivered circRNAs can be synthesized in vitro using self-splicing permuted introns (e.g., self-splicing Group I or Group II intron) from in vitro transcribed constructs. Unlike linear RNAs, circular RNAs are more resistant to the degradation by exonuclease and have a longer half-life than their corresponding linear counterparts. A circular RNA can be a circular RNA that encodes a polypeptide of interest (e.g., an immunogen or a therapeutic polypeptide, including but not limited to a chimeric antigen receptor (CAR) or a T cell receptor (TCR)). In some embodiments, circular RNAs generated using the introns disclosed herein have significantly improved drug-like properties compared to linear mRNA therapeutics, including enhanced longevity as protein production vectors.

[099] Circularization efficiency. As used herein, the term “circularization efficiency” in the context of RNA circularization refers to a measurement of the resultant circular RNA generated as a proportion of its non-circular starting material (e.g., a linear precursor RNA). A higher circularization efficiency indicates that a greater proportion of the initial noncircular starting material was successfully circularized into circular RNA.

[0100] Corresponding to: As used herein, the term “corresponding to” refers to a nucleic acid sequence or an amino acid sequence at particular positions of an intron, or the corresponding positions in another intron. A sequence corresponding to the sequence at particular positions of an intron may comprise a corresponding substitution or a variant, e.g., the substituted nucleotides or amino acids do not naturally occur at the corresponding positions. The substituted nucleotides or amino acids may be the corresponding residues in another intron (e.g., Group I or II intron).

[0101] Delivery. As used herein, “delivery” refers to the act or manner of delivering a circular RNA, a construct, a linear RNA precursor, a cell comprising a circular RNA, a construct or a linear RNA precursor, or a composition comprising a circular RNA, a construct or a linear RNA precursor, a protein, cargo and/or payload.

[0102] Encapsulate'. As used herein, the term “encapsulate” means to enclose, surround, or encase. As it relates to the formulation of the compositions of the disclosure, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.9% or greater than 99.999% of the pharmaceutical composition of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulated” means that less than 50%, 40%, 30%, 20%, 10%, or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or greater than 99.99% of the pharmaceutical composition of the present disclosure are encapsulated in the delivery vehicle (e.g., a lipid nanoparticle (LNP)).

[0103] Encode'. As used herein, the term “encode” or “encoding” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.

[0104] Enhance'. As used herein, the terms “enhance” and “enhancement” refers to an increase of at least about 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more of a reference; the reference may be a biological function of a nucleic acid or protein and a gene expression level, etc.

[0105] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA sequence from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0106] Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element. Features of the polypeptides encoded by the present circular polynucleotide, such as surface manifestations, local conformational shape, folds, loops, halfloops, domains, half-domains, sites, termini or any combination thereof.

[0107] Formulation'. As used herein, a “formulation” includes at least one compound, substance, entity, moiety, cargo or payload, and a delivery agent.

[0108] Fragment'. A “fragment,” as used herein, refers to a portion. For example, an intron fragment may comprise a portion of the full intron sequence. Fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells. [0109] Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

[0110] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). A sequence can be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to a reference sequence. In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[OHl] Intron: Introns are non-coding sequences of DNA or RNA. Following transcription, new, immature strands of messenger RNA, called pre-mRNA, may contain, amongst other sequences, both introns and exons (exons are any sequence of DNA or RNA that encode for proteins). The pre-mRNA molecule goes through a modification process called splicing during which the noncoding introns are cut out, and only the coding exons remain. Splicing produces a mature messenger RNA molecule that is then translated into a protein. In the context of the present invention, the Group I or Group II introns are selfsplicing introns. As used herein, the term “ self-splicing intron" refers to introns that can act as ribozymes to autocatalytically splice them out from the parent RNA in the absence of any added protein or RNA. An autocatalytic or self-splicing intron can be a Group I intron or a Group II intron.

[0112] Ionizable Lipid. As used herein, “ionizable lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH.

[0113] Lipid Nanoparticle '. As used herein, “lipid nanoparticle” or “LNP” refers to a delivery vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, PEG- modified lipids).

[0114] Liposome '. As used herein, “liposome” generally refers to a vesicle composed of lipids (e.g., amphiphilic lipids) arranged in one or more spherical bilayers or bilayers.

[0115] Modified. As used herein, “modified” or, as appropriate “modification” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and/or functionally. With respect to nucleic acid molecules (e.g., DNA and RNA), the A, G, C, T (if DNA), or U (if RNA) nucleotides are modified. With respect to polypeptides, the term “modification” refers to a modification as compared to the canonical set of 20 amino acids.

[0116] mRNA: As used herein, the term “messenger RNA” (mRNA) means a polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0117] Non-Cationic Lipid. As used herein, “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid.

[0118] Pharmaceutical Composition'. As used herein, the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

[0119] PEG'. As used herein “PEG” means any polyethylene glycol or other polyalkylene ether polymer.

[0120] Spacer'. As used herein, the term “spacer” refers to any contiguous nucleotide sequence (e.g., of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. The spacer can be a 5’ spacer or 3’ spacer. In some embodiments, the nucleic acid for making a circular RNA of the present invention comprises a 5’ spacer that is located between the upstream intron fragment and a sequence of interest (i.e., to be circularized). In some embodiments, the 5’ spacer can be inserted between the upstream intron fragment and the IRES. In some embodiments, the nucleic acid for making a circular RNA of the present invention comprises a 3’ spacer that is located between a sequence of interest (to be circularized) and the downstream intron fragment. The 5’ and 3’ spacer sequences may be 10 nucleotides to 100 nucleotides in length, or 20 nucleotides to 50 nucleotides in length. In some embodiments, the 5’ spacer is at least 10 nucleotides in length. In some embodiments, the 5’ spacer sequence is at least 15 nucleotides in length. In some embodiments, the 5’ spacer is at least 20 nucleotides in length. In some embodiments, the 5’ spacer sequence is at least 30 nucleotides in length. In some embodiments, a 3’ spacer is located between a sequence to be circularized and the downstream intron sequence.

[0121] Sterol'. As used herein, “sterol” is a subgroup of steroids consisting of steroid alcohols.

[0122] Structural Lipid. As used herein, “structural lipid” refers to sterols and lipids containing sterol moieties.

[0123] Transcription'. As used herein, the term “transcription” refers to the formation or synthesis of an RNA molecule by an RNA polymerase using a DNA molecule as a template. [0124] Translation'. As used herein, the term “translation” refers to the formation of a polypeptide molecule by a ribosome based upon an RNA template.

[0125] Treat'. As used herein, the term “treat,” or “treating,” refers to a prophylactic or therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, an autoimmune disorder, a genetic disease) in a subject, including a human subject. The effect of treatment can include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e., not worsening) the state of the disease or disorder, or preventing the spread of the disease or disorder as compared to the state or the condition of the disease or disorder in the absence of the therapeutic treatment. [0126] Unmodified. As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.

[0127] Vector: As used herein, a “vector” is any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise viral parent or reference sequences. Such parent or reference viral sequences may serve as an original, second, third, or subsequent sequence for engineering vectors. In non-limiting examples, such parent or reference viral sequences may comprise any one or more of the following sequences: a polynucleotide sequence encoding a polypeptide or multi-polypeptide, which sequence may be wild-type or modified from wild-type and which sequence may encode full-length or partial sequence of a protein, protein domain, or one or more subunits of a protein; a polynucleotide comprising a modulatory or regulatory nucleic acid which sequence may be wild-type or modified from wild-type; and a transgene that may or may not be modified from wild-type sequence.

DETAILED DESCRIPTION

Nucleic Acid Molecules and Circular RNAs

[0128] The present invention provides nucleic acid molecules and methods for synthesizing circular RNAs. The nucleic acid molecules use new self-splicing intron sequences that can efficiently circularize a nucleic acid sequence. In some embodiments, the nucleic acid molecules are optimized to increase circularization efficiency. Accordingly, the present invention also provides circular RNAs synthesized using the nucleic acid molecules and methods disclosed herein and methods of use of circular RNAs.

[0129] The circular RNAs are distinguished from linear polynucleotides (e.g., mRNA) in their functional and/or structural design features which serve to, as evidenced herein, overcome existing problems of effective polypeptide production using nucleic acid-based methodologies.

[0130] In some aspects, provided herein is a nucleic acid molecule for making a circular RNA, comprising, from 5’ to 3’ end, (i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, (ii) a nucleic acid sequence of interest, and (iii) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same Group I or Group II intron as the upstream intron sequence, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are each derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0131] In some embodiments of the nucleic acid molecule, the 3’ intron splicing fragment and the 5’ intron splicing fragment retain a catalytic core of the Group I or Group II intron.

[0132] In some embodiments of the nucleic acid molecule, the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from variants or truncations of any one of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0133] In some embodiments, the circular RNAs described herein comprise additional features to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, half-life, translation efficiency, immune evasion, and other functions and/or activities.

[0134] In one aspect, the present invention provides a nucleic acid molecule for making a circular RNA. In some embodiments, the nucleic acid molecule is a DNA construct (e.g., a vector) that is transcribed and circularizes into a circular RNA. In some embodiments, the nucleic acid molecule is a linear precursor RNA polynucleotide (e.g., a linear mRNA) that circularizes into a circular RNA. The nucleic acid molecule described herein comprises different elements essential for circular RNA synthesis and function. In some embodiments, the present invention provides a circular RNA that is made from a nucleic acid molecule or by a method described herein. [0135] In some embodiments, the nucleic acid molecule for making a circular RNA comprises self-splicing intron sequences and a sequence of interest (i.e., to be circularized). In some embodiments, the sequence of interest is a non-coding sequence.

[0136] In some embodiments, the sequence of interest is a coding sequence.

[0137] In some embodiments, the sequence of interest encodes a protein.

[0138] In some embodiments, the protein is a chimeric antigen receptor (CAR), a T cell receptor, a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody. In some embodiments, the protein is a chimeric antigen receptor (CAR). In some embodiments, the protein is a T cell receptor. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an enzyme replacement protein. In some embodiments, the protein is an antigen. In some embodiments, the protein is an antibody

[0139] In some embodiments, the protein is an antigen derived from a pathogen, or specific to a tumor.

[0140] In some embodiments, the sequence of interest encodes a CAR. In some embodiments, the CAR comprises an antigen binding domain, a hinge, a transmembrane domain, and one or more intracellular signaling domains.

[0141] In some embodiments, wherein self-splicing of the upstream and downstream Group I or Group II intron sequences makes a circular RNA. A rational design of a synthetic circular RNA polynucleotide cassette includes at least two self-splicing intron sequences flanking the sequence to be circularized. The upstream and downstream intron sequences are self-spliced to generate a circular RNA comprising the RNA sequence of interest. In some embodiments, the nucleic acid molecule comprises one or more additional sequences that facilitate circularization and functions of circular RNA. For example, a 5’ spacer and/or 3’ spacer (e.g., 3’ untranslated region (UTR)) sequences are included between the intron sequences and the sequence of interest (i.e., to be circularized). In some embodiments, one or more homology elements are included. In some embodiments, a 5’ inner homology element and a 3’ homology element are included between the intron sequences and the sequence of interest (i.e., to be circularized). In some embodiments, at least one internal ribosome entry site (IRES) is added and linked to the sequence of interest when the sequence is a coding sequence.

[0142] In some embodiments, provided herein is a circular RNA that is made from the nucleic acid molecule of the present disclosure.

[0143] In some embodiments, provided herein is a composition comprising the circular RNA of the present disclosure. [0144] In some embodiments, the nucleic acid molecule for making a circular RNA comprises, from 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ intron splicing fragment of a Group I or Group II intron, a sequence of interest (e.g., one or more exons), and a downstream intron sequence corresponding to a 5’ intron splicing fragment of a Group I or Group II intron, wherein the upstream and downstream intron sequences are from the same Group I or Group II intron.

[0145] In some embodiments, the nucleic acid molecule that encodes a circular RNA comprises, from 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ intron splicing fragment of a Group I or Group II intron, a 5’ spacer, a sequence of interest (e.g., one or more exons), a 3’ UTR, and a downstream intron sequence corresponding to a 5’ intron splicing fragment of a Group I or Group II intron, wherein the upstream and downstream intron sequences are from the same Group I or Group II intron.

[0146] In some embodiments, the nucleic acid molecule described herein does not include 5’ or 3’ homology arm. In some embodiments, the nucleic acid molecule does not include outer homology elements.

[0147] In some embodiments, the nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence of interest and/or a 3’ untranslated region (UTR) sequence between the sequence of interest and the downstream intron sequence.

[0148] In some embodiments, the 5’ spacer sequence comprises an inner homology element.

[0149] In some embodiments, the 5’ spacer sequence does not comprise an inner homology element.

[0150] In some embodiments, the nucleic acid molecule does not include the 5’ spacer sequence.

[0151] In some embodiments, the nucleic acid molecule further comprises an internal ribosome entry site (IRES) between the 5’ spacer sequence and the sequence of interest. [0152] In some embodiments, the nucleic acid molecule comprises a 3’ UTR between the sequence of interest and the downstream intron sequence.

[0153] In some embodiments, the nucleic acid molecule for making a circular RNA comprises, from 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ intron splicing fragment of a Group I or Group II intron, a 5’ spacer, a IRES, a sequence of interest (e.g. , one or more exons), a 3 ’ UTR, and a downstream intron sequence corresponding to a 5’ intron splicing fragment of a Group I or Group II intron, wherein the upstream and downstream intron sequences are from the same Group I or Group II intron.

[0154] In some embodiments, the nucleic acid molecule for making a circular RNA comprises, from 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ intron splicing fragment of a Group I or Group II intron, a 5’ spacer comprising a 5’ inner homology element, a IRES, a sequence of interest (e.g., one or more exons), a 3’ UTR, a 3’ inner homology element and a downstream intron sequence corresponding to a 5’ intron splicing fragment of a Group I or Group II intron, wherein the upstream and downstream intron sequences are from the same Group I or Group II intron. The 5’ and 3’ inner homology elements (UTEs) form base paring (also referred to as “internal homology regions”).

[0155] In some embodiments, the nucleic acid molecule that encodes a circular RNA comprises, from 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ intron splicing fragment of a Group I or Group II intron, an IRES, a sequence of interest (e.g., one or more exons), a 3’ UTR, a downstream intron sequence corresponding to a 5’ intron splicing fragment of a Group I or Group II intron, wherein the upstream and downstream intron sequences are from the same Group I or Group II intron. In some embodiments, the nucleic acid does not include 5’ or 3’ homology arm (also referred to as “outer homology element (OHE)”).

Self-splicing Introns

[0156] In some embodiments, the nucleic acid molecule described herein comprises permuted self-splicing intron sequences. The permutation generates an upstream (z.e., 5’ end) intron sequence corresponding to a 3’ intron splicing fragment of a self-splicing Group I or Group II intron and a downstream (z.e., 3’ end) intron sequence corresponding to a 5’ intron splicing fragment of the self-splicing intron. The intron sequences flank the RNA sequence of interest to be circularized.

[0157] It has been known that Group I introns have autocatalytic activity (acting as ribozymes), z.e., are self-splicing. Group I introns splice themselves out without assistance from the spliceosome or other proteins, results in joining of the flanking exons and circularization of the intervening intron to produce an intronic circRNA. Group I introns can be found naturally within the rRNA, tRNA, and mRNA genes of bacteria and non-metazoan eukaryotes. A general discussion of the catalytic activity of Group I introns can be found in the review article by Hausner et al., (Mobile DNA, vol 5(8) (2014)). [0158] Group II introns constitute another ribozyme self-splicing system with a mechanism similar to that of both Group I introns and pre-mRNA introns. Group II introns are naturally found in the bacteria and organelle genomes of lower eukaryotes. A general discussion of the catalytic activity of Group II introns can be found in the review article by Pyle (Annu Rev Biophys. 5(45): 183-205) (2016)).

[0159] Though the self-splicing function of Group I and Group II introns has been well- known, only a few Group I and Group II introns have been tested in permuted intron-exon (PIE) methods for synthesizing a circular RNA.

[0160] The inventors of the present application selected a number of new Group I intron sequences and developed permutation strategies based on structural analysis of the candidate Group I introns that allowed for efficient circularization. The results showed that three Group I introns: Twort-ORF142 intron, Anabaena-spiroides-trnL intron, and Oscillator ia-splendida- trnL intron, can efficiently circularize a linear RNA sequence. The inventors also identified particular permutation sites (z.e., the positions at which the intron sequence is split or cut) for circularization. In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence and a downstream intron sequence generated from the cut site or splitting position (the terms “cut site”, “permutation site”, and “splitting position” are used interchangeably herein), wherein the cut site retains the structural integrity of the intron. As used herein, the term “catalytic core” refers to an intron region (e.g., the internal stem structure) needed for the intron to self-splice, which is well-understood in the art.

Typically, the catalytic core of an intron, for example, a Group I intron, is a highly conserved small region of about 70 nucleotides composed of paired regions (e.g., P1-P6) that form elongated domains (e.g., helical domains). More detailed description of the catalytic core of an intron can be found in, e.g., Michel and Westhof, J. Mol. Bio., 1990, 216: 585-610; Luptak and Doudna, Nucleic Acids Research, 2004, 32(7): 2272-80, the contents of which are incorporated by reference herein. In some embodiments, the cut site is designed to retain the catalytic core of the intron, which is a region of nucleotides containing sequences and structures needed for the intron to self-splice. In some embodiments, a suitable cut site is chosen outside the catalytic core of the intron such that the structural integrity of the intron is retained, and the resulting upper intron fragments and lower intron fragments allow for RNA circularization.

[0161] Accordingly, in some embodiments, the present disclosure provides a nucleic acid molecule for making circular RNA comprising an upstream intron sequence and a downstream intron sequence corresponding to a 3’ splicing intron fragment and a 5’ splicing intron fragment derived from a Group I intron or from a Group II intron, respectively.

Exemplary Group I Intron Sequences

[0162] In some embodiments, the nucleic acid molecule described herein comprises permuted intron sequences corresponding to a Twort-ORF142 intron, an Anabaena-spiroides- trnL intron, or an Oscillator ia-splendida-trnL intron.

[0163] Exemplary Group I introns include but are not limited to the Twort-ORF142 intron comprising SEQ ID NO: 7, the Anabaena-spiroides-trnL intron comprising SEQ ID NO: 8, and the Oscillator ia-splendida-trnL intron comprising SEQ ID NO: 9.

[0164] In some embodiments, the upstream and downstream intron sequences within the nucleic acid molecule for making a circular RNA correspond to a 3’ splicing fragment and a 5’ splicing fragment of the Twort-ORF142 intron, respectively.

[0165] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’ and 5’ intron splicing fragments of SEQ ID NO: 7.

[0166] The Twort-ORF142 intron sequence includes nucleotides 1055-1336 of the open reading frame Twort-ORF142 (unknown gene) in the genome of Staphylococcus phage Twort (GenBank: AF132670.1) (Landthaler and Shub, PNAS, 1999, 96 (12), 7005-7010): AACTACTGAAAGCATAAATAATTGTGCCTTTATACAGTAATGTATATCGAAAAAT CCTCTAATTCAGGGAACACCTAAACAAACTAAGATGTAGGCAATCCTGAGCTAA GCTCTTAGTAATAAGAGAAAGTGCAACGACTATTCCGATAGGAAGTAGGGTCAA GTGACTCGAAATGGGGATTACCCTTCTAGGGTAGTGATATAGTCTGAACATATAT GGAAACATATAGAAGGATAGGAGTAACGAACCTATTCGTAACATAATTGAACTT TTAGTTATTT (SEQ ID NO: 7).

[0167] In some embodiments, the nucleic acid molecule for making a circular RNA comprises the upstream intron sequence comprising a sequence corresponding to positions 119 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 118 of SEQ ID NO: 7. In one non-limiting example, the upstream intron sequence corresponds to positions 119 to 282 of SEQ ID NO; 7, and the downstream intron sequence corresponds to positions 1 to 118 of SEQ ID NO: 7.

[0168] In some embodiments, the nucleic acid molecule for making a circular RNA comprises the upstream intron sequence of SEQ ID NO: 1 and the downstream intron sequence of SEQ ID NO: 2. [0169] In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and a downstream intron sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2.

[0170] In some embodiments, the upstream intron sequence comprises the sequence of SEQ ID NO: 1, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 2.

[0171] In some embodiments, the upstream intron fragment comprises a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and the downstream intron fragment comprises a sequence corresponding to positions 1 to 146 of SEQ ID NO: 7. In one nonlimiting example, the upstream intron sequence corresponds to positions 147 to 282 of SEQ ID NO: 7, and the downstream intron sequence corresponds to positions 1 to 146 of SEQ ID NO: 7.

[0172] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 140 to 282 of SEQ ID NO. 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7. In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1 to 146 of SEQ ID NO: 7.

[0173] In some embodiments, the upstream intron fragment comprises the sequence of SEQ ID NO: 12 and the downstream intron fragment comprises the sequence of SEQ ID NO: 13.

[0174] In some embodiments, the upstream intron sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. In some embodiments, the downstream intron sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

[0175] In some embodiments, the nucleic acid molecule for making a circular RNA comprises the upstream intron sequence comprising a sequence corresponding to positions 140 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7. In one non-limiting example, the upstream intron sequence corresponds to positions 140 to 282 of SEQ ID NO: 7, and the downstream intron sequence corresponds to positions 1 to 139 of SEQ ID NO: 7. [0176] In some embodiments, the upstream intron fragment comprises the sequence of SEQ ID NO: 10 and the downstream intron fragment comprises the sequence of SEQ ID NO: 11.

[0177] In some embodiments, the upstream intron sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In some embodiments, the downstream intron sequence is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11.

[0178] In some embodiments, the upstream and downstream intron sequences within the nucleic acid molecule for making a circular RNA correspond to a 3’ splicing fragment and a 5’ splicing fragments of the Oscillator ia-splendida-trnL intron, respectively.

[0179] In some embodiments, the upstream and downstream intron sequences are derived from the Oscillator ia-splendida-trnL intron of SEQ ID NO. 8.

CGGACTTAGAAAACTGAGCCTTATTGGAGAAATCCATTAAGTGACCGCTCTCAA ATTCAGGGAAACCTAACTCTGGTAACAGACAAGGCAATCCTGAGCCAAGCCGAA ATTTCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGTAAAGCCGAGGGT AAAGGGAGAGTCCAATTCTCAAAACCAGAATTCTGGCAGCAGCGAAAGTTGCGG GAGAATGAAAATCCG (SEQ ID NO. 8)

[0180] In some embodiments, the nucleic acid molecule for making a circular RNA comprises the upstream intron sequence comprising a sequence corresponding to positions 112-231 of the Oscillator ia-splendida-trnL intron of SEQ ID NO. 8, and the downstream intron sequence comprising a sequence corresponding to positions 1-111 of the Oscillatoria- splendida-trnL intron of SEQ ID NO. 8. In one non-limiting example, the upstream sequence corresponds to positions 112-231 of the Oscillator ia-splendida-trnL intron of SEQ ID NO. 8; and the downstream intron sequence corresponds to positions 1 to 111 of the Oscillatoria- splendida-trnL intron of SEQ ID NO: 8.

[0181] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’ and 5’ splicing fragments of SEQ ID NO: 8.

[0182] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 112-231 of SEQ ID NO: 8, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-111 of SEQ ID NO: 8.

[0183] In some embodiments, the nucleic acid molecule for making a circular RNA comprises the upstream intron sequence of SEQ ID NO: 3 and the downstream intron sequence of SEQ ID NO: 4. [0184] In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, and a downstream intron sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

[0185] In some embodiments, the upstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4.

[0186] In some embodiments, the upstream intron sequence comprises the sequence of SEQ ID NO: 3, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 4.

[0187] In some embodiments, the upstream and downstream intron sequences within the nucleic acid molecule for making a circular RNA correspond to a 3’ splicing fragment and a 5’ splicing fragments of the Anabaena-spiroides-trnL intron, respectively.

[0188] In some embodiments, the upstream and downstream intron sequences are derived from the Anabaena-spiroides-trnL intron of SEQ ID NO: 9.

CGGACTTAAATAATTGAGCCTTAGAGAAGAAATTCTTTAAGTGGATGCTCTCAAA CTCAGGGAAACCTAAATCTAGCTATAGACAAGGCAATCCTGAGCCAAGCCGAAG TAGTAATTAGTAAGTTAACAACAGATAACTTACAGCTAATCGGAAGGTGCAGAG ACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTC TCAAAGCCAATAGGCAGTAGCGAAAGCTGCGGGAGAATGAAAATCCG (SEQ ID NO: 9)

[0189] In some embodiments, the nucleic acid molecule described herein comprises the upstream sequence comprising a sequence corresponding to positions 127-264 of the Anabaena-spiroides-trnL intron of SEQ ID NO: 9, and the downstream intron sequence comprising a sequence corresponding to positions 1-126 of the Anabaena-spiroides-imL intron of SEQ ID NO: 9. In one non-limiting example, the upstream intron sequence corresponds to positions 127 to 264 of the Anabaena-spiroides-trnL intron of SEQ ID NO: 9 and the downstream sequence corresponds to positions 1- 126 of the Anabaena-spiroides- trnL intron of SEQ ID NO: 9.

[0190] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’ and 5’ splicing fragments of SEQ ID NO: 9.

[0191] In some embodiments, the upstream intron sequence comprises a sequence corresponding to positions 127-264 of SEQ ID NO: 9, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-126 of SEQ ID NO: 9. [0192] In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5, and a downstream intron sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. [0193] In some embodiments, the nucleic acid molecule for making a circular RNA comprises the upstream intron sequence of SEQ ID NO: 5 and the downstream intron sequence of SEQ ID NO: 6.

[0194] In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence and a downstream intron sequence generated from a cut site or splitting position (“cut site” and “splitting position” are used interchangeably herein), wherein the cut site retains the structural integrity of the intron. As used herein, the term “catalytic core” refers to an intron region (e.g., the internal stem structure) needed for the intron to self-splice, which is well-understood in the art. Typically, the catalytic core of an intron, for example, a Group I intron, is a highly conserved small region of about 70 nucleotides composed of paired regions (e.g., P1-P6) that form elongated domains (e.g., helical domains). More detailed description can be found in, e.g., Michel and Westhof, J. Mol. Bio., 1990, 216: 585-610; Luptak and Doudna, Nucleic Acids Research, 2004, 32(7): 2272-80, the contents of which are incorporated by reference herein. In some embodiments, the cut site is designed to retain the catalytic core of the intron, which is a region of nucleotides containing sequences and structures needed for the intron to self-splice. In some embodiments, a suitable cut site is chosen outside the catalytic core of the intron such that the structural integrity of the intron is retained, and the resulting upper intron fragments and lower intron fragments allow for RNA circularization.

[0195] In some embodiments, the nucleic acid molecule for making a circular RNA comprises an upstream intron sequence and a downstream intron sequence generated from a cut site or splitting position, wherein the cut site is between any two residues within a region defined by Pl, P2, P3, P4, P5, P5a, P6, P6a, P7, P7.1, P7.2, P8, P9, P9.0, P9.1, P10 or P12 as shown in FIG. 3. In some embodiments, the VI cut site is between any two residues in a P7 region. In some embodiments, the V2 cut site is between any two residues in a P6a region. In some embodiments, the V3 cut site is between any two residues in a P7.1 region.

[0196] In some embodiments, the VI, V2 and/or V3 cut sites are between any two residues within a loop. In some embodiments, the VI, V2 and/or V3 cut sites are not within an internal stem structure of the intron. In some embodiments, a VI site is between adenine and uracil (A/U), as shown in FIG. 3. [0197] In some embodiments, a V2 site is between any two residues in a loop comprising AGUAAU as shown in FIG. 3. In some embodiments, a V2 cute site is between adenine and guanine (A/G) within the loop of the intron. In some embodiments, a V2 site is between guanine and uracil (G/U) within the loop of the intron. In some embodiments, a V2 cut site between uracil and adenine (U/A) within the loop of the intron. In some embodiments, a V2 sit is between two adenines within the loop of the intron. In some embodiments, a V2 site is between adenine at position 5 of the loop and uracil at position 6 of the loop. In some embodiments, a V2 cut site between uracil at position 3 of the loop and adenine at position 4 (U/A) of the loop. In some embodiments, a V2 cut site is at an equivalent position in a loop comprising a complementary loop sequence to one depicted in FIG. 3. In some embodiments, a V2 site is at an equivalent position in a loop comprising one or more variations in the loop sequence depicted in FIG. 3. In some embodiments, a V2 cut site is at an equivalent position in a loop comprising 1, 2, 3, 4, 5 or 6 variations in the loop sequence depicted in FIG. 3. In some embodiments, a V2 site is at a position between a stem residue and the first or last residue of the loop as shown in FIG. 3.

[0198] In some embodiments, a V3 site is between any two residues in a loop comprising GAUA. In some embodiments, a V3 site is between guanine and adenine (G/A) within a loop of the intron. In some embodiments, a V3 cut site is between adenine and uracil (A/U) within a loop of the intron. In some embodiments, a V3 site is between uracil and adenine (U/A) within a loop of the intron. In some embodiments, a V3 site is at an equivalent position in a loop comprising a complementary loop sequence to one depicted in FIG. 3. In some embodiments, a V3 cut site is at an equivalent position in a loop comprising one or more variations in the loop sequence depicted in FIG. 3. In some embodiments, a V3 cut site is at an equivalent position in a loop comprising 1, 2, 3 or 4 variations in the loop sequence depicted in FIG. 3. In some embodiments, a V3 cut site is at a position between a stem residue and the first or last residue of the loop as shown in FIG. 3.

[0199] In some embodiments, the nucleic acid molecules comprise intron sequence fragments listed in Table 1.

Table 1: Exemplary intron sequence fragments

5 ’ Spacer

[0200] In some embodiments, the nucleic acid molecule for making a circular RNA may comprise a 5’ spacer between the upstream intron fragment and the sequence of interest (i.e., to be circularized), or the IRES.

[0201] The 5 ’spacer sequence comprises a random sequence that increases circularization efficiency.

[0202] The 5’ spacer sequence may be of any length (e.g., 10 to 100 nucleotides, 10 to 90 nucleotides, 10 to 80 nucleotides, 10 to 70 nucleotides, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 100 nucleotides, 20 to 90 nucleotides, 20 to 80 nucleotides, 20 to 70 nucleotides, 20 to 60 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 30 nucleotides, 30 to 100 nucleotides, 30 to 90 nucleotides, 30 to 80 nucleotides, 30 to 70 nucleotides, 30 to 60 nucleotides, 30 to 50 nucleotides, 30 to 40 nucleotides, 40 to 100 nucleotides, 40 to 90 nucleotides, 40 to 80 nucleotides, 40 to 70 nucleotides, 40 to 60 nucleotides, 40 to 50 nucleotides, 50 to 100 nucleotides, 50 to 90 nucleotides, 50 to 80 nucleotides, 50 to 70 nucleotides, 50 to 60 nucleotides, 60 to 100 nucleotides, 60 to 90 nucleotides, 60 to 80 nucleotides, 60 to 70 nucleotides, or 50 nucleotides). For example, in some embodiments, the length of the 5’ spacer is selected to optimize translation of the protein-coding nucleic acid sequence.

[0203] In some embodiments, the 5’ spacer sequence is between 20 and 50 nucleotides in length. In some embodiments, the 5’ spacer sequence is between 30 and 100 nucleotides in length. In certain embodiments, the 5’ spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length. [0204] In some embodiments, the 5’ spacer sequence includes a 5’ inner homology element. As used herein, the terms “internal homology region” and “inner homology element (IHE)” are used interchangeably. In some embodiments, the internal homology element is about 5-50 nucleotides in length. In some embodiments, the internal homology elements about 5-30 nucleotides in length. In some embodiments, the internal homology region is about 10-25 nucleotides in length. In some embodiments, the internal homology element is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the 5’ internal homology element is located at the 5’ end of the 5’ spacer sequence. The internal homology element forms base-pairing with an internal homology region, e.g., at the 3’ end (3’ inner homology element). In some embodiments, the nucleic acid described herein comprising 5’ and 3’ inner homology elements that are 75%, 80%, 85%, 90%, 95%, or 100% complementary to each other.

[0205] In some embodiments, the 5’ spacer sequence comprises a polyA sequence. The polyA sequence may comprise 15-30 As. In some embodiments, the polyA sequence comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 As. In other embodiments, the 5’ spacer sequence comprises a polyA-C sequence.

Internal Ribosome Entry Site (IRES)

[0206] In some embodiments, the nucleic acid molecule described herein comprises an internal ribosome entry site (IRES) sequence. As used herein, the term “internal ribosome entry site” or “IRES” refers to an RNA sequence or structural element ranging in size from 10 nucleotides to 1,000 nucleotides or more which is capable of initiating translation of a polypeptide in the absence of a normal RNA cap structure. An IRES element may engage a eukaryotic ribosome for translation, or initiate cap-independent translation and protein synthesis.

[0207] In some embodiments, the IRES has a sequence of an IRES, or is a functional fragment or variant thereof.

[0208] The IRES sequence may be derived from a viral genome or is a cellular IRES. In one embodiment, the nucleic acid molecule described herein comprises a viral IRES.

[0209] In some embodiments, the IRES may include but is not limited to, the encephalomyocarditis virus (EMCV) IRES, polio virus IRES, Kaposi sarcoma-associated herpesvirus (KSHV) vFLIP IRES, or hepatitis C virus (HCV) IRES. In some embodiments, the IRES has a sequence of an IRES from Taura syndrome virus, Triatoma virus, Theiler’s encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPAI, Human AMLI/RUNXI, Drosophila antennapedia, Human AQP4, Human ATIR, Human BAG-I, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEFI, Mouse HIFI alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2/c-sis, Human p53, Human Pim-I, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae Y API, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus EID, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT00I, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SHI, Salivirus FHB, Salivirus NG- JI, Human Parechovirus 1, Crohivirus B, Y c-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus El 4, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVAI0, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GTI 10, GBV-C KI 737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picoma-like Virus, CRPV, Apodemus, Apodemus Agrarius Picomavirus, Caprine Kobuvirus, Canine Kobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, or EV24.

[0210] In some embodiments, the IRES that can be included in a circular RNA is a Type 1 IRES.

[0211] In some embodiments, the IRES that can be included in a circular RNA is a viral IRES sequence.

[0212] In some embodiments, the IRES is an enterovirus IRES. In some embodiments, the IRES is a human rhinovirus (HRV) IRES.

[0213] In some embodiments, the IRES that can be included in a circular RNA is a non- viral IRES sequence, including but not limited to IRES sequences from yeast, the human angiotensin II type 1 receptor IRES, fibroblast growth factor IRESs (e.g., FGF-1 IRES and FGF-2 IRES), vascular endothelial growth factor IRES, and insulin-like growth factor 2 IRES.

[0214] In some embodiments, the IRES that can be included in a circular RNA is a synthetic IRES sequence. A “synthetic IRES” is an IRES that is modified relative to a wildtype IRES in order to modulate its structure and/or activity. For example, in some embodiments, an IRES that is modified to incorporate an aptamer sequence is a synthetic IRES.

[0215] In some embodiments, the IRES sequence in the circular RNA comprises at least one RNA secondary structure element or feature.

[0216] The IRES may be of any length or size. For example, the IRES may be about 100 nucleotides to about 1,000 nucleotides in length (e.g., about 150, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 750, about 800, about 850, or about 900 nucleotides in length, or a range defined by any two of the foregoing values). In some embodiments, the IRES may be about 200 nucleotides to about 800 nucleotides in length (e.g., about 150, about 200, about 210, about 220, about 240, about 260, about 280, about 320, about 340, about 360, about 380, about 420, about 440, about 460, about 480, about 500, about 520, about 540, about 560, about 580, about 600, about 620, about 640, about 660, about 680, about 700, about 720, about 740, about 760, about 780, or about 800 nucleotides in length, or a range defined by any two of the foregoing values). In some embodiments, the IRES may be about 200 to about 400, about 400 to about 600, about 600 to about 700, or about 600 to about 800 nucleotides in length. In some embodiments, the IRES is about 210 nucleotides in length. In some embodiments, the IRES may be about 100 to about 3,000 nucleotides in length.

[0217] In some embodiments, the IRES sequence may be operably linked to a proteincoding sequence. Different IRES elements (and exonic elements in general) affect the strength of protein expression as well as the cell/tissue specificity. Selection of an IRES element depends on the purpose of protein expression.

[0218] In some embodiments, the IRES is “in-frame” with respect to the protein-coding nucleic acid sequence, that is, the IRES is positioned in the circular RNA molecule in the correct reading frame for the encoded protein. In other embodiments, the IRES may be “out of frame” with respect to the protein-coding nucleic acid sequence, such that the position of the IRES disrupts the open reading frame (ORF) of the protein-coding nucleic acid sequence. In other embodiments, the IRES may overlap with one or more ORFs of the protein-coding nucleic acid sequence.

3 ’ UTR

[0219] In some embodiments, the nucleic acid molecule provide herein comprises 3’ UTR. In some embodiments, the 3’ UTR is also a 3’ spacer sequence. There is a growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the nucleic acid molecules and circular RNAs made from the nucleic acid constructs. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organ sites.

[0220] The 3’ UTR may be derived from human beta-globin, human alpha-globin, xenopus beta-globin, xenopus alpha-globin.

[0221] In some embodiments, the circRNA includes a region to initiate translation. This region may include any translation initiation sequence or signal including a Start codon. As a non-limiting example, the region includes a Start codon. In some embodiments, the Start codon may be “ATG,” “ACG,” “AGG,” “ATA,” “ATT,” “CTG,” “GTG,” “TTG,” “AUG,” “AUA,” “AUU,” “CUG,” “GUG,” or “UUG”. [0222] In some embodiments, the circRNA includes a region to stop translation. This region may include any translation termination sequence or signal including a Stop codon. As a non-limiting example, the region includes a Stop codon. In some embodiments, the Stop codon may be “TGA,” “TAA,” “TGA,” “TAG,” “UGA,” “UAA,” “UGA” or “UAG .” [0223] In some embodiments, the regions to initiate or terminate translation may independently range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. Additionally, these regions may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.

[0224] In some embodiments, a masking agent may be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.

[0225] In some embodiments, the start codon may be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon which is not the start codon. Translation of the polynucleotide may begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon. The polynucleotide sequence where the start codon is removed may further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the polynucleotide.

[0226] In some embodiments, the 3’ UTR sequence comprises a 3’ inner homology element at the 3’ end of the 3’ UTR. In other embodiments, a 3’ inner homology element is added downstream of the 3’ UTR. The 3’ inner homology element pairs with the inner homology element in the 5’ spacer. In some embodiments, the 3’ internal homology element is about 5-50 nucleotides in length. In some embodiments, the 3’ inner homology element is 5-30 nucleotides in length. In some embodiments, the 3’ internal homology region is about 10-25 nucleotides in length. In some embodiments, the 3’ inner homology element has the same length as the inner homology element in the 5’ spacer. In some embodiments, the inner homology element is 10, 15, 20, or 25 nucleotides in length. In some embodiments, the 3’ inner homology element is at least 75%, 80%, 85%, 90%, 95%, or 100% complementary to the inner homology element in the 5’ spacer.

[0227] In some embodiments, the nucleic acid molecule does not include inner homology elements.

Homology arms [0228] As discussed herein, the inventors generated four different embodiments of RNA constructs (as shown in FIG. 2A-FIG. 2D). In some embodiments, the inventors of the present disclosure surprisingly found that the nucleic acid molecule that does not include 5’ or 3’ homology arm (HA) had higher circularization efficiency (as shown in FIG. 8B). As used herein, the terms “homology arm” and “outer homology element (OHE)” are used interchangeably. A 5’ homology arm (HA) is also referred to a 5’ outer homology element (OHE). A 3’ homology arm (HA) is also referred to a 3’ outer homology element (OHE). Surprisingly, the results indicated that 5’ and 3’ homology arms (i.e., outer homology elements (OHEs) are not required for RNA circularization.

[0229] Homology arms are complementary sequences (e.g., 10-50 nucleotides) that pair with each other at the ends of a linear nucleic acid (e.g., a linear precursor mRNA). The 5’ homology arm is located at the 5’ end of the upstream intron sequence (i.e., before and adjacent to or within the upstream intron sequence). The 3’ homology arm is located at the 3’ end of the downstream intron sequence (i.e., after and adjacent to or within the downstream intron sequence). Though it has been reported that 5’ and 3’ homology arms are required for RNA circularization, the present disclosure demonstrates that, surprisingly, as exemplified in FIG. 8B, a nucleic acid that does not include 5’ or 3’ homology arm has increased circularization efficiency.

[0230] In some embodiments, the nucleic acid molecule comprises, from the 5’ to 3’ end, an upstream intron sequence corresponding to a 3’ splicing fragment of Twort-ORF142 intron comprising the sequence of SEQ ID NO: 7, a 5’ spacer comprising a 5’ inner homology element, optionally an IRES, a RNA sequence of interest, a 3’UTR, a 3’ inner homology element, and a downstream intron sequence corresponding to the 5’ splicing fragment of Twort-ORF142 intron comprising the sequence of SEQ ID NO: 7, wherein the nucleic acid molecule does not comprise 5’ or 3’ homology arm. As a non-limiting example, the 5’ inner homology element comprises the sequence of accacacaaatggtcgccga, and the 3’ inner homology element comprises its complement.

RNA sequence of interest

[0231] The circular RNAs and nuclei acid molecules for making circular RNAs comprise a sequence of interest, i.e., a nucleic acid sequence element with a biological function, including but not limited to encoding a polypeptide of interest, a CAR, a TCR, a regulatory element of gene expression, a therapeutic nucleic acid molecule, and a guide RNA, etc. In some embodiments, the sequence of interest may comprise two or more biological functions. Accordingly, the circular RNAs may be bifunctional. As the name implies, bifunctional circular RNAs are those having or capable of at least two functions. These molecules may also by convention be referred to as multi-functional (e.g., have more than two functions).

Protein of interest

[0232] In some embodiments, the sequence of interest encodes a polypeptide of interest (i.e., a protein of interest). In some embodiments, the sequence of interest may encode two or more polypeptides. The two or more polypeptides may have the same amino acid sequences; alternatively, the two or more polypeptides may have different amino acid sequences. The polypeptide of interest may be one known in the art and/or described herein. In this context, the circular RNA may act as a messenger RNA (mRNA), having the same encoding function as its liner mRNA counterpart.

[0233] A polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned. As used herein, the term “polypeptides of interest” refer to any polypeptide which is selected to be encoded in the primary construct of the present disclosure. As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0234] The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.

[0235] In general, the coding nucleic acid sequence may be sufficient to encode a polypeptide of at least 10 amino acids in length, e.g., 10 to 5000 amino acids in length, or 10- 1000 amino acids in length, or 50-2000 amino acids in length, or 30-3000 amino acids in length, or 100-1000 amino acids in length, or 100-3000 amino acids in length, or 200-1000 amino acids in length, or 200-500 amino acids in length, or 500-5000 amino acids in length, or 500-4000 amino acids in length, or 500-1500 amino acids in length, or 1000-5000 amino acids in length.

[0236] Generally, the coding nucleic acid sequence element may be greater than 30 nucleotides in length, e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2250, 2,500, and 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides.

[0237] In some embodiments, the sequence of interest may encode a therapeutic protein or polypeptide. The therapeutic protein may be an enzyme, a replacement therapy protein, etc. According to the present disclosure, one or more therapeutic proteins or peptides currently being marketed or in development may be encoded by the circular RNAs of the present disclosure.

[0238] In some embodiments, the therapeutic polypeptide is a recombinant or chimeric polypeptide. As a non-limiting example, the recombinant polypeptide is a chimeric antigen receptor (CAR).

[0239] In some embodiments, the therapeutic polypeptide is a cytokine (e.g., IL2, IL12, IL15, IL21, IL27 etc.)

[0240] In some embodiments, the therapeutic polypeptide is a transcription factor.

[0241] In some embodiments, the sequence of interest may encode an antigen of interest. The antigen may be an antigen that causes infection such as viral antigen and a bacterial antigen. The antigen may also be a cancer antigen such as a neoantigen and an antigen that is specific or associated with a cancer (e.g., a tumor associated antigen (TAA)). [0242] As used herein, a “neoantigen” refers to a class of tumor antigens which arises from tumor-specific mutations in an expressed protein.

[0243] In some embodiments, the encoded polypeptide may be an antibody, a heavy chain of an antibody, a light chain of an antibody, a variable region of a heavy chain of an antibody, a variable region of a light chain of an antibody, a Fab fragment, and the like.

[0244] As used herein, the term “antibody” is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multiple-specific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”). Antibodies are primarily amino acid-based molecules but may also comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.). Non-limiting examples of antibodies or fragments thereof include VH and VL domains, scFvs, Fab, Fab’, F(ab’)2, Fv fragment, diabodies, linear antibodies, single chain antibody molecules, multiple-specific antibodies, bispecific antibodies, intrabodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, codon-optimized antibodies, tandem scFv antibodies, bispecific T-cell engagers, mAb2 antibodies, chimeric antigen receptors (CAR), tetravalent bispecific antibodies, biosynthetic antibodies, native antibodies, miniaturized antibodies, unibodies, maxibodies, antibodies to senescent cells, antibodies to conformers, antibodies to disease specific epitopes, or antibodies to innate defense molecules.

[0245] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. [0246] The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0247] As used herein the term, “antibody fragment” refers to any portion of an intact antibody. In some embodiments, antibody fragments comprise antigen binding regions from intact antibodies. Examples of antibody fragments may include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multiple-specific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Compounds and/or compositions of the present invention may comprise one or more of these fragments. For the purposes herein, an "antibody" may comprise a heavy and light variable domain as well as an Fc region.

[0248] As used herein, the term “antibody variant” refers to a biomolecule resembling an antibody in structure and/or function comprising some differences in their amino acid sequence, composition or structure as compared to a native antibody.

[0249] In some embodiments, the polypeptides of interest encoded may also include but are not limited to, biologies, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. As used herein, a “biologic” is a polypeptide-based molecule produced by the methods provided herein and which may be used to treat, cure, mitigate, prevent, or diagnose a serious or life-threatening disease or medical condition.

[0250] In some embodiments, the polypeptide of interest is a recombinant protein. In some cases, the recombinant protein is a chimeric antigen receptor (CAR). The CAR comprises an antigen binding domain, a hinge, a transmembrane domain and at least one intracellular signaling domain. In some embodiments, the CAR comprises an antigen binding domain, a hinge, a transmembrane domain, at least one co-stimulating signaling domain and an activation signaling domain.

[0251] The antigen binding domain of the CAR recognizes antigen. Numerous antigen binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics and T cell receptors. In some embodiments, the antigen binding domain is ab antigen binding fragment derived from an antibody specific to an antigen of interest. Such as a single-chain variable fragment (scFv) and a single domain antibody VHH. [0252] In some embodiments, the antigen binding domain recognizes a tumor antigen, such as CD 19, CD20, CD22, BCMA and other tumor associated antigens.

[0253] The hinge region (also known as spacer or linker region) is a region with the CAR to connect the antigen binding domain with the transmembrane domain and spatially separate the antigen-binding domain from the intracellular domains within the CAR. A flexible hinge allows the antigen-binding domain to orient in different directions to facilitate binding and effective formation of the CAR-antigen complex. The hinge domain may comprise about 20- 100 amino acids in length. In some embodiments, the hinge domain comprises at least 25, 30, 35, 40, 45, 50, or 60 amino acids. In some embodiments, the hinge domain comprises a sequence derived from an IgG Fc region (e.g., CHI, CH2 and/or CH3 regions), an IgG hinge or CD8 stalk region.

[0254] The transmembrane domain of the CAR may be any protein structure which is thermodynamically stable in a membrane. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the CAR described herein. As non-limiting examples, the CAR comprises a transmembrane domain derived from CD28, CD3^, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137/4-1BB, CD154, ICOS/CD278, GITR/CD357, TNFRSF16, TNFRSF19, or NKG2D.

[0255] The intracellular portion of the CAR comprises at least one co-stimulatory domain and an activation signaling domain. In some embodiments, the intracellular activation signaling domain is derived from CD3 zeta.

[0256] In some embodiments, one or more costimulatory signaling domains are inserted between the transmembrane domain and the CD3zeta intracellular domain. The costimulatory domain may be derived from, but not limited to, CD28, 0X40, CD27, 4-1BB/CD137, CD2, CD7, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), CD8 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF1.4), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, 11.2 beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD 103, IT GAL, LFA-1, ITGAM, ITGAX, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 or CD83.

Non-coding functional sequences

[0257] In some embodiments, the circular RNAs and nucleic acid molecules for making circular RNAs described herein may comprise one or more nucleic acid sequences of interest that have a regulatory function, i.e., the sequence is a non-coding sequence but has a biological function and/or activity (e.g., non-coding functions), e.g., interactions with other types of non-coding RNA molecules, primarily microRNAs, long noncoding RNAs, and RNA-binding proteins.

[0258] In one embodiment, the circular RNA of the present disclosure comprises a nucleic acid sequence acting as a miRNA sponge, which competes endogenous RNA. As used herein, the term “miRNA sponge” refers to a circular polynucleotide comprising a single-stranded non-coding polynucleotide with repeat copies of at least one specific microRNA binding site to hold microRNA molecules of interest. The miRNA sponge acts as an artificial microRNA inhibitor, when expressed in a cell, would decrease the cellular level of the microRNA of interest.

[0259] The miRNA sponge sequence may comprise at least one miRNA response element (MRE) that binds to a miRNA and negatively regulates its activity. As used herein, the term “miRNA response element (MRE)” refers to a target site (i.e., a short nucleic acid fragment) that binds to a miRNA. In some embodiments, the circular polynucleotide may comprise two or more MREs. The number of MREs in the circular polynucleotide is variable and relates to the length of the circular polynucleotide. As non-limiting examples, the circular polynucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more MREs. The multiple MREs may have the same nucleic acid sequences and bind to the same miRNA; or alternatively, the MREs have different nucleic acid sequences and bind to different miRNAs, such as 2, 3, 4, 5, or more different miRNAs.

[0260] In some embodiments, the miRNA sponge sequence may comprise 1-150 conserved miRNA target sites to bind to a miRNA to sponge the miRNA, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, or 150 miRNA target sites to sponge the miRNA.

[0261] In some embodiments, the circular RNA described herein may regulate more than one miRNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more miRNAs.

[0262] The circular RNA described herein may be used to affect target miRNA activities, i.e., by acting as miRNAs sponge, circular RNAs may down-regulate miRNA activity and/or up-regulate the expression of miRNA target genes.

[0263] In some embodiments, the circular RNA comprises a nucleic acid sequence that binds to one or more RNA binding proteins (RBPs) acting as a protein sponge.

[0264] In some embodiments, the circular RNA comprises a nucleic acid sequence that interacts with one or more protein to enhance protein function.

[0265] In some embodiments, the circular RNA comprises a nucleic acid sequence that acts as scaffold to mediate complex formation between specific enzymes and substrates. [0266] In some embodiments, the circular RNA comprises a nucleic acid sequence that binds to one or more protein to recruit proteins to specific locations.

[0267] In some embodiments, the circular RNA may comprise one or more long noncoding RNA (IncRNA, or lincRNA), a small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA) and/or a portion thereof.

[0268] These functional non-coding sequences may be included in the circular RNA alone and used for their functions. Alternatively, these functional non-coding sequences may be included in the circular RNA encoding a polypeptide of interest.

Signaling nucleotides

[0269] In some embodiments, the circular RNAs and nucleic acid molecules for making circular RNAs may also encode additional features which may facilitate the trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5' terminus of the coding region or the N- terminus polypeptide encoded, respectively. In some embodiments, addition of these sequences results in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported. [0270] As described herein, the circular RNAs may comprise regions that partially or substantially not translatable, e.g., having a noncoding region. Such noncoding regions are different from the non-coding functional sequences and may located in any region of the circular RNA. Those non-coding regions include but are not limited to the linker, the spacer and/or the flanking regions. The noncoding regions may locate in more than one region of the circular RNA.

Constructs and vectors

[0271] In some embodiments, the nucleic acid molecule described herein is a DNA construct. The construct comprises an upstream intron sequence and a downstream intron sequence which flanks exonic sequence (e.g., a protein coding RNA sequence).

[0272] In some embodiments, the nucleic acid molecule is a DNA vector. In some embodiment, viral vectors may be used to package the constructs for making circular RNAs. The viral vectors may be AAV vectors.

[0273] In other embodiments, the construct is a non-viral vector. Exemplary non-viral vectors may include plasmids, cosmids and artificial chromosomes.

[0274] In some embodiments, the construct includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides.

[0275] The constructs and vectors provided herein can be made using standard techniques of molecular biology. For example, the various elements of the vectors provided herein can be obtained using recombinant methods, or by deriving the polynucleotides from a vector known to include the same. The various elements of the vectors provided herein can also be produced synthetically, rather than cloned, based on the known sequences. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into the complete sequence.

Linear RNA

[0276] In some embodiments, the nucleic acid molecules described herein are linear RNA polynucleotides (e.g., a linear mRNA). The linear RNA polynucleotide can be circularized in vitro or in vivo to produce a circular RNA.

[0277] In some embodiments, the nucleic acid molecule is a linear RNA.

[0278] In some embodiments, the linear RNA is unmodified.

[0279] In some embodiments, the linear RNA is modified.

[0280] In some embodiments, the linear RNA comprises one or more modified nucleotides N 1 -methylpseudouridine.

[0281] In some embodiments, the linear RNA comprises one or more modified nucleotides 5-methoxyuridine.

[0282] In some embodiments, the linear RNA comprises one or more modified nucleotides m5C.

[0283] In some embodiments, the linear RNA polynucleotide is synthesized by in vitro transcription (IVT) of a DNA construct described herein. For example, the linear RNA polynucleotide can be generated by incubating a vector described herein under conditions permissive of transcription of the precursor RNA encoded by the vector. In some embodiments, a linear RNA is synthesized by incubating a vector described herein that comprises an RNA polymerase promoter upstream of the upstream intron sequence and/or expression sequences with a compatible RNA polymerase enzyme under conditions permissive of in vitro transcription. In some embodiments, the vector is incubated inside of a cell by a bacteriophage RNA polymerase or in the nucleus of a cell by host RNA polymerase II.

[0284] In some embodiments, the resulting linear RNA can be used to generate circular RNA by incubating it in the presence of magnesium ions and guanosine nucleotide or nucleoside at a temperature at which RNA circularization occurs (e.g., between 30 °C and 60 °C). Circular RNAs

[0285] In accordance with the present disclosure, provided herein include circular RNAs. The circular RNA can be made from a nucleic acid molecule described herein. In some embodiments, the circular RNA is synthesized by the Group I or II intron sequences mediated self-splicing of the nucleic acid molecule described herein. For an example, an IVT linear RNA polynucleotide is circularized to produce the circular RNA.

[0286] In some embodiments, the circular RNA of the present disclosure has increased stability in vivo.

[0287] In some embodiments, the circular RNA of the present disclosure may have reduced immunogenicity.

[0288] In some embodiments, the circular RNA of the present disclosure has enhanced translation efficiency. In some embodiments, the circular RNA has a translation efficiency at least at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 35 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 75 fold, at least 80 fold, at least 85 fold, at least 90, at least 95 fold, or at least 100 fold greater than a linear RNA counterpart.

Method of Making Circular RNA

[0289] In some embodiments, provided herein is a method for making a circular RNA using the nucleic acid molecule of the present disclosure.

[0290] In some aspects, provided herein is a method for making a circular RNA comprising circularizing a nucleic acid molecule comprising, from 5’ to 3’ end, (i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, (ii) a nucleic acid sequence of interest, and (iii) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same Group I or Group II intron as the upstream intron sequence, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and wherein transcribing the nucleic acid molecule to a linear precursor RNA and circularizing the linear precursor RNA into the circular RNA occurs in one reaction. [0291] In some embodiments, the nucleic acid molecule does not include a 5’ or 3’ homology arm.

[0292] In some embodiments, the nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence of interest and/or a 3’ untranslated region (UTR) sequence between the sequence of interest and the downstream intron sequence.

[0293] In some embodiments, the 5’ spacer sequence comprises an inner homology element.

[0294] In some embodiments, the 5’ spacer sequence does not comprise an inner homology element.

[0295] In some embodiments, the nucleic acid molecule does not include the 5’ spacer sequence.

[0296] In some embodiments, the nucleic acid molecule further comprises an internal ribosome entry site (IRES) between the 5’ spacer sequence and the RNA sequence.

[0297] In some embodiments, the nucleic acid molecule is a DNA vector.

[0298] In some embodiments, the nucleic acid molecule is a linear RNA.

[0299] In some embodiments, the method comprises incubating the nucleic acid molecule at a temperature at which RNA circularization occurs.

[0300] In some embodiments, the temperature is about 30°C to about 60°C, including every degree in between this range. In some embodiments, the temperature is 30°C to 60°C, including every degree in between this range.

[0301] In some embodiments, the upstream and downstream intron sequences each correspond to the 3’and 5’ intron splicing fragments of SEQ ID NO: 7.

[0302] In some embodiments, provided herein is a method of the present disclosure, wherein the nucleic acid comprises: (i) the upstream intron sequence comprising a sequence corresponding to positions 119 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1-118 of SEQ ID NO: 7; (ii) the upstream intron sequence comprising a sequence corresponding to positions 140 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7; or (iii) the upstream intron sequence comprising a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 146 of SEQ ID NO: 7.

[0303] In some embodiments, the nucleic acid comprises the upstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 1, and the downstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO. 2.

[0304] In some embodiments, the circularization occurs at 35°C to 42°C.

[0305] In some embodiments of the method, the nucleic acid comprises the upstream and downstream intron sequences each corresponding to the 3 ’and 5’ splicing fragments of SEQ ID NO. 8.

[0306] In some embodiments of the method, the nucleic acid comprises the upstream intron sequence comprising a sequence corresponding to positions 112-231 of SEQ ID NO: 8, and the downstream intron sequence comprising a sequence corresponding to positions 1-111 of SEQ ID NO: 8.

[0307] In some embodiments of the method, the nucleic acid comprises the upstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 3, and the downstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 4.

[0308] In some embodiments, the circularization occurs at a temperature between 35°C to 45°C.

[0309] In some embodiments, the nucleic acid comprises the upstream and downstream intron sequences each corresponding to the 3’ and 5’ splicing fragments of SEQ ID NO. 9. [0310] In some embodiments, the nucleic acid comprises the upstream intron sequence comprising a sequence corresponding to positions 127-264 of SEQ ID NO: 9, and the downstream intron sequence comprising a sequence corresponding to positions 1-126 of SEQ ID NO: 9.

[0311] In some embodiments, the nucleic acid comprises the upstream intron sequence comprising a sequence at least 85%, 90%, 95%, 99%, or 99% identical to SEQ ID NO: 5, and the downstream intron sequence comprising a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

[0312] In some embodiments of the method, wherein the circularization occurs at 35°C to 42°C.

[0313] In some embodiments, provided herein is a method wherein the RNA of interest encodes a chimeric antigen receptor (CAR), a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.

[0314] In some embodiments of the method, the RNA of interest encodes an antigen. [0315] In some embodiments, the method makes intact circular RNA. [0316] In some embodiments, provided herein is a circular RNA made by the method of the present disclosure.

Modifications

[0317] In some embodiments, the linear RNA is unmodified.

[0318] In some embodiments, the linear RNA is modified.

[0319] In some embodiments, the linear RNA comprises one or more modified nucleotides N1 -methylpseudouridine and/or 5-methoxyuridine.

[0320] Modifications may be introduced into the circular RNAs and nucleic acid molecules described herein. The circular RNAs of the present disclosure may include one, two, three, or more modifications. The modifications may be various distinct modifications. In some embodiments, the modifications may locate at various regions and fragments of the circular RNAs, including but not limited to, the coding region(s), the untranslated region(s), intron sequences, the flanking region(s), and/or the terminal or tailing regions.

[0321] The modifications which render the nucleic acid molecules, when introduced to a cell, more resistant to degradation in the cell and/or more stable in the cell as compared to unmodified polynucleotides. The modifications may also increase the biological functions of nucleic acid molecules as compared to unmodified polynucleotides, such as binding to an RBP or another polynucleotide.

[0322] The modifications may be structural and/or chemical modifications. The chemical modification may be a nucleotide and/or nucleoside modification including a nucleobase modification and/or a sugar modification, and a backbone linkage modification (i.e., the intemucleoside linkage, e.g., a linking phosphate, a phosphodiester linkage, and a phosphodiester backbone). The structural modification may include a secondary structural modification, and a tertiary structural modification.

[0323] Modifications according to the present disclosure may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof.

[0324] In some embodiments, one, two, or more (optionally different) nucleoside or nucleotide modifications may be incorporated to the circular RNA. As described herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or a pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). Five primary/canonical nucleobases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) are the fundamental units of nucleic acid molecules, in which Adenine and guanine, referred to purine bases, have a fused-ring skeletal structure derived of purine while uracil, and thymine, derived of pyrimidine, are referred to pyrimidine bases. As described herein, “nucleotide” is defined as a nucleoside including a phosphate group or other backbone linkage (internucleoside linkage).

[0325] In some embodiments, the circular RNA comprises at least one modification described herein. In other embodiments, the circular RNA comprises two, three, four, or more (optionally different) chemical modifications described herein. The modifications may be combinations of nucleobase (purine and/or pyrimidine), sugar and backbone (intemucleoside) linkage modifications. The modifications may be located on one or more nucleotides of the circular RNA. In some embodiments, all the nucleotides of the circular RNA are chemically modified. In some embodiments, all the nucleotides of the nucleic acid sequence with a biological function are chemically modified.

[0326] The circular RNA described herein may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, T/U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 85% to 95%, from 85% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[0327] In some embodiments, the polynucleotides are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the polynucleotides are at least 75% modified, e.g., at least 75% of the nucleotides are modified. It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linkage) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification. [0328] In some embodiments, the polynucleotides are at least 10% modified in only one component of the nucleotide, with such component being the nucleobase, sugar, or linkage between nucleosides. For example, modifications may be made to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleobases, sugars, or linkages of a polynucleotide described herein.

[0329] The circular RNA described herein can be designed with a patterned array of sugar, nucleobase or linkage modifications. In some embodiments, the polynucleotides can comprise modifications to maximize stability.

[0330] The modified nucleosides and nucleotides can include a modified nucleobase. Examples of nucleobases in RNA include, but are not limited to, adenine(A), guanine(G), cytosine(C), and uracil(U). Examples of nucleobases in DNA include, but are not limited to, adenine(A), guanine(G), cytosine(C), and thymine(T).

[0331] In some embodiments, the modified nucleobase is a modified uracil(U). Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (y), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4- thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uridine (ho⁵U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5 -iodo-uridine (I⁵U) or 5 -bromo-uridine (br⁵U)), 3- methyl-uridine (m³U), 5 -methoxy -uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5 -carboxymethyl -uridine (cm⁵U), 1 -carb oxy methyl - pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5 -m ethoxy carbonylmethyl-uri dine (mcm⁵U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U), 5 -aminomethyl -2 -thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine (mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U), 5- methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5- carboxymethylaminomethyl-uridine (cmnm⁵U), 5 -carboxymethylaminomethyl -2-thio-uridine (cmnm⁵s²U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm⁵U), 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm⁵s²U), 1 -taurinomethyl-4- thio-pseudouridine, 5 -methyl -uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1- methylpseudouridine (m ¹ \|/), 5-methyl -2 -thio-uridine (m⁵s²U), pseudouracil (y), 1 -methyl -4- thio-pseudouridine (m's ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m³\|/), 2-thio- 1 -methyl -pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, Nl-methyl-pseudouridine (also known as 1 -methylpseudouridine (m¹!]/)), 3- (3-amino-3-carboxypropyl)uridine (acp³U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp³ y), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5- (isopentenylaminomethyl)-2 -thio-uridine (inm⁵s²U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m⁵Um), 2'-O-methyl-pseudouridine (ym), 2-thio-2'-O- methyl-uridine (s²Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm⁵Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2'-O- methyl-uridine (cmnm⁵Um), 3,2'-O-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)- 2'-O-methyl-uridine (inm⁵Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F- uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E- propenylamino)uridine.

[0332] In some embodiments, the modified nucleobase is a modified cytosine(C). Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (ac⁴C), 5- formyl-cytidine (CC), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1 -methyl -pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl- pseudoisocytidine, lysidine (k^C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O- dimethyl-cytidine (m⁵Cm), N4-acetyl-2'-O-methyl-cytidine (ac⁴Cm), N4,2'-O-dimethyl- cytidine (m⁴Cm), 5-formyl-2'-O-methyl-cytidine (CCm), N4,N4,2'-O-trimethyl-cytidine (n Crn), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0333] In some embodiments, the modified nucleobase is a modified adenine(A). Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyl -adenosine (nfA), 2-methyl- adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms²m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis- hydroxyisopentenyl)adenosine (io⁶ A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), N6-methyl-N6-threonylcarbamoyl -adenosine (m⁶t⁶A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶2A), N6-hydroxynorvalylcarbamoyl- adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A), N6- acetyl-adenosine (ac⁶A), 7-methyl -adenine, 2-methylthio-adenine, 2-methoxy-adenine, a- thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m⁶Am), N6,N6,2'-O-trimethyl-adenosine (m Am), l,2'-O-dimethyl-adenosine (m'Am), 2'-0- ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido- adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0334] In some embodiments, the modified nucleobase is a modified guanine(G). Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1- methyl-inosine (m¹!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (o?yW), hydroxy wybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl -queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G⁺), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine (m⁷G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (nfG), N2-methyl -guanosine (m²G), N2,N2- dimethyl-guanosine (m²2G), N2,7-dimethyl-guanosine (m^2,7G), N2, N2,7-dimethyl -guanosine (m^2,2’⁷G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl- guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m²Gm), N2,N2-dimethyl-2'-O-methyl- guanosine (nAGm), 1 -methyl -2'-O-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-O-methyl- guanosine (m^2,7Gm), 2'-O-methyl-inosine (Im), l,2'-O-dimethyl-inosine (irflm), and 2'-O- ribosylguanosine (phosphate) (Gr(p)).

[0335] In some embodiments, the nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. The nucleobase and/or analog may be each be independently selected from adenine, cytosine, guanine, uracil, naturally-occurring and synthetic derivatives of a base, including but not limited to pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3- deazaguanine, deazaadenine, 7-deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a]l,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5- d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.

[0336] The circular polynucleotide of the present disclosure may comprise a nucleoside modification. One or more atoms of a pyrimidine nucleobase may be replaced or substituted, for example, with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or groups.

[0337] As non-limiting examples, the uracil nucleosides of the circular RNA described herein are all modified. The modifications may be the same or different. In some embodiments, the guanine nucleosides of the circular polynucleotide of the present disclosure are all modified. The modifications may be the same or different. In some embodiments, the guanine nucleosides of the circular polynucleotide of the present disclosure are all modified. The modifications may be the same or different. In some embodiments, the cytosine nucleosides of the circular polynucleotide of the present disclosure are all modified. The modifications may be the same or different. In some embodiments, the adenine nucleosides of the circular polynucleotide of the present disclosure are all modified. The modifications may be the same or different.

[0338] In one embodiment of the disclosure, the circular RNA described herein is modified to comprise N6-methyladenosine (m6A) nucleotides.

[0339] Modifications of the modified nucleosides and nucleotides can be present in the sugar subunit. In some embodiments, the circular RNA described herein comprises at least one sugar modification. Generally, RNA includes the sugar subunit: ribose, which is a 5- membered ring having an oxygen atom.

[0340] In one example, the 2’ hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'OH-position include, but are not limited to, H, halo, optionally substituted Cl -6 alkyl; optionally substituted Cl -6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-Cl-6 alkoxy, optionally substituted Cl-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG)- O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); and “locked” nucleic acids (LNA) in which the 2'-hydroxyl is connected by a C 1-6 alkylene or Cl -6 heteroalkylene bridge to the 4’ -carbon of the same ribose sugar, where exemplary bridges include methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0341] Other exemplary sugar modifications include replacement of the oxygen atom (O) in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multi cyclic forms (e.g., tri cyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3'— >2')) , and peptide nucleic acid (PNA, where 2-amino-ethyl- glycine linkages replace the ribose and phosphodiester backbone).

[0342] The sugar subunit can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, polynucleotides as described herein, including circRNAs, can include nucleotides containing, e.g., arabinose, as the sugar.

[0343] Nonlimiting examples of the sugar modification may include the modifications provided in Table 2. The polynucleotides of the present disclosure can have one or more nucleotides carrying a modification as provided in Table 2. In some embodiments, each of the nucleotides of a polynucleotide described herein carries any one of the modifications as provided in Table 2, or none of the modifications as provided in Table 2.

Table 2. Nucleotide Sugar Modifications

[0344] In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -O- Methoxy ethyl, referred to as 2’-0Me. In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -F, referred to as 2’-F. In some embodiments, the sugar modification can be one or more locked nucleic acids (LNAs). In some embodiments, the polynucleotides can be fully 2’-MOE-sugar modified.

[0345] In some embodiments, modifications (e.g., one or more modifications) are present in the intemucleoside linkage (the linking phosphate or the phosphodiester linkage or the phosphodiester backbone). In the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably.

[0346] Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonates phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0347] The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides through the unnatural phosphorothioate backbone linkages.

Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.

[0348] In some embodiments, the circular RNA described herein comprise at least one phosphorothioate linkage, methylphosphonate linkage between nucleotides, 5 ’-(E)- vinylphosphonate (5 ’-A- VP), a phosphate mimic, as a modification.

[0349] The intemucleoside linkages of the polynucleotides may be partially or fully modified.

[0350] Modified nucleotides incorporated the circular polynucleotides of the present disclosure may include for example, 2’-O-Methyl-modified or 2’ -O-Methoxy ethyl -modified nucleotides (2’-0Me and 2’ -MOE modifications, respectively), an alpha-thio-nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5'- O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thiophosphate)- pseudouridine.

[0351] Additional modifications to the circular RNA described herein include, but are not limited to, any modifications as described in PCT Publication WO2017070626, including, for example, modification or deletion of nucleotides (or codons) encoding one or more N-linked glycosylation site in a translated polypeptide. Modifications may also comprise any modifications as described in PCT Publication WO2018200892. The circular polynucleotides of the present disclosure may further comprise features or modifications as described in PCT patent application publications W02020255063, WO2020182869, W02016011222, W02016011226, W02016005004, W02016000792, WO2015176737, WO2015085318, WO2015048744, and WO2015034925, and United States patent application publications US20200254086, US20200206362, US20180311336 and US20180303929; the contents of each of which are incorporated herein by reference in their entireties. [0352] Different sugar modifications, nucleobase modifications, and/or intemucleoside linkages (e.g., backbone structures) may be introduced at various positions in a polynucleotide described herein. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.

Codon optimization

[0353] The circular RNAs and nucleic acid molecules for making circular RNAs, their regions or parts or subregions may be codon optimized. In some embodiments, the coding sequences of the circular RNAs are codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include, but are not limited to, match codon frequencies in target and host organisms to ensure proper folding, alter GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide.

[0354] In some embodiments, a codon optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted in order to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, and/or (x) systematic variation of codon sets for each amino acid. [0355] In some embodiments, a codon optimized polynucleotide may minimize ribozyme collisions and/or limit structural interference between the expression sequence and the IRES. [0356] Codon optimization tools, algorithms and services are known in the art, nonlimiting examples include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In some embodiments, the coding sequence is optimized using optimization algorithms.

Pharmaceutical compositions

[0357] Provided by the present disclosure include compositions such as pharmaceutical compositions comprising at least one circular RNA or nucleic acid molecule for making a circular RNA as described herein. Compositions described herein may be formulated for administration to a particular target cell, a target tissue, or a target organ and/or a subject. [0358] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington'. The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[0359] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., circular RNAs) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0360] A pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0361] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1 and 30%, between 5 and 80%, between 10 and 50%, between 20 and 90%, at least 70% (w/w), or at least 80% (w/w) active ingredient.

[0362] In some embodiments, the formulations described herein may contain at least one circular RNA molecule. In some embodiments, the formulations may contain one, two, three, four or five circular RNAs with different sequences. In one embodiment, the formulation contains at least two circular RNAs. In one embodiment, the formulation contains at least three circular RNAs. In another embodiment, the formulation contains at least four circular RNAs. In yet another embodiment, the formulation contains at least five circular RNAs.

[0363] The pharmaceutical compositions and formulations of the present disclosure can be formulated with one or more excipients to increase the stability of circular RNA; increase cell penetration; permit the sustained, controlled or delayed release; alter the biodistribution (e.g., target the nucleic acid vaccine composition to specific tissues or cell types); increase the translation of encoded protein in vivo; and/or alter the release of encoded protein in vivo.

[0364] In addition to traditional excipients, excipients of the present disclosure can include, without limitation, lipids, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nucleic acid molecules, cells, organelles, explants, nanoparticle mimics and combinations thereof.

[0365] In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle, degree of loading, polynucleotide to lipid/lipidoid ratio, nature of polynucleotides such as sequence contents, single-stranded or double-stranded, linear or circular, length and modifications, particle sizes and charges, and administration routes, etc.

[0366] The present disclosure contemplates the formulation and use in delivering at least one circular RNA compositions and at least one pharmaceutically acceptable carrier, such as circular RNAs encoding proteins including antigen proteins for nucleic acid vaccines. Complexes, micelles, liposomes or particles can be prepared containing any suitable lipids and lipidoids and therefore, can result in an effective delivery of the circular polynucleotide compositions following the injection of a formulation via localized and/or systemic routes of administration, e.g., by various means including, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), sub-retinal, intranasal, or intracerebroventricular (ICV) administration.

Lipid nanoparticles (LNPs)

[0367] In some embodiments, the circular RNAs, nucleic acid molecules and compositions thereof described herein may be formulated in a delivery vehicle, e.g., a lipid nanoparticle (LNP). In some embodiments, an RNA is formulated in lipid nanoparticles (LNPs). LNP components are selected based on the desired target (e.g., a specific cell type to be delivered to, in the patient), cargo (e.g., circRNA molecules), size, and/or other desired features. LNP components include, for example, ionizable lipids, helper lipids, sterols, and/or PEG-lipids. The relative amounts, or molar ratios, of ionizable lipid, helper lipid, cholesterol, and PEG-lipid are optimized for a given target or administration route. In some embodiments, the LNPs do not contain a targeting ligand. In some embodiments, the LNPs contain a targeting ligand. In some embodiments, LNPs are used to target specific cells using endogenous or exogenous ligands by encapsulating circular RNA by methods known in the art. In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof that is conjugated to the surface of the engineered nanoparticle, such as a lipid nanoparticle. In some embodiments, the conjugation is via enzymatic or chemical methods. Endocytosis of LNPs destabilizes the endosomal membrane and release circular RNAs into the target cell cytoplasm.

[0368] As used herein, the term “delivery vehicle” encompasses various delivery vehicles, including “lipid nanoparticle”, “nanoparticle” or grammatical equivalents. In general, LNPs can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.

[0369] The LNPs for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from 10-1000 nm. The nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,

195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,

285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370,

375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460,

465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550,

555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640,

645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730,

735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820,

825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910,

915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm, or less than 100 nm, less than 150 nm, less than 200 nm, less than 250 nm, less than 300 nm, less than 350 nm, less than 400 nm, less than 450 nm, less than 500 nm, less than 550 nm, less than 600 nm, less than 650 nm, less than 700 nm, less than 750 nm, less than 800 nm, less than 850 nm, less than 900 nm, less than 950 nm or less than 1000 nm.

[0370] In some embodiments, the lipid nanoparticles for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from about 1 to about 100 nm, such as but not limited to, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, and/or from about 5 nm to about 100 nm.

[0371] In some embodiments, the lipid nanoparticles for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from about 10 to about 100 nm, such as, but not limited to, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 30 nm to about 80 nm, about 30 nm to about 90 nm, about 30 nm to about 100 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, about 40 nm to about 70 nm, about 40 nm to about 80 nm, about 40 nm to about 90 nm, about 40 nm to about 100 nm, about 50 nm to about 60 nm, about 50 nm to about 70 nm about 50 nm to about 80 nm, about 50 nm to about 90 nm, about 50 nm to about 100 nm, about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm to about 100 nm, about 80 nm to about 90 nm, about 80 nm to about 100 nm and/or about 90 nm to about 100 nm.

[0372] In some embodiments, the lipid nanoparticles for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may have a diameter from about 50 nm to about 100 nm.

[0373] LNPs useful herein are known in the art and generally comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more polyethylene glycol (PEG) modified lipids. In some embodiments, a LNP comprises no more than three distinct lipid components. The components of the LNP may be selected based on the desired target, tropism, cargo (e.g., a circular RNA), size, or other desired feature or property. The relative amounts (ratio) of ionizable lipid, helper lipid, cholesterol and PEG-modified lipids substantially affect the efficacy of lipid nanoparticles and may be optimized for a given application and administration route.

[0374] In general, the circular RNAs, nucleic acid molecules and compositions thereof described herein may be formulated using LNPs into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.

Ionizable lipids

[0375] The LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein comprises at least one ionizable (i.e., cationic) lipid. [0376] In some embodiments, the LNPs may contain one or more cationic lipids selected from C12-200, DLin-KC2-DMA, HGT4003, HGT5000, HGT5001, MC3,DLinDMA, DLinkC2DMA, cKK-E12, ICE, , DODAC, DDAB, DMRIE DOSPA, DOGS, DODAP, DODMA, DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, and combinations thereof.

[0377] Other suitable cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23- dien- 10-amine, (17Z,20Z)-N,N-dimemylhexacosa- 17,20-dien-9-amine, ( 1Z, 19Z)-N5N- dimethylpentacosa-1 6, 19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5- amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N- dimethyltricosa- 14, 17-dien-6-amine, (15Z, 18Z)-N,N-dimethyltetracosa-l 5, 18-dien-7-amine, ( 18Z,2 lZ)-N,N-dimethylheptacosa- 18,21 -di en- 10-amine, ( 15Z, 18Z)-N,N-dimethyltetracosa- 15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N- dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N-dimethylheptacosa- 18 ,21 -dien-8 - amine, (17Z,20Z)-N,N-dimethylhexacosa- 17,20-dien-7-amine, (16Z,19Z)-N,N- dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10- amine, (21 Z ,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimetylheptacos- 18-en- 10-amine, ( 17Z)-N,N -dimethylhexacos- 17 -en-9-amine, ( 19Z,22Z)-N,N- dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-l 0-amine, (20Z,23Z)-N- ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(1 lZ,14Z)-l-nonylicosa-l 1 , 14-dien-l-yl] pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-l 0-amine, (15Z)-N,N-dimethyl eptacos-15- en-1 0-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17- en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20- en-1 0-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos- 16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine, (13Z,16Z)- N,N-dimethyl-3-nonyldocosa-13,16-dien-l-amine, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl] eptadecan-8-amine, 1 -[( 1 S,2R)-2-hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine, N,N-dimethyl-l-[(l S ,2R)-2-octylcyclopropyl]nonadecan-10- amine, N,N-dimethyl-21-[(lS,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-l- [(lS,2S)-2-{[(lR,2R)-2-pentylcyc!opropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N- dimethyl-l-[(lS,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(lR,2S)-2- undecy!cyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(lS,2R)-2- octylcyclopropyl]heptyl} dodecan-1 -amine, l-[(lR,2S)-2-hepty lcyclopropyl]-N,N- dimethyloctadecan-9-amine, 1 -[(1 S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6- amine, N,N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-l- [(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl-l- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, l-{2-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-l-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-l- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l-yloxy]propan-2-amine, l-{2- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-l-[(octyloxy)methyl]ethyl}azetidine, (2S)-1- (hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, (2S)-1- (heptyloxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-2-amine, N,N- dimethyl-l-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N- dimethyl- 1 -[(9Z)-octadec-9-en- 1 -yloxy]-3 -(octyloxy)propan-2-amine; (2S)-N,N-dimethyl- 1 - [(6Z,9Z,12Z)-octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1- [(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1 - (hexyloxy)-3-[(l lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethylpropan-2-amine, 1- [(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1- [( 13Z, 16Z)-docosa-13 , 16-dien-l-yloxy]-N,N-dimethyl-3 -(octyloxy )propan-2-amine, (2S)- 1 - [(13Z,16Z)-docosa-13,16-dien-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1- [(13Z)-docos-13-en-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, l-[(13Z)-docos- 13 -en- 1 -yloxy ] -N,N-dimethyl -3 -(octyloxy)propan-2-amine, 1 - [(9Z)-hexadec-9-en- 1 -yloxy ] - N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(l -metoylo ctyl)oxy]-3- [(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-2-amine, (2R)-l-[(3,7-dimethyloctyl)oxy]- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N-dimethyl-l- (octyloxy)-3-({8-[(lS,2S)-2-{[(lR,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-l-{[8-(2- oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,2OZ,23Z)-N,N- dimethylnonacosa-ll,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0378] Other suitable cationic lipids which may be used in the compositions and methods of the present disclosure include ionizable cationic lipids described in PCT Patent Application Publication Nos. W02012040184, WO2011153120, WO2011149733, WO201 1090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865 and W02008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No. US20100036115 and US20120202871; the contents of each of which are herein incorporated by reference in their entirety.

[0379] In some embodiments, the cationic lipid may be synthesized by methods known in the art and/or as described in PCT Patent Application Publication Nos. W02012040184, WO201 1153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724 and W0201021865; the contents of each of which are herein incorporated by reference in their entirety.

[0380] In some embodiments, the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a plurality of cationic lipids, such as a first and a second cationic lipid. The first cationic lipid can be selected on the basis of a first property and the second cationic lipid can be selected on the basis of a second property. The first and second properties may be complementary.

[0381] In some embodiments, the nanoparticles described herein may comprise at least one cationic polymer described herein and/or known in the art.

[0382] In some embodiments, the compositions of the present disclosure include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the lipid nanoparticle. In some embodiments, the compositions of the present disclosure include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total lipid content in the lipid nanoparticle. In some embodiments, the compositions of the present disclosure include one or more cationic lipids that constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the lipid nanoparticle. In some embodiments, the compositions of the present disclosure include one or more cationic lipids that constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol %, of the total lipid content in the lipid nanoparticle.

[0383] In some embodiments, the ionizable lipid comprises a compound disclosed in WO 2021/141969 (Hamilton et al.), the contents of which are incorporated by reference herein in their entireties. In some embodiments, the ionizable lipid comprises a compound of Formula (I) of WO 2021/141969.

(Formula (I))

[0384] In some embodiments, R¹ in Formula (I) comprises C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation. For example, in some embodiments R¹ comprises a C9-C20 alkenyl with 2 units of unsaturation, such as, without limitation, a C17 alkenyl with 2 units of unsaturation.

[0385] In some embodiments, X³, X⁵, and X⁶ in Formula (I) are independently absent.

[0386] In some embodiments, X¹ is -O-. In some embodiments, X¹ is absent. -(CH)a

[0387] In some embodiments, X² is . In some embodiments, X² is -(CH2)_a- or - CH(OH)-. In some embodiments, a is an integer between 0 and 6. In some embodiments, a is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, a is 0 and X² is absent. In some embodiments, a is 1.

[0388] In some embodiments, X⁷ is independently hydrogen or hydroxyl. In some embodiments, X⁷ is hydroxyl. In some embodiments, X⁷ is hydrogen.

[0389] In some embodiments, X⁴ is a 6-membered heterocyclyl optionally substituted with 1 or 2 Ci-Ce alkyl groups. In some embodiments, the heterocyclyl comprises at least one nitrogen. For example, some embodiments, X⁴ is piperidinyl. In some embodiments, X⁴ is ethylpiperidinyl.

[0390] In some embodiments, A¹ and A² are independently C5-C12 alkyl or C5-C12 alkenyl with 1-3 units of unsaturation. In some embodiments, A¹ and A² are independently C5-C12 alkenyl with 1 unit of unsaturation. In some embodiments, A¹ is Cs alkenyl with 1 unit of unsaturation. In some embodiments, A² is Cs alkenyl with 1 unit of unsaturation.

[0391] In some embodiments, nl is an integer between 1 and 6. In some embodiments, nl is 1, 2, 3, 4, 5, or 6. In some embodiments, nl is 2.

[0392] In some embodiments, the ionizable lipid comprises a compound disclosed in WO 2022/140252 Al (Patwardhan et al. the contents of which are incorporated by reference herein in their entireties. In some embodiments, the ionizable lipid comprises a compound of Formula (III-a-i) of WO 2022/140252, or its N-oxide: ffi-

(Formula (III-a-i))

[0393] In some embodiments, R¹ is hydrogen.

[0394] In some embodiments, L¹ is C2-C6 heteroalkylenyl comprising at least 1 heteroatom. In some embodiments, the heteroatom is oxygen. For example, in some embodiments, L¹ is a C4 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2CH2-. In some embodiments, L¹ is a C3 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-. [0395] In some embodiments, each R is independently Ce-Cn alkyl or Ce-Cn alkenyl with 1-3 units of unsaturation.

[0396] In some embodiments, each L is independently C1-C5 alkylenyl.

[0397] In some embodiments, each L² is independently C4-C8 alkylenyl.

[0398] In some embodiments, the ionizable lipid comprises a compound of Formula (I”- a) of WO 2022/140252 Al (Patwardhan et al.).

(Formula (I” -a-iii))

[0399] In some embodiments, R¹ is hydrogen.

[0400] In some embodiments, L¹ is C2-C6 heteroalkylenyl comprising at least 1 heteroatom. In some embodiments, the heteroatom is oxygen. In some embodiments, L¹ is a C3 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-.

[0401] In some embodiments, each R is independently C6-C12 alkyl or C6-C12 alkenyl with 1-3 units of unsaturation.

[0402] In some embodiments, R” is C6-C12 alkyl.

[0403] In some embodiments, each L is independently C1-C5 alkylenyl.

[0404] In some embodiments, each L² is independently C4-C8 alkylenyl.

[0405] Exemplary ionizable lipids include 3-((((l-ethylpiperidin-3- yl)methoxy)carbonyl)oxy)-2-(((4-(((Z)-oct-5-en- 1 -yl)oxy)-4-(((Z)-oct-5-en- 1 - yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-di enoate ( Compound 1).

(Compound 1) (3-hydroxypropyl)azanediyl)bis(heptane-7,l-diyl) bis(4,4-bis(((E)-oct-5-en-l- yl)oxy)butanoate) (Compound 2);

(Compound 2)

((2-hydroxyethyl)azanediyl)bis(hexane-6,l-diyl) bis(6,6-bis(hexyloxy)hexanoate)

(Compound 3).

(Compound 3) nonyl 8-((6-((4,4-bis(octyloxy)butanoyl)oxy)hexyl)(2-hydroxyethyl)amino)octanoate (Compound 4).

(Compound 4) nonyl 8-((2-hydroxyethyl)(6-((4-(((Z)-oct-5-en-l-yl)oxy)-4-(((Z)-oct-5-en-l- yl)oxy)butanoyl)oxy)hexyl)amino)octanoate (Compound 5).

(Compound 5) Or Compound 6

(Compound 6)

Non-cationic lipids/helper lipids

[0406] The LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise one or more non-cationic lipids (helper lipids). The helper lipids in LNPs may contribute to their stability and delivery efficiency, and/or mitigate the toxicity owing to the cationic lipids. A “non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid.

[0407] Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-0- monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), or a mixture thereof.

[0408] In some embodiments, the non-cationic lipid is a phospholipid such as a synthetic phospholipid, including but not limited to, DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC and DEPC; DMPG, DPPG, DSPG and POPG; DMPA, DPPA and DSP A; DMPE, DPPE, DSPE and DOPE; DOPS; and polyglycerin attached phospholipids (PG phospholipid). The phospholipid may be selected based on administration routes, e.g., DPPC, POPC and POPG used in LNPs for injection and DOPC, POPC and DDPC used in LNPs for pulmonary delivery. In some embodiments, the phospholipid may be a purified lipid from a natural source.

[0409] In some embodiments, the LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise one or more neutral helper lipids such as dioleoyl phosphoethanolamine (DOPE), prostaglandins, eicosanoids, glycerides, glycosylated diacyl glycerols, oxygenated fatty acids, NAGly and PAHSA. The neutral lipid is a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.

[0410] In some embodiments, the non-cationic lipid may comprise a molar ratio of about 5% to about 90%, or about 10 % to about 70% of the total lipid present in a LNP. In some embodiments, the percentage of non-cationic lipid in a LNP may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

Cholesterol- derived lipids

[0411] The LNP for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein comprises one or more cholesterol derived lipids. The cholesterol derived lipids can be a cholesterol, a naturally occurring cholesterol analogue, or a synthetic cholesterol like compound and the cholesterol derivatives. In some embodiments, a naturally occurring cholesterol analog may be selected from those by Patel et al., (Nature Communications, 2020; 983); the contents of which are incorporated herein by reference in their entirety. In some embodiments, the LNPs comprise one or more cholesterol derivatives, e.g., PtdChol.

[0412] In some embodiments, the cholesterol-based lipid may comprise a molar ration of about 2% to about 30%, or about 5% to about 20% of the total lipid present in a LNP. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.

PEG-modified lipids

[0413] The LNP described herein comprises one or more PEG modified lipids, such as PEG polymers and PEGylated lipids.

[0414] Suitable PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of Ce- C20 length. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipidnucleic acid composition to the target tissues, or they may be selected to rapidly exchange out of the formulation in vivo. Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). The PEG-modified may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the LNP.

[0415] In some embodiments, LNPs for delivering the circular RNAs, nucleic acid molecules and compositions thereof described herein may include at least one of the PEGylated lipids described in PCT Patent Application Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.

[0416] In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations.

[0417] In some embodiments, the LNP comprises PEG-c-DOMG. In some embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DPG (1,2-Dipalmitoyl-sn- glycerol, methoxypolyethylene glycol), or PEG-DMG 2000 (l,2-dimyristoyl-sn-glycero-3- phophoethanolamine-N-[methoxy(polyethylene glycol)-2000). As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin- DMA, DSPC and cholesterol in a molar ratio of 2:40: 10:48 (see e.g., Geall et al., PNAS, 2012, 109(36): 14604-14609; herein incorporated by reference in its entirety).

[0418] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.

[0419] In some embodiments, the LNP for delivering circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a cleavable lipid such as those described in PCT Patent Application Publication No. WO2012170889, the contents of which are herein incorporated by reference in their entirety.

[0420] In some embodiments, the LNP for delivering circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a conjugated lipid. In a non-limiting example, the conjugated lipid may have a formula such as described in US Pub. No. US 20120264810 to Lin et al., the contents of which are incorporated herein by reference in their entirety. The conjugate lipid may form a lipid particle which further comprises a cationic lipid, a neutral lipid, and a lipid capable of reducing aggregation.

[0421] In some embodiments, the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a mixture of cationic compounds and neutral lipids. As a non-limiting example, the cationic compounds may be formula (I) disclosed in PCT Patent Application Publication No: WO 1999010390 to Ansell et al., the contents of which are incorporated herein by reference in their entireties, and the neutral lipid may be selected from the group consisting of diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide and sphingomyelin.

[0422] In some embodiments, the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; the contents of which are herein incorporated by reference in their entireties.

[0423] In some embodiments, the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.

[0424] In some embodiments, the LNP for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may be encapsulated in the lipid formulation to form a stable nucleic acid-lipid particle (SNALP) such as described in US Pat. No. US8,546,554 to de Fougerolles et al., the contents of which are incorporated here by reference in their entirety. In one non-limiting example, the SNALP includes 40% 2,2- Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (Lipid A), 10% di oleoylphosphatidylcholine (DSPC), 40% cholesterol, 10% polyethylene glycol (PEG)-C- DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 nucleic acid/lipid ratio. [0425] In some embodiments, the LNPs for formulating the circular RNAs, nucleic acid molecules, and compositions thereof described herein may comprise an endosomal membrane destabilizer as disclosed in US Pat. No. US 7,189,705 to Lam et al., the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the endosomal membrane destabilizer may be a Ca²⁺ ion.

[0426] In some embodiments, the LNPs for formulating the circular RNAs, nucleic acid molecules and compositions thereof described herein may comprise a charged lipid or an amino lipid. As used herein, the term “charged lipid” is meant to include those lipids having one or two fatty acyl or fatty alkyl chains and a quaternary amino head group. The quaternary amine carries a permanent positive charge. The head group can optionally include an ionizable group, such as a primary, secondary, or tertiary amine that may be protonated at physiological pH. The presence of the quaternary amine can alter the pKa of the ionizable group relative to the pKa of the group in a structurally similar compound that lacks the quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine). In a nonlimiting example, the charged lipid used in any of the formulations described herein may be any charged lipid described in EP2509636 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety. In some embodiments, a charged lipid is referred to as an “amino lipid.” In a non-limiting example, the amino lipid may be any amino lipid described in US Pub. No. US20110256175 to Hope et al., the contents of which are incorporated herein by reference in their entirety. For example, the amino lipids may have the structure disclosed in Tables 3-7 of Hope, such as structure (II), DLin-K-C2-DMA, DLin-K2- DMA, DLin-K6-DMA, etc. In another non-limiting example, the amino lipids may be any amino lipid described in US 20110117125 to Hope et al., the contents of which are incorporated herein by reference in their entirety, such as a lipid of structure (I), DLin-K- DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLin-S-DMA, etc. In another non-limiting example, the amino lipid may have the structure (I), (II), (III), or (IV), or 4-(R)-DLin-K- DMA (VI), 4-(S)-DLin-K-DMA (V) as described in PCT Patent Application Publication No. W02009132131 to Manoharan et al., the contents of which are incorporated herein by reference in their entirety.

[0427] In some embodiments, the LNPs for formulating the circular polynucleotide compositions of the present disclosure may comprise reverse head group lipids, e.g., formulated with a zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the head group, such as a lipid having structure (A) or structure (I) described in PCT Patent Application Publication No. WO2011056682 to Leung et al., the contents of which are incorporated herein by reference in their entirety.

[0428] In some embodiments, the lipid components of the LNP to nucleic acid ratio (mass/mass ratio) (e.g., lipids to circular polynucleotide compositions ratio) may be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3: 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1, or 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1. 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1. 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 21 : 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1, 29: 1, 30: 1, 31 : 1, 32: 1, 33: 1, 34: 1, 35: 1, 36: 1, 37: 1, 38: 1, 38: 1, 39: 1, 40: 1, 41 : 1, 42: 1, 43: 1, 44: 1, 45: 1, 46: 1, 47: 1, 48: 1, 49: 1, or 50: 1.

[0429] In some embodiments, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or W02008103276. As a nonlimiting example, the circular polynucleotide compositions of the present disclosure may be encapsulated in any of the lipid nanoparticle (LNP) formulations described in WO201 1127255 and/or W02008103276; the contents of each of which are herein incorporated by reference in their entirety.

Other nanoparticles and delivery agents

[0430] In some embodiments, other delivery vehicles are used to deliver circular RNAs, nucleic acid molecules and compositions thereof described herein. The vehicles may include other lipid-based particles such as lipidoids, liposomes, lipoplexes, micelles, multilamellar vesicle (MLV), unicellular vesicle (SUV), polymer-based nanoparticles and exosomes.

[0431] In some embodiments, compositions described herein may also be constructed or altered such that their properties are suitable for different administration routes, such as parenteral (intravenously, intramuscularly or subcutaneously), oral, rectal, opthalmic and/or topical administration.

[0432] In some embodiments, compositions of the present disclosure can be formulated for controlled release and/or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a therapeutic outcome. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. In some embodiments, the compositions may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery.

Engineered exosomes

[0433] Exosomes are tiny vesicles smaller than 50 nm secreted by mature reticulocytes, which are associated with transferrin receptors and function in antigen presentation during the regulation of immune cells. In some embodiments, engineered exosomes act as cargo carriers and deliver small hydrophilic or lipophilic molecules, including some therapeutic drugs to cells, participating in the regulation of many major diseases. Exosomes can improve bioavailability of some drugs when taken orally, reducing the total dose required for administration, and minimizing side effects.

Viral like particles (VLPs)

[0434] In some embodiments, RNA therapeutics discussed herein are delivered using viral delivery particles. Viral particles include recombinant viruses and viral like particles (VLPs). As used herein, the term Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non -infectious because they contain no viral genetic material. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs can be produced from different viruses, such as adeno-associated viruses, retroviruses, lentiviruses and vesiculoviruses. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. VLPs possess diverse applications in therapeutics, immunization, and diagnostics. VLPs have been synthesized in a wide range of expression systems, including prokaryotic (bacteria) and eukaryotic (insect cells, mammalian cell lines, plant cells, or yeast) expression systems. The functionality of VLPs can be increased through modifying their exterior or interior surface by displaying the heterologous epitopes of interest using different methods like peptide conjugation, genetic fusion, and chemical crosslinking.

[0435] In some embodiments, the VLP is derived from a Vesiculovirus. In some embodiments, the VLP is derived from VSV (Indiana vesiculovirus, formerly Vesicular stomatitis Indiana virus (VSIV or VSV). In some embodiments, the virus like particle comprises a mutated VSV-G protein. VSV-G protein is a single transmembrane glycoprotein (G) which plays a critical role during the initial steps of virus infection, it is responsible for virus attachment to specific receptor, LDL-R. In the cell, G protein triggers the fusion between the viral and endosomal membranes, which releases the viral genome in the cytosol for the subsequent steps of infection. In some embodiments, VSV-G protein is mutated to abolish its binding to LDL-R receptor. For example, a VSV-G envelope protein may be a mutated at one or more of any one of H8, K47, Y209, and/or R354. In some embodiments, a VLP may comprise a mutated VSV-G protein described in the PCT patent application Publication No. WO2019057974; the contents of which are incorporated herein by reference in their entireties. In some aspects, the VLP for delivery RNA therapeutics is a viral particle disclosed in the PCT Publication NOs. WO2020236263 and WO2023107886; the contents of each of which are incorporated herein by reference in their entireties.

[0436] In some embodiments, the virus like particle is pseudotyped. As a non-limiting example, the virus like particle is VSV-G-pseudotyped lentiviruses (VSV-G-LVs).

[0437] In some embodiments, the viral particle for delivering RNA therapeutics is a retrovirus, a recombinant AAV, or an adenovirus.

Administration

[0438] The present disclosure encompasses the delivery of circular RNAs, nucleic acid molecules and compositions thereof described herein for any therapeutic, prophylactic, pharmaceutical, diagnostic or research use. In some embodiments, the circular RNAs and compositions thereof described herein are loaded to delivery vehicles such as those formulation components discussed herein in order to be administered to target cells, tissues, organs and/or subjects. The formulated circular RNA compositions are delivered to the cell using routes of administration known in the art and described herein.

[0439] In some embodiments, the circular RNAs and compositions are delivered to a cell “naked.” As used herein in, “naked” refers to delivering the compositions described herein free from agents which promote transfection. The naked circular RNA compositions are delivered to the cell using routes of administration known in the art and described herein. [0440] As described herein, the circular RNA compositions are also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.

[0441] The compositions of the present disclosure may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra- amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in a way which allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. Routes of administration disclosed in International Publication WO 2013/090648 filed December 14, 2012, the contents of which are incorporated herein by reference in their entirety, may be used to administer the circular polynucleotide-based compositions of the present disclosure.

[0442] The compositions described herein can be formulated into a dosage form and for a route of administration as described herein, such as liquid dosage forms, injectable preparations, pulmonary forms, and solid dosage.

Methods of use thereof

[0443] In some embodiments, provided herein is a method for expressing a protein of interest in a subject comprising delivering to the subject the circular RNA of the present disclosure.

[0444] In some aspects, provided herein is a method of expressing a protein of interest in a subject comprising delivering to the subject a circular RNA transcribed from a nucleic acid, wherein said nucleic acid comprises the following elements: (i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, (ii) 5’ spacer, (iii) an internal ribosome entry sequence (IRES), (iv) a sequence that encodes the protein of interest, (v) a 3 ’ UTR sequence, (vi) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same intron as the upstream intron sequence, and wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and wherein the nucleic acid does not include a 5’ or 3’ homology arm.

[0445] In some embodiments, the circular RNA is formulated in a delivery vehicle.

[0446] In some embodiments, the delivery vehicle is a lipid nanoparticle.

[0447] In some embodiments, the lipid nanoparticle is conjugated to a targeting moiety.

[0448] In accordance, the circular RNAs, nucleic acid molecules and compositions thereof are used for therapy. In some embodiments, the circular RNAs, nucleic acid molecules and compositions thereof are used for treating a disease, e.g., an autoimmune disease, an infectious disease, a genetic disorder and a cancer, in a subject, including a human subject. In accordance, the present disclosure provides a method for treating or preventing a disease using circular RNAs, nucleic acid molecules and compositions thereof described herein. [0449] As a non-limiting example, the circular RNAs, nucleic acid molecules and compositions thereof are used for treating or preventing cancer. Cancer includes a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. Examples of cancers that may be treated by the methods disclosed herein include, but are not limited to, cancers of the immune system including lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example , bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, multiple myeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, epidermoid cancer, squamous cell cancer, T cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and combinations of said cancers. In some embodiments, the methods disclosed herein may be used to reduce the tumor size of a tumor derived from, for example, sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Kaposi's sarcoma, sarcoma of soft tissue, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinomna, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (for example adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the renal pelvis, CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma). The particular cancer may be responsive to chemo- or radiation therapy or the cancer may be refractory. A refractory cancer refers to a cancer that is not amenable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.

[0450] In some embodiments, the circular RNAs, nucleic acid molecules and compositions thereof are used for treating or preventing an infectious disease, such as a viral infection. The circular RNAs, nucleic acid molecules and compositions thereof may be used as vaccines.

[0451] In some embodiments, the circular RNAs, nucleic acid molecules and compositions thereof are used for treating or preventing a genetic disorder.

[0452] In some embodiments, the circular RNAs, nucleic acid molecules and compositions thereof are used for inducing an immune response in a subject. The immune response includes the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast, cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0453] In some embodiments, the compositions of the present disclosure are used to treat an autoimmune disease, including systemic lupus erythematosus (SLE)/lupus nephritis. [0454] In some embodiments, the compositions of the present disclosure are used to induce an immune response for therapeutic or prophylactic purposes.

[0455] In some embodiments, the compositions of the present disclosure are used as vaccines to prevent infection, e.g., viral infections. [0456] In some embodiments, the compositions of the present disclosure are used as cancer vaccines. In this regard, the circular RNA comprises an antigen coding nucleic acid sequence. The antigen is a tumor associated antigen (TAA) or a fragment thereof.

[0457] In some embodiments, the compositions of the present disclosure are used for protein replacement therapy.

[0458] In some embodiments, the compositions described herein are co-administered with one or more additional therapeutic agents (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the compositions described herein can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the therapeutic compositions described herein, and the one or more additional therapeutic agents can be administered simultaneously.

[0459] In some embodiments, the subject is a mammal. In some embodiments, the mammal referred to herein can be any mammal, including, but not limited to, mice and hamsters, rabbits, cats, dogs, pigs and primates. Preferably, the mammal is a human (e.g., a patient).

EXAMPLES

Example 1: Permuted Intron-Exon (PIE) constructs that allow RNA circularization

[0460] This example illustrates the nucleic acid construct designs that were used to test RNA circularization and efficiency.

[0461] As illustrated in FIG. 1, plasmid parts (Part 0 for the upstream intron sequence (US intron); Part 1 for 5’ spacer; Part 2 for IRES, Part 345 for gene coding sequence, which is also referred to herein as “the sequence of interest”; Part 6 for 3’ UTR; and Part 7 for the downstream intron sequence (DS intron)) were synthesized by cloning polymerase chain reaction (PCR) products or pre-made DNA fragments. The nucleic acid constructs for making circular RNA were assembled by cloning parts 1-7 into an entry vector including a T7 promotor and a T7 terminator via a Golden Gate reaction according to the method described in Chen et al (Nature Biotechnology, 2023; 41(2): 262-272).

[0462] Four different embodiments of the nucleic acid constructs were generated, as shown in FIGs. 2A-D. FIG. 2A shows an RNA transcript construct comprising 5’ and 3’ inner homology elements (i.e., UTEs), and 5’ and 3’ homology arms (i.e., outer homology arms, also known as outer homology elements (OHEs))(OHE/IHE). FIG. 2B shows an RNA transcript construct including 5’ and 3’ inner homology elements (i.e., IHE) only (-/IHE), without outer homology arms. FIG. 2C shows an RNA transcript construct that lacks both inner homology elements, and 5’ and 3’ homology arms (-/-). FIG. 2D shows an RNA transcript construct including 5’ and 3’ homology arms only (OHE/-), without inner homology elements.

[0463] The nucleic acid constructs were transformed into E. coli. Colonies were picked, mini-prepped, and sequenced to confirm the assembled constructs.

[0464] The sequence encoding a green fluorescent protein (GFP) was cloned into the construct as Part 345 (gene coding sequence), and the IRES sequence from iHRVB3 was used as Part 2.

Example 2: Co-transcriptional circularization

[0465] The plasmids were linearized and used as linearized transcription templates for in vitro transcription (IVT) to generate linear RNA precursors. In vitro transcription (IVT) was performed in the presence of magnesium (Mg+2) only, or Mg+2 and a guanine nucleotide (Guanosine Monophosphate (GMP)), in addition to nucleoside triphosphates (ATP, CTP, GTP and UTP). The ratio of GMP to GTP (Guanosine Triphosphate) was about 5: 1. Mg+2 was also a component of the IVT reaction buffer. Mg+2 and guanine nucleotide (GMP or GTP) were cofactors for the autocatalytic reaction of self-splicing Group I intron. IVT synthesis was initiated with the addition of T7 RNA polymerase. The self-splicing reaction circularized the linear RNA transcript during the IVT reaction.

[0466] Additionally, after IVT, the reaction mixtures were conditioned for post-refolding ribozyme activation to increase the yield of circular RNA products. The splicing reactions, IVT, and refolding were analyzed by gel electrophoresis to measure circularization of the RNA precursors.

[0467] This study screened new group I intron sequences and used permutation strategies for efficient circularization of linear RNA sequences.

[0468] Viral and cyanobacterial Group I introns were selected and permuted to generate a pair of an upstream (US) intron sequence comprising a 3’ splicing site and a downstream (DS) intron sequence comprising a 5’ splicing site; the corresponding US intron and DS intron sequences were cloned into circular constructs in Example 1 to test their circularization efficiency. The permutation sites were determined computationally based on structural elements that are critical for self-splicing (e.g., Perriman and Ares, RNA, 1998, 4: 1047- 1054.) The permuted US intron and DS intron sequences derived from the intron of T4 bacteriophage td gene (T4-td) used in Chen et al. (Nature Biotechnology, 2023; 41(2): 262- 272) was used as a comparison for circularization efficiency of the screened introns.

[0469] Screening of viral and cyanobacterial introns provided three group I intron sequences that demonstrated high circularization efficiency: Twort-ORF142 intron (SEQ ID NO: 7), Oscillator ia-splendida-trnL intron (SEQ ID NO: 8), and Anabaena-spiroides-trnL intron (SEQ ID NO: 9). FIG. 3 illustrates the predicted secondary structure of the Twort- ORF142 intron (SEQ ID NO: 7) and three different permutation sites (the split sites of the US and DS fragments), VI, V2, and V3, within the intron sequence.

[0470] FIGs. 4A -4C illustrate exemplary upstream intron sequence comprising a 3’ splicing site and downstream intron sequence comprising a 5’ splicing site from Twort- ORF142 intron (SEQ ID NO: 7), Oscillatoria-splendida-trnL intron (SEQ ID NO: 8), and Anabaena-spiroides-trnL intron (SEQ ID NO: 9).

[0471] The permuted intron sequences of Twort-ORF142 intron (as shown in FIG. 4A), Oscillatoria-splendida-trnL intron (as shown in FIG. 4B), and Anabaena-spiroides-trnL intron (as shown in FIG. 4C) all resulted in circularized RNA by direct co-transcriptional circularization (FIG. 5A - FIG. 5C, upper panels). All three intron fragment pairs generated circular RNAs, more efficiently or comparably to the T4-td intron sequence (Chen et al., Nature Biotechnology, 2023; 41(2): 262-272). Similarly, all three new intron sequence pairs had higher or comparable circularization efficiency post-refolding ribozyme activation (FIG. 5A - FIG. 5C, lower panels), as compared to the T4-td intron. The results showed that the three new group I introns (e.g., Twort-ORF142 intron, Oscillatoria-splendida-trnL intron, and Anabaena-spiroides-trnL intron) resulted in less accumulation of nicked circRNAs during in vitro transcription and post refolding. Fewer undesirable byproducts of IVT and circularization reactions (e.g, dsRNA, linear RNA, nicked circRNA) were observed. As shown in FIG. 5A - FIG. 5C, Twort-ORF142 intron (FIG. 5A), Oscillatoria-splendida-trnL intron (FIG. 5B), and Anabaena-spiroides-trnL intron (FIG. 5C) show less nicked circular RNAs (the bands on top of the labeled “circle” bands in FIG. 5A - FIG. 5C). The nicked circRNA band represents single nicks that occur at random positions in an intact circRNA. Less nicked circular RNAs from new Group I introns indicated purer intact circRNAs. Three different permuted sites within the Twort-ORF142 intron (VI, V2 and V3 as shown in FIG. 3) were tested for circularization efficiency. The upstream and downstream intron sequences of each permutation are listed in Table 1 (VI (SEQ ID NO: 10 and 11; V2 (SEQ ID NOs: 1 and 2; and V3 (SEQ ID NOs: 12 and 13)). FIG. 6A is a representative gel image showing circular RNA formed by constructs comprising those intron sequences. By initial co- transcriptional circularization reaction, all three permutation strategies circularized RNA, and generated about 20-30% circular RNA over total RNAs (including circular RNAs and linear RNAs) (FIG. 6B). As shown in FIG. 6C, after post-refolding ribozyme activation, circular RNA from the construct comprising the intron fragments permuted at position V2 (Twort_ORF142_v2) increased to over 75% circular RNA over total RNAs (including circular RNAs and linear RNAs) (FIG. 6C).The new intron sequences were further tested for self-splicing and nicking activities at different temperatures ranging from 30°C to 60°C. FIG. 7A - FIG. 7D are representative gel images showing RNA circularization of the new intron sequences at different temperatures (post- refolding and ribozyme activation). Similarly, the “% spliced” circRNA over total RNAs for each new intron post refolding ribozyme activation was measured and the “% nicked RNA (post-refolding ribozyme activation) for each intron was measured and subtracted. The overall % intact circRNA was measured for each intron sequence at each temperature (as shown in Table 3).

Table 3: Overall intact circRNA yield (% circRNA over total RNAs (post-refolding ribozyme activation) at different temperature ranges

[0472] The data showed that different self-splicing intron sequences have different optimal activation temperatures (Table 4).

Table 4: Optimal temperature for intron self-splicing activity

Example 4: Permuted Intron-exon (PIE) constructs without homology arms [0473] This study tested circularization efficiency of the constructs that removed the 5’ and 3’ homology arms (“-/OHE” (as illustrated in FIG. 2B-FIG. 2C).

[0474] The four construct designs described in Example 1 (as illustrated in FIG. 2A-FIG. 2D): (1) both OHE/H E (“OHE/IHE”, FIG. 2A), (2) UTE only, no OHE (“-/IHE”, FIG. 2B), (3) no OHE or IHE (“-/-“, FIG. 2C), and (4) OHE only, no IHE (“OHE/-”, FIG. 2D) were prepared and co-transcriptionally circularized. Each construct included “US” and “DS” sequences from Twort-ORF142 intron or Oscillator ia-splendida-trnL intron. FIG. 8A and FIG. 8B show RNA circularization efficiency of using the upstream and downstream intron sequences from Twort-ORF142 intron or from Oscillator ia-splendida-trnL intron. FIG. 8A is a representative gel image showing circular RNA formed by initial co-transcriptional circularization (IVT) and after post-refolding ribozyme activation. The circularization efficiency was measured for each construct. As shown in FIG. 8B, the PIE method using TwortORF142 intron including only the inner homology elements without 5’ and 3’ homology arms (i.e., “IHE/-”) had high RNA circularization: 70.5% circularization by direct IVT, and 81.7% circularization after post-refolding ribozyme activation. For the Twort- ORF142 intron, the construct that only included inner homology element sequences without 5’ and 3’ homology arms (i.e., “IHE/-”) had higher percentages of circularization as compared to the constructs including 5’ and 3’ homology arms, i.e., outer homology elements (OHEs) (FIG. 8B).

[0475] Table 5 lists exemplary upstream and downstream sequences used in precursor RNA constructs.

Table 5: Exemplary upstream and downstream sequences used in precursor RNA constructs

EQUIVALENTS AND SCOPE

[0476] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims:

Claims

1. A nucleic acid molecule for making a circular RNA, comprising, from 5’ to 3’ end, i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, ii) a nucleic acid sequence of interest, and iii) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same Group I or Group II intron as the upstream intron sequence, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are each derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

2. The nucleic acid molecule of claim 1, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment retain a catalytic core of the Group I or Group II intron.

3. The nucleic acid molecule of claim 1, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from variants or truncations of any one of SEQ ID NO:

7, SEQ ID NO: 8, or SEQ ID NO: 9.

4. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule does not include a 5’ or 3’ homology arm.

5. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence of interest and/or a 3’ untranslated region (UTR) sequence between the sequence of interest and the downstream intron sequence.

6. The nucleic acid molecule of claim 5, wherein the 5’ spacer sequence comprises an inner homology element.

7. The nucleic acid molecule of claim 5, wherein the 5’ spacer sequence does not comprise an inner homology element.

8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule does not include the 5’ spacer sequence.

9. The nucleic acid molecule of any one of the preceding claims, wherein the nucleic acid molecule further comprises an internal ribosome entry site (IRES) between the 5’ spacer sequence and the sequence of interest.

10. The nucleic acid molecule of any one of the preceding claims, wherein the nucleic acid molecule comprises a 3’ UTR between the sequence of interest and the downstream intron sequence.

11. The nucleic acid molecule of any one of claims 1-10, wherein the upstream and downstream intron sequences each correspond to the 3’ and 5’ intron splicing fragments of SEQ ID NO: 7.

12. The nucleic acid molecule of claim 11, wherein the upstream intron sequence comprises a sequence corresponding to positions 119 to 282 of SEQ ID NO. 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-118 of SEQ ID NO: 7.

13. The nucleic acid molecule of claim 11, wherein the upstream intron sequence comprises a sequence corresponding to positions 140 to 282 of SEQ ID NO. 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7.

14. The nucleic acid molecule of claim 11, wherein the upstream intron sequence comprises a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1 to 146 of SEQ ID NO: 7.

15. The nucleic acid molecule of any one of claims 1-12, wherein the upstream intron sequence comprises a sequence at least 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2.

16. The nucleic acid molecule of claim 15, wherein the upstream intron sequence comprises the sequence of SEQ ID NO: 1, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 2.

17. The nucleic acid molecule of any one of claims 1-10, wherein the upstream and downstream intron sequences each correspond to the 3’ and 5’ splicing fragments of SEQ ID NO: 8.

18. The nucleic acid molecule of claim 17, wherein the upstream intron sequence comprises a sequence corresponding to positions 112-231 of SEQ ID NO: 8, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-111 of SEQ ID NO: 8.

19. The nucleic acid molecule of claim 17 or 18, wherein the upstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 4.

20. The nucleic acid molecule of claim 19, wherein the upstream intron sequence comprises the sequence of SEQ ID NO: 3, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 4.

21. The nucleic acid molecule of any one of claims 1-10, wherein the upstream and downstream intron sequences each correspond to the 3’ and 5’ splicing fragments of SEQ ID NO: 9.

22. The nucleic acid molecule of claim 21, wherein the upstream intron sequence comprises a sequence corresponding to positions 127-264 of SEQ ID NO: 9, and wherein the downstream intron sequence comprises a sequence corresponding to positions 1-126 of SEQ ID NO: 9.

23. The nucleic acid molecule of claim 21 or 22, wherein the upstream intron sequence comprises a sequence at least 85%, 90%, 95%, 99%, or 99% identical to SEQ ID NO: 5, and wherein the downstream intron sequence comprises a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

24. The nucleic acid molecule of claim 23, wherein the upstream intron sequence comprises the sequence of SEQ ID NO: 5, and wherein the downstream intron sequence comprises the sequence of SEQ ID NO: 6.

25. The nucleic acid molecule of any one of claims 1-24, wherein the nucleic acid molecule is a DNA vector.

26. The nucleic acid molecule of any one of claims 1-24, wherein the nucleic acid molecule is a linear RNA.

27. The nucleic acid molecule of claim 26, wherein the linear RNA is unmodified.

28. The nucleic acid molecule of claim 26, wherein the linear RNA is modified.

29. The nucleic acid molecule of claim 28, wherein the linear RNA comprises one or more modified nucleotides N1 -methylpseudouridine.

30. The nucleic acid molecule of claim 28, wherein the linear RNA comprises one or more modified nucleotides 5-methoxyuridine.

31. The nucleic acid molecule of claim 28, wherein the linear RNA comprises one or more modified nucleotides m5C.

32. The nucleic acid molecule of any one of claims 1-31, wherein the sequence of interest is a non-coding sequence.

33. The nucleic acid molecule of any one of claims 1-31, wherein the sequence of interest is a coding sequence.

34. The nucleic acid molecule of claim 33, wherein the sequence of interest encodes a protein.

35. The nucleic acid molecule of claim 34, wherein the protein is a chimeric antigen receptor (CAR), a T cell receptor, a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.

36. The nucleic acid molecule of claim 35, wherein the protein is an antigen derived from a pathogen, or specific to a tumor.

37. The nucleic acid molecule of claim 35, wherein the CAR comprises an antigen binding domain, a hinge, a transmembrane domain, and one or more intracellular signaling domains.

38. The nucleic acid molecule of any one of the preceding claims, wherein self-splicing of the upstream and downstream Group I or Group II intron sequences makes a circular RNA.

39. A circular RNA that is made from the nucleic acid molecule of any one of claims 1-38.

40. A composition comprising a circular RNA of claim 39.

41. A composition comprising the circular RNA of claim 40 formulated in a delivery vehicle.

42. The composition of claim 41, wherein the delivery vehicle is a lipid nanoparticle.

43. The composition of claim 41, wherein the lipid nanoparticle is conjugated to a targeting moiety.

44. A method for making a circular RNA using the nucleic acid molecule of any one of claims 1-38.

45. A method for making a circular RNA comprising circularizing a nucleic acid molecule comprising, from 5’ to 3’ end, i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment, ii) a nucleic acid sequence of interest, and iii) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same Group I or Group II intron as the upstream intron sequence, wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and wherein transcribing the nucleic acid molecule to a linear precursor RNA and circularizing the linear precursor RNA into the circular RNA occurs in one reaction.

46. The nucleic acid molecule of claim 45, wherein the nucleic acid molecule does not include a 5’ or 3’ homology arm.

47. The method of claim 46, wherein the nucleic acid molecule further comprises a 5’ spacer sequence between the upstream intron sequence and the sequence of interest and/or a 3’ untranslated region (UTR) sequence between the sequence of interest and the downstream intron sequence.

48. The nucleic acid molecule of claim 47, wherein the 5’ spacer sequence comprises an inner homology element.

49. The nucleic acid molecule of claim 47, wherein the 5’ spacer sequence does not comprise an inner homology element.

50. The nucleic acid molecule of claim 47, wherein the nucleic acid molecule does not include the 5’ spacer sequence.

51. The method of any one of claims 45-50, wherein the nucleic acid molecule further comprises an internal ribosome entry site (IRES) between the 5’ spacer sequence and the RNA sequence.

52. The method of any one of claims 45-51, wherein the nucleic acid molecule is a DNA vector.

53. The method of any one of claims 45-51, wherein the nucleic acid molecule is a linear RNA.

54. The method of any one of claims 45-53, wherein the method comprises incubating the nucleic acid molecule at a temperature at which RNA circularization occurs.

55. The method of claim 54, wherein the temperature is about 30°C to 60°C.

56. The method of any one of claims 45-55, wherein the upstream and downstream intron sequences correspond to the 3 ’and 5’ intron splicing fragments of SEQ ID NO: 7, respectively.

57. The method of claim 56, wherein the nucleic acid comprises: i) the upstream intron sequence comprising a sequence corresponding to positions 119 to 282 of SEQ ID NO:7, and the downstream intron sequence comprising a sequence corresponding to positions 1-118 of SEQ ID NO: 7; ii) the upstream intron sequence comprising a sequence corresponding to positions 140 to 282 of SEQ ID NO. 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 139 of SEQ ID NO: 7; or iii) the upstream intron sequence comprising a sequence corresponding to positions 147 to 282 of SEQ ID NO: 7, and the downstream intron sequence comprising a sequence corresponding to positions 1 to 146 of SEQ ID NO:7.

58. The method of claim 57, wherein the nucleic acid comprises the upstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 1, and the downstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO. 2.

59. The method of any one of claims 56-58, wherein the circularization occurs at 35°C to 42°C.

60. The method of any one of claims 45-55, wherein the nucleic acid comprises the upstream and downstream intron sequences each corresponding to the 3 ’and 5’ splicing fragments of SEQ ID NO. 8.

61. The method of claim 60, wherein nucleic acid comprises the upstream intron sequence comprising a sequence corresponding to positions 112-231 of SEQ ID NO: 8, and the downstream intron sequence comprising a sequence corresponding to positions 1-111 of SEQ ID NO: 8.

62. The method of claim 60 or 61, wherein the nucleic acid comprises the upstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 3, and the downstream intron sequence that is at least 85%, 90%, 95%, 99%, or 100% identical to the sequence of SEQ ID NO: 4.

63. The method of any one of claims 60-62, wherein the circularization occurs at a temperature between 35°C to 45°C.

64. The method of any one of claims 45-55, wherein the nucleic acid comprises the upstream and downstream intron sequences each corresponding to the 3’ and 5’ splicing fragments of SEQ ID NO. 9.

65. The method of claim 64, wherein the nucleic acid comprises the upstream intron sequence comprising a sequence corresponding to positions 127-264 of SEQ ID NO: 9, and the downstream intron sequence comprising a sequence corresponding to positions 1-126 of SEQ ID NO: 9.

66. The method of claim 64 or 65, wherein the nucleic acid comprises the upstream intron sequence comprising a sequence at least 85%, 90%, 95%, 99%, or 99% identical to SEQ ID NO: 5, and the downstream intron sequence comprising a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6.

67. The method of any one of claims 64-66, wherein the circularization occurs at 35°C to 42°C.

68. The method of any one of claims 45-67, wherein the RNA of interest encodes a chimeric antigen receptor (CAR), a therapeutic protein, an enzyme replacement protein, an antigen, or an antibody.

69. The method of claim 68, wherein the RNA of interest encodes an antigen.

70. The method of claim 53, wherein the linear RNA is unmodified.

71. The method of claim 53, wherein the linear RNA is modified.

72. The method of claim 71, wherein the linear RNA comprises one or more modified nucleotides N1 -methylpseudouridine and/or 5-methoxyuridine.

73. The method of any one of claims 45-72, wherein the method makes intact circular RNA.

74. A circular RNA made by the method of any one of claims 45-72.

75. A method for expressing a protein of interest in a subject comprising delivering to the subject the circular RNA of claim 74.

76. A method of expressing a protein of interest in a subject comprising delivering to the subject a circular RNA transcribed from a nucleic acid, wherein said nucleic acid comprises the following elements:

(i) an upstream Group I or Group II intron sequence corresponding to a 3’ intron splicing fragment,

(ii) 5’ spacer,

(iii) an internal ribosome entry sequence (IRES),

(iv) a sequence that encodes the protein of interest,

(v) a 3 ’ UTR sequence, and (vi) a downstream Group I or Group II intron sequence corresponding to a 5’ intron splicing fragment of the same intron as the upstream intron sequence, and wherein the 3’ intron splicing fragment and the 5’ intron splicing fragment are derived from any one of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and wherein the nucleic acid does not include a 5’ or 3’ homology arm.

77. The method of claim 75 or 76, wherein the circular RNA is formulated in a delivery vehicle.

78. The method of claim 77, wherein the delivery vehicle is a lipid nanoparticle.

79. The method of claim 78, wherein the lipid nanoparticle is conjugated to a targeting moiety.

80. The method of any one of claims 75-79, wherein the subject is a human subject.