Compositions and methods for preparing capped circular RNA molecules

Abstract

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

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 355,456, filed on June 24, 2022, and U.S. Provisional Application No. 63 / 480,291, filed on January 17, 2023, the disclosures of which are hereby expressly incorporated by reference herein.

[0002] Incorporation by Reference of Electronically - Provided Sequence Listing This application includes a sequence listing submitted as an electronic text file named "21 - 1401 - WO_SequenceListing.xml", created on June 26, 2023, and having a size of 24,183 bytes. The information contained in this electronic file is hereby incorporated by reference in its entirety herein.

Background Art

[0003] Background RNA therapeutics have recently been rapidly evolving as a field, as evidenced by the recent clinical validation of successful mRNA vaccines against SARS-CoV-2. See Polack et al., 2020, Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 383, 2603-2615 (Non-Patent Document 1), Lombardi et al., 2021, Mini Review Immunological Consequences of Immunization With COVID-19 mRNA Vaccines: Preliminary Results. Front. Immunol. 12, 657711 (Non-Patent Document 2). The inherent programmability and relative ease of production of mRNA underlie its potential to replace traditional protein-based therapeutics. See Sahin et al, 2014, mRNA-based therapeutics-developing a new class of drugs, “Nature reviews Drug discovery 13:759-780 (Non-Patent Document 3). In addition to the demonstrated clinical use exemplified by COVID vaccines, mRNA has been experimentally used to express angiogenic factors and generate vaccines against influenza and Zika virus (Zangi et al., 2013, Nature biotechnology 31:898 (Non-Patent Document 4), Bahl et al., 2017, Molecular Therapy 25:1316-1327 (Non-Patent Document 5), Richner et al., 2017, Cell 168:1114-1125 (Non-Patent Document 6)).

[0004] mRNA is also a newly emerging therapeutic modality due to its ability to rapidly produce the protein of interest (POI) in vivo. Some of the main advantages of mRNA as a platform are its programmability, ability for transient nature, ease of production, and lack of risk of genomic integration compared to DNA-based therapeutic approaches. In eukaryotic cells, canonical mRNA is linear and contains a 5’ 7-methylguanosine cap (m 7 G) and a 3’ poly(A) tail, both of which are essential for efficient translation in the cell. Recent studies have described that circular mRNA (circRNA) has an increased half-life in cells compared to linear mRNA due to their reduced sensitivity to exonucleases. However, cap-independent translation by circRNA is not as efficient as cap-dependent translation by linear RNA.

[0005] Thus, in this technical field, among other uses, there is still a need for reagents and methods for generating and using compositions comprising circular RNA molecules, particularly circular mRNA molecules, that encode polypeptides such as therapeutic proteins, which bring about useful, particularly therapeutically useful, phenotypic effects on recipient cells.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

[0007] The present invention preferably provides a composition, a reagent, and a method comprising an RNA molecule encoding a polypeptide, wherein the RNA molecule is a circular RNA molecule, particularly a circular mRNA molecule.

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

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

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

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

[0012] In certain embodiments, type 2 and type 1 capped circular RNA molecules comprise pseudouridine, N 1 -methylpseudouridine (m 1 Ψ), 6-methyladenosine (m 6A) It contains one or more modified nucleotides such as 5-methylcytidine, inosine, or any derivatives thereof. In some embodiments, the modified nucleotide includes locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethoxy (2-MOE) sugar backbone, or any derivatives thereof.

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

[0014] In certain embodiments, the type 1 and type 3 capped circular RNA molecules include circular RNAs containing multiple mRNA regions encoding multiple polypeptides. In these embodiments, the type 1 and type 3 capped circular RNA molecules can also include a plurality of RNA oligonucleotides containing a 5' end with a cap structure and a 3' end portion, and a plurality of derivatized nucleotides at positions 5' to each of the mRNA regions encoding a peptide or polypeptide in the circular RNA, and each 3' end of each of the plurality of RNA oligonucleotides is covalently bonded to each of the plurality of derivatized nucleotides. In some embodiments, each mRNA region encoding a peptide or polypeptide includes a 3' polyA sequence, and the polypeptide encodes Cas9, a base editor, or derivatives thereof, or a therapeutic protein.

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

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

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

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

[0019] The present invention also provides a method for generating a type 3 capped circular RNA molecule of this aspect of the present invention, comprising the steps of generating a circular RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region, synthesizing an RNA oligonucleotide comprising a 5′ end containing a cap structure and a 3′ end containing a moiety reactive with the derivatized nucleotide, and reacting the derivatized nucleotide with the 3′ end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the circular RNA, wherein the synthesis of the circular RNA oligonucleotide further comprises the steps of synthesizing an RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme recognition site, and twister ribozyme sequences on both the 5′ and 3′ ends, reacting the RNA oligonucleotide with an enzyme to generate a derivatized nucleotide within the hairpin structure, and using the twister ribozyme sequences to circularize the RNA oligonucleotide.

[0020] The capped circular RNA molecules provided by the present invention advantageously increase the stability and translation efficiency of the peptides and polypeptides encoded thereby. They can be used, inter alia, to facilitate the therapeutic replacement of polypeptide variants encoded by gene polymorphisms, particularly such polymorphisms associated with genetic diseases. The capped circular RNA molecules provided by the present invention can advantageously provide transient expression of the encoded peptide or polypeptide, thus providing a therapeutic flexibility that could not be achieved by conventional gene replacement therapies. The capped circular RNA molecules provided by the present invention have the advantages of other circular RNA processes, including resistance to exonucleases and higher ribosome loading. The capped circular RNA molecules provided by the present invention further advantageously provide translation initiation that is not limited to internal ribosome entry sites (IRES) or translation enhancing elements (TEE), thereby providing a more robust cap-dependent translation initiation. Another advantage of the capped circular RNA molecules provided by the present invention is to replace protein-based therapeutics (i.e., proteins that must be delivered, specifically introduced into target cells in a functional manner, and targeted to appropriate intracellular niches, see Lagasse et al., 2017, F1000 Research 6:113; doi:10.12688 / f1000research.9970.1) with the delivery of RNA encoding the required peptide or polypeptide in a form (capped and circular) that is resistant to exonucleolytic degradation and provides robust expression due to the presence of the eukaryotic cap.

[0021] These and other features, objects, and advantages of the present invention will be better understood from the following description. In the description, reference is made to the accompanying drawings, which form a part hereof and in which are shown, by way of illustration and not of limitation, embodiments of the present invention. The description of the preferred embodiments is not intended to limit the present invention to cover all modifications, equivalents, and alternatives. Accordingly, the appended claims should be referred to in order to interpret the scope of the present invention.

Brief Description of the Drawings

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[0023] Detailed Description A more detailed description of the compositions, reagents, and methods of the invention is provided herein, which is provided to explain and enhance the claims set forth below, but is not intended to replace or be a substitute for them.

[0024] All publications, including but not limited to patents and patent applications cited herein, are hereby incorporated by reference in their entirety as if fully set forth herein.

[0025] Definitions As used herein, the articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means at least one element and may include more than one element.

[0026] "About" is used to provide flexibility to numerical range endpoints by providing that a given value can vary "slightly above" or "slightly below" the endpoint without affecting the desired result. The term "about" associated with a numerical value means that the numerical value can vary by up to plus or minus 5% of the numerical value.

[0027] Throughout this specification, unless the context requires otherwise, the words "comprise" and "include" and variations thereof (e.g., "comprises", "comprising", "includes", "including") are to be interpreted as including the stated integer or step, or group of integers or steps, but not as excluding any other integer or step, or group of integers or steps.

[0028] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0029] The recitation of a range of values herein is merely intended to serve as a concise method of referring individually to each separate value falling within the range, unless otherwise indicated herein. Further, each separate value is incorporated herein as if it were individually recited herein. For example, if a range is described as 1 to 50, values such as 2 to 4, 10 to 30, or 1 to 3 are intended to be explicitly recited in this disclosure. These are merely examples of what is specifically intended, and all possible combinations of numerical values between and including the recited minimum value and the recited maximum value are to be considered as explicitly recited in this disclosure.

[0030] Unless otherwise indicated, all technical and scientific terms used in accordance with this disclosure are to be understood as having the same meaning as commonly understood by one of ordinary skill in the art. Unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular.

[0031] This application discloses and claims compositions, reagents, and methods comprising capped circular RNA molecules, particularly preferably circularized mRNA molecules encoding peptides or polypeptides. As provided herein, the cap used in the capped circular RNA molecules of the invention can comprise 7-methylguanosine (m 7 G), but in addition, among others, the cap analogs described in U.S. Patent Application No. 2020 / 0055891 to Walczak et al., Holstein et al., 2016, Angew Chem. Int. Ed. Engl. 55: 10899-10903, Walczak et al., 2017, Chem. Sci. 8: 260-267, Muttach et al., 2017, J. Org. Chem. 13: 2819-2832) can be incorporated into the circular RNA molecule precursor to produce the capped circular RNA molecules provided herein.

[0032] Capped circular mRNA (QRNA) Despite the progress of circular RNA (circRNA) engineering, current constructs rely on IRES (internal ribosome entry site) or TEE (translation enhancing element)-mediated translation, which are embodiments that enable cap-independent translation (Figure 1A). Linear mRNA is the predominant form of translation in cells (Sonenberg and Hinnebusch, 2009, Cell 136: 731-745) and is generally more efficient than cap-independent translation (Koch et al., 2020, Nat. Struct. Mol. Biol. 27: 1095-1104), and can undergo cap-dependent translation through interaction with eIF4E and other eukaryotic translation initiation factors (Figure 1B).

[0033] As described herein, a "capped circular mRNA" is a circular mRNA characterized by one or more covalent bonds to one or more cap structures (or derivatives thereof). Circular mRNAs can contain all of the canonical elements of linear mRNAs: (1) a cap, (2) a 5' UTR (untranslated region), (3) a protein coding region (CDS), (4) a 3' UTR, and (5) a poly(A) tail. By circularizing these features into a capped circular RNA, it is intended to enhance the half-life (increased nuclease resistance) of the canonical circular mRNA while retaining the benefits of efficient cap-dependent translation such as that of linear mRNAs.

[0034] The RNA embodiments and methods disclosed herein utilize the exonuclease resistance feature of circRNAs while leveraging a strong m7G-cap-dependent translation initiation mechanism. Such features can be achieved through chemical conjugation of a capped oligonucleotide to a circRNA via click chemistry such as copper-catalyzed azide-alkyne cycloaddition (CuAAC) or tetrazine-trans-cyclooctene inverse electron demand Diels-Alder reaction (IEDDA) (Figure 1C). As shown in Figures 1D and 1E, the present invention contemplates two general structures of capped circular messenger RNA (QRNA). Type 1 QRNA and Type 2 QRNA. In Type 1 QRNA, a circular poly-phosphodiester backbone is present and capping is achieved via chemical ligation of a short capped oligonucleotide to an internal handle of the circular mRNA through click chemistry. The 5' cap can contain, for example, 7-methylguanylate that enables efficient translation of the mRNA or an alternative general mRNA cap structure as shown in Mccaffreyanton, 2019, Genetic Engineering & Biotechnology News. 39. In Type 2 QRNA, a continuous mRNA poly-phosphodiester backbone is present and circularization is achieved via chemical conjugation between the 3'-end and the 5'-UTR of the mRNA through click chemistry.

[0035] The various components of circRNA are shown in Figure 2A. The 5’ capping and 3’ poly(A) tailing steps are useful for the production of active synthetic mRNA, and these modifications prevent mRNA degradation and promote translation initiation in eukaryotic cells. As used herein, “capping” means the modification at the 5’ end of mRNA by the addition of a “cap” molecule such as a 7-methylguanosine (m 7 G) cap. Other cap structures and cap modifications described below can be used to optimize translation efficiency.

[0036] Enzymes that can catalyze the reaction of binding a cap molecule to mRNA include, but are not limited to, the Vaccinia capping system (Figure 2C) containing 2’-O-methyltransferase, tRNA guanine transglycosylase (TGT), Faustovirus capping enzyme, and T4-RNA ligase. Capping can also occur during the synthesis of mRNA, which is called co-transcriptional capping.

[0037] As used herein, the term "molecular handle" or "handle" refers to a chemical group that is attached to a nucleotide on an mRNA and forms a covalent bond to another molecule distinct from the mRNA, enabling this other molecule to be attached to the mRNA. The covalent bond can be formed via various suitable functional cross-linking reactions. In some embodiments described herein, the cross-linking reaction is click chemistry. As used herein, the term "click handle" refers to a molecule on an mRNA that can covalently bond to another molecule via a click chemistry reaction. Examples of handles include, but are not limited to, alkyne or azide (when CuAAC is used in click chemistry), or trans-cyclooctene or tetrazine (when IEDDA is used in click chemistry), or hydrazone or oxime, or any equivalent structure thereof. Other cross-linking chemistries are contemplated, including thio-ene and thiol-yne reactions (Escorihuela et al., 2014, Bioconjug. Chem. 25:618-627), phosphate-amine based reactions (El-Sagheer and Brown, 2017, Chem. Commun. 53:10700-10702, Kalinowski et al., 2016, Chembiochem. 17:1150-1155) (shown in FIGS. 5A and 5B, respectively), thiol-yne, amino-yne, and hydroxyl-yne reactions (Worch et al., 2021, Chem Rev. 121(12):6744-6776), as well as other bioconjugation reactions (Gassensmith, https: / / chem.libretexts.org / Bookshelves / Organic_Chemistry / Supplemental_Modules_(Organic_Chemistry) / Reactions / Introduction_to_Bioconjugation , accessed June 23, 2023).

[0038] As used herein, the term "hairpin" or "hairpin oligonucleotide" refers to a single-stranded oligonucleotide having sequences of complementary base pairs at both ends that can form a "stem and loop" structure.

[0039] As used herein and understood in the art, the term "click chemistry" is intended to encompass chemical methods for joining chemical components together that are "easy to perform, have high yields, require no purification or minimal purification, and are versatile in joining diverse structures without the prerequisite of protecting steps", including, but not limited to, joining nucleotides to polypeptides and amino acids to peptides and polypeptides (see, e.g., Hein et al., 2006, Pharm. Res. 10:2216-2230). In current chemical synthesis practice, four major reactions are employed: 1) cycloaddition (including, for example, the most widely used copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes), 2) nucleophilic ring opening (including ring systems containing strained heterocyclic electrophiles), 3) non-aldol carbonyl chemistry (including, for example, hydrazone / oxime ether formation), and 4) carbon multiple bond addition (including, for example, certain Michael additions and the formation of various three-membered rings, particularly by epoxidation). Click chemistry has been found to be particularly useful for macromolecular substances such as proteins and nucleic acids, as exemplified herein.

[0040] As used herein, the term "equivalent structure" means any molecule that is sufficiently structurally similar and performs the same function in a chemical reaction.

[0041] As used herein, the terms "derivatization" or "functionalization" mean the modification of a nucleotide that results in some functional consequences for its chemical properties or reactivity, or both. Both terms are understood to be equivalent to the extent that certain embodiments of capped circular RNA molecules have functions by virtue of the benefits of their derivatization, particularly with respect to the crosslinking-dependent cyclization embodiments provided herein. In some embodiments, the derivatized nucleotide is a nucleotide that is modified to contain a chemical group / handle and can participate in a crosslinking reaction.

[0042] As used herein, the term "QRNA" is intended as a general term meaning capped circular messenger RNA. Various species of circular RNA molecules, particularly the circular mRNA molecules disclosed herein, are specifically encompassed by this term, but these examples are not intended to be limiting.

[0043] In some embodiments, the synthetic pathways of type 1 and type 3 QRNAs enable multiple oligonucleotides containing 5' caps to bind to circular RNA. For example, the circular RNA can contain multiple derivatized nucleotides that can covalently bind to multiple oligonucleotides containing 5' caps. Alternatively, a single circular RNA backbone can encode multiple TGT sites to enable the simultaneous binding of multiple oligonucleotides containing 5' caps to the circular RNA.

[0044] In some embodiments, the capped circular RNA molecule contains an mRNA region encoding one or more peptides or polypeptides.

[0045] Cap modification To optimize the translation efficiency of QRNA, several modifications of the cap structure are contemplated herein. These modifications include including multiple cap structures (cap 0, 1, and 2, Shanmugasundaram et al., 2022, Chem Rec. 22(8):e202200005), N as a terminal modification adjacent to the mRNA cap 6 ,2'-O-dimethyladenosine (m 6including (Sun et al., 2021, Nat Commun. 12(1):4778), using a cap structure with a modified triphosphate bridge (Sun et al., 2021, Nat Commun. 12(1):4778, Wojtczak et al., 2018, J Am Chem Soc. 140(18):5987 - 5999), incorporating locked nucleic acid (LNA)-modified cap analogs (Kore et al., 2009, J Am Chem Soc. 131(18):6364 - 5), introducing cap analogs with alternative functionalities such as light reactivity and click groups (Klocker et al., 2022, Nat Chem. 14(8):905 - 913, Nowakowska et al., 2014, Org. biomol. Chem. 12:4841 - 4847), hydrophobic cap analogs (WO2017066782A1), and others (Wojcik et al., 2021, Pharmaceutics 13(11):1941, Grudzien et al., RNA 10(9):1479 - 1487, Grzela et al., 2023, RNA 29(2):200 - 216).

[0046] In some embodiments, the methyl group in the 7 - methylguanosine (m 7 G) cap structure can be modified to produce 7 - benzylguanosine (Bn 7 G), 7 - chlorobenzylguanosine (ClBn 7 G), and chlorobenzyl - O - ethoxyguanosine (ClBnOEt 7 G). The introduction of one or more locked nucleic acids (LNA), 2'-methoxy (2OMe), and 2 - methoxyethoxy (2MOE) into the m 7 G structure significantly increases mRNA translation. In some embodiments, the cap structure includes, but is not limited to, m 7 G - LNA, LNAm 7 G - LNA, LNAm 7 G - LNAx6, LNAm 7 G - 2OMex6. In some embodiments, the cap structure is m7 Imidazolide diphosphate (m 7 GDP-Im).

[0047] Nucleotide modification In some embodiments, as disclosed and recognized herein, modifying the incorporation of nucleotide type / nucleotide identity, specifically, adenosine (A), guanosine (G), 6-methyladenosine (m 6 A), or non-canonical inosine (I) in mRNA, preferably at the +1 position, is beneficial and increases translation efficiency. In some embodiments, the replacement of some or all uridine residues with N 1 -methylpseudouridine (m 1 Ψ) in mRNA also improves translation. Nucleotides are numbered according to their positions immediately downstream of the cap structure. For example, the cap structure found at the 5' end of eukaryotic mRNA consists of 7-methylguanosine (m 7 G) moiety linked to the first nucleotide (+1 position) of the transcript via a 5'-5' triphosphate bridge.

[0048] Other modified nucleotides include, but are not limited to, pseudouridine, 5-methylcytidine, 2-thiouridine, 5-methoxyuridine, 4-acetylcytidine, xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, digoxigeninylated adenine, digoxigeninylated cytosine, digoxigeninylated guanine, digoxigeninylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouridine, 2-thiocytosine, 2-thiouridine, 5-methyluracil, 4-thiothymidine, 4-thiouridine, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propynylaminocytosine, 5-propynylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propynylaminoadenine, 7-deaza-7-propynylaminoguanine, 8-azadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, ara-adenine, ara-cytosine, ara-guanine, ara-uracil, biotin-16-7-deaza-7-propynylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine3-5-propynylaminocytosine, cyanine3-6-propynylaminouracil, cyanine3-aminoallylcytosine, cyanine3-aminoallyluracil, cyanine5-6-propynylaminocytosine, cyanine5-6-propynylaminouracil, cyanine5-aminoallylcytosine,Cy5-aminoallyl uracil, Cy7-aminoallyl uracil, Dabsyl-5-3-aminoallyl uracil, Desthiobiotin-16-aminoallyl-uracil, Desthiobiotin-6-aminoallyl cytosine, Isoguanine, N1-Ethylpseudouracil, N1-Methoxymethylpseudouracil, N1-Methyladenine, N1-Methylpseudouracil, N1-Propylpseudouracil, N2-Methylguanine, N4-Biotin-OBEA-cytosine, N4-Methylcytosine, N6-Methyladenine, O6-Methylguanine, Pseudoisocytosine, Pseudouracil, Thienocytosine, Thienoguanine, Thienouracil, Xanthosine, 3-Deazaadenine, 2,6-Diaminoadenine, 2,6-Diaminoguanine, 5-Carboxamido-uracil, 5-Ethynyluracil are included, and N6-Isopentenyladenine (i6A), 2-Methyl-thio-N6-isopentenyladenine (ms2i6A), 2-Methylthio-N6-methyladenine (ms2m6A), N6-(cis-Hydroxyisopentenyl)adenine (io6A), 2-Methylthio-N6-(cis-Hydroxyisopentenyl)adenine (ms2io6A), N6-Glycylcarbamoyladenine (g6A), N6-Threonylcarbamoyladenine (t6A), 2-Methylthio-N6-threonylcarbamoyladenine (ms2t6A), N6-Methyl-N6-threonylcarbamoyladenine (m6t6A), N6-Hydroxynorvalylcarbamoyladenine (hn6A), 2-Methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-Dimethyladenine (m62A), and N6-Acetyladenine (ac6A) are also contemplated at the +1 and other positions.,

[0049] In some embodiments, the modified phosphate backbone can be phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphonate (BP), methylphosphonate, or guanidinopropyl phosphoramidate.

[0050] In some embodiments, the introduction of locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), and 2-methoxyethoxy (2-MOE) to the ribose sugar backbone increases mRNA translation. The addition of multiple 2-OMe and 2-MOE modified bases further increases translation. LNA particularly increased expression at the +1 position.

[0051] In some embodiments, the modified sugar can be 2-thioribose, 2,3-dideoxyribose, 2-amino-2-deoxyribose, 2'deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2,3-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene linkage, 2'-O,4'-C-amino linkage ribose, or 2'-O,4'-C-thio linkage ribose.

[0052] These backbone modifications have been shown to affect the nuclease resistance properties of RNA, so stereoisomeric structures are also considered (Iwamoto et al., 2017, Nat. Biotech. 35:845-851, Jahns et al., 2022, Nucleic Acids Res. 50(3):1221-1240).

[0053] Modifications of nucleotides on conventional circRNAs are limited because not all of them are compatible with internal ribosome entry sites (IRES). QRNA translation does not require an IRES and is thus acceptable for a wider range of percentages of modified nucleotides. These modifications can be spiked into the circular backbone at varying percentages (m6A is typically spiked at 5%). Also, "stem" oligos containing a cap, or 5' / 3' UTRs and tails, may be able to tolerate higher percentages of modification. Alternatively, these modifications can be present at different percentages along different regions of the circular RNA backbone (e.g., 5' UTR, or 3' UTR, or CDS, or near the cap structure, or combinations thereof). Furthermore, the "stem" oligos of type 1 QRNA (oligonucleotides containing a cap) are chemically synthesized and potentially acceptable for more complex structures that are difficult to enzymatically incorporate, such as locked nucleic acids (LNAs), 2'-O-methyl nucleotides, peptide nucleic acids (PNAs), morpholinos, and various internal chemical linkers provided herein.

[0054] Peptides and polypeptides encoded by QRNA The polypeptides encoded by the capped circular RNA molecules provided by the present invention include any therapeutically useful polypeptides for the treatment or intervention of any disease process related to or dependent on hereditary or acquired, polymorphic or mutant polypeptide species as a result of environmental disorders or damage. QRNA can encode multiple polypeptides, such as self-amplifying mRNA cassettes, or multiple therapeutic peptides or polypeptides. In some embodiments, the capped circular RNA molecule includes an mRNA region encoding one or more peptides or polypeptides. The multiple polypeptides include multiple copies of the same polypeptide or multiple copies of different polypeptides.

[0055] An IRES, or a self-cleaving peptide such as a T2A sequence, can be present between multiple polypeptide coding sequences on the QRNA. Alternatively, an RNA oligonucleotide containing a cap residue site is located in front of each polypeptide coding sequence, which will ultimately result in a QRNA having multiple cap residue-containing RNA oligonucleotides, ensuring that all coding sequences are efficiently translated.

[0056] Peptides encoded by the capped circular RNA molecules of the present invention can include, but are not limited to, therapeutic peptides or antigenic peptides, particularly antigenic peptides suitable for presentation by antigen-presenting cells to humoral (B cell) or cellular (T cell) immune system cells. In certain embodiments, these antigenic peptides are suitable and effective for use as vaccines. In other embodiments, the antigenic peptides are suitable or effective for suppressing the immune response, for example, in autoimmune diseases or transplant patients. In additional embodiments, the antigenic peptides are suitable and effective for inducing a specific anti-tumor immune response in tumor cells or in attracting cytotoxic natural (natural killer cells) or engineered (e.g., CAR-T) cells. Therapeutic peptides encoded by the capped circular RNA molecules of the present invention can include, but are not limited to, human parathyroid hormone, filgrastim, oxytocin, somatostatin, calcitonin, glucagon, insulin, liraglutide, vasopressin, etc. (see Fosgerau & Hoffman, 2015, Drug Discovery Today 20:122-128, al Musaimi et al., 2021, Pharmaceuticals (Basil) 14:145, Wang et al., 2022, Signal Transduct. and Targeted Therap. 7:1-27).

[0057] In some embodiments, peptides encoded by the capped circular RNA molecules of the present invention can include, but are not limited to, Cas9 or derivatives (Rothgangl et al., 2021, Nat. Biotechnol. 39:949-957) and adenine base editors or other base editors (Gaudelli et al., 2017, Nature 551:464-471), or RNA base editors for the delivery of genome or epigenome editing therapies.

[0058] In some embodiments, the peptides encoded by the capped circular RNA molecules of the invention can be selected from any of several target categories including, but not limited to, biopharmaceuticals, antibodies, vaccines, therapeutic proteins or peptides, cell membrane permeable peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane-bound proteins, nuclear proteins, proteins associated with human diseases, or target moieties.

[0059] Synthesis of QRNA Type 2: The invention also provides a method for generating a type 2 capped circular RNA molecule of this aspect of the invention, comprising synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure, an mRNA encoding a peptide or polypeptide, a derivatized nucleotide located between the cap structure and the mRNA region encoding the polypeptide, and a 3'-end containing moiety, and reacting the derivatized nucleotide with the 3'-end portion to form a covalently linked capped circular RNA molecule.

[0060] Type 1: The invention also provides a method for generating a type 1 capped circular RNA molecule of this aspect of the invention, comprising generating a circular RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region, synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end containing a moiety reactive with the derivatized nucleotide, and reacting the derivatized nucleotide with the 3'-end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the circular RNA. In certain embodiments, the derivatized nucleotide comprises a moiety capable of reacting with the 3'-end portion by bioconjugation chemistry, and the bioconjugation chemistry is click chemistry. Additionally, the circular RNA is generated by ribozyme-mediated splicing, enzymatic ligation, or click chemistry-mediated cyclization.

[0061] Type 3: The present invention also provides a method for generating a capped circular RNA molecule of this aspect of the present invention, the method comprising generating a circular RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region, synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end containing a moiety reactive with the derivatized nucleotide, and reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the circular RNA, wherein the synthesis of the circular RNA oligonucleotide further comprises synthesizing an RNA oligonucleotide comprising an mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme recognition site, and twister ribozyme nucleotides on both the 5' and 3' ends, reacting the RNA oligonucleotide with an enzyme to generate a derivatized nucleotide within the hairpin structure, and circularizing the RNA oligonucleotide using the twister ribozyme sequence.

[0062] The derivatized nucleotides in these three types of QRNA can be generated using different strategies as demonstrated in FIGS. 2D-2I, 4A, and 10D (type 1), FIGS. 3A and 3B (type 2), and FIG. 1F (type 3). That is, the derivatized nucleotides can be specifically targeted by having a hairpin structure containing a specific enzyme recognition site. Enzymes described in some examples are tRNA guanine transferase (TGT). In other examples, the derivatized nucleotide is generated by substitution of a single cytidine with azido-cytidine.

[0063] Circularization of the RNA molecules in Type 1 and Type 3 can be achieved by ligation with T4 ligase, RtcB ligase, or ribozyme-mediated splicing. In these embodiments, the 5' and 3' ends of the linear oligonucleotide contain appropriate moieties to participate in the enzymatic reaction to form circular RNA. Alternatively, click chemistry moieties are also contemplated for circularization. In some embodiments, additional splint probes containing complementary sequences to the 5' and 3' ends of the linear oligonucleotide can be used as demonstrated in Figure 2E or Figure 2G to bring the two ends in proximity and facilitate circularization.

[0064] Pharmaceutical compositions for delivery and methods therefor The present invention provides pharmaceutical compositions comprising the capped circular RNA molecules of the present invention, particularly circularized mRNA molecules. In certain embodiments, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable excipient, and in certain other embodiments, one or more additional therapeutic agents.

[0065] In some embodiments, the composition is suitable for administration to a human subject in need thereof. In the context of the present disclosure, "active ingredient" generally refers to the capped circular RNA molecules described herein, particularly circularized mRNA molecules, and any additional therapeutic agents provided therewith.

[0066] It is generally understood by those skilled in the art that the compositions described herein are also suitable for administration to any non-human subject. Those skilled in the art of veterinary medicine will understand that the pharmaceutical compositions described herein may be suitable for administration to mammals including, but not limited to, primates, cattle, pigs, horses, sheep, goats, cats, dogs, mice, rats, whales, and other mammals. Those skilled in the art of veterinary medicine will also understand that the pharmaceutical compositions described herein may be suitable for administration to chickens, ducks, geese, turkeys, and other domesticated birds, as well as wild birds, particularly those species of birds that are particularly endangered. In addition, those skilled in the art of veterinary medicine will understand that the pharmaceutical compositions described herein may be suitable for administration to a variety of fish including commercially available or wild salmon, tuna, cod, sardines, zebrafish, sharks, and the like.

[0067] The pharmacological compositions described herein can be prepared by any method known or developed in the fields of pharmacology, immunology, virology, or generally biotechnology.

[0068] In some embodiments, the formulation of the pharmacological compositions described herein can include a unit dose of at least one derivatized RNA, particularly a circular mRNA molecule, in addition to at least one other pharmaceutically acceptable excipient. Such excipients can include, but are not limited to, solvents, dispersants, buffers, diluents, surfactants, emulsifiers, isotonic agents, preservatives, thickeners, lubricants, oils, and the like.

[0069] In some embodiments, the pharmacological composition can include a delivery mechanism that further includes lipid nanoparticles. The size of the lipid nanoparticles can be modified to counter an immunogenic response from the subject or to enable an increase in efficacy and pharmacological activity.

[0070] In other embodiments, the pharmacological composition can include a delivery mechanism further comprising a lipidoid as described above in the art. See Akinc et al., 2008, Nat Biotechnol. 26:561-596, Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920, Akinc et al., 2009, Mol Ther. 17:872-879, Love et al., 2010. Proc Natl Acad Sci USA 107:1864-1869, Leuschner et al., 2011, Nat Biotechnol. 29:1005-1010, all of which are hereby incorporated by reference in their entirety. Lipidoids broadly refer to lipid nanoparticles, liposomes, lipid emulsions, lipid micelles, and the like. Lipidoids containing a pharmacological composition comprising derivatized RNA can be administered parenterally by means including, but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, via dialysis fluid, intrathecal injection, or intracranial injection.

[0071] One of ordinary skill in the art will also recognize that there are other nucleotide delivery mechanisms such as the use of virus-like, or virus-derived particles. See Rohovie et al., 2016, Bioengineering & Translational Med. 2(1):43-57. Virus-like particles can contain viral coat proteins or viral capsids. Such particles can be PEGylated or further annealed to compounds that avoid phagocytic clearance. In addition, the surface of virus-like particles can be further functionalized to provide cell-specific targeting, promote extravasation, promote radiolabeling, improve permeability across cell boundaries, or transport across the blood-brain barrier via transcellular transport. Virus-like particles can be derived from animal viruses, bacteriophages, or plant viruses. Examples of suitable viruses for the derivation of virus-like particle delivery mechanisms include, but are not limited to, among other suitable viruses, Cowpea chlorotic mottle virus, Cowpea mosaic virus, Hepatitis B virus (core), Enterobacteria phage MS2, Salmonella typhimurium P22, Enterobacteria phage Qβ. The derivatized RNA payload can be loaded onto virus-like particles by electrostatic adsorption or any other suitable method known to one of ordinary skill in the art.

[0072] Various exemplary embodiments of the compositions and methods according to the present invention are described herein in the following non-limiting examples. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the present invention will become apparent to one of ordinary skill in the art from the foregoing description and the following examples, and will fall within the scope of the appended claims.

Examples

[0073] Example 1. Synthesis of Type 1 Capped Circular RNA The synthesis of type 1 QRNA can be divided into two main steps: the synthesis of a 5'-capped oligonucleotide with a 3'-click chemistry handle, and the synthesis of a circular RNA containing a click chemistry handle corresponding to its untranslated region (the "UTR", i.e., outside of the portion of the RNA that encodes, among other things, a protein or peptide). The 5'-capped oligo is synthesized from an oligonucleotide generated by solid-phase synthesis (having a click handle and a 5'-phosphate incorporated therein), followed by chemically capping the RNA using N7-methylated GDP imidazolide (Figure 2B) (Abe et al., 2022, ACS Chem. Biol. 17:1308-1314), or it can be enzymatically capped using a capping enzyme, such as the vaccinia capping enzyme, together with a guanine methyltransferase (Figure 2C) (Shuman in Progress in Nucleic Acid Research and Molecular Biology, 50:101-129 (Cohn & Moldave, Eds. Academic Press, 1995, Kyrieleis et al., 2014, Structure 22:452-465). CircRNAs containing click chemistry handles were synthesized in multiple ways. In a non-limiting example, a linear mRNA having click handles in its 5' / 3'-UTR was generated through chemical or enzymatic synthesis, and this transcript was designed to contain complementary sequences in its 5' / 3'-UTR to enable cyclization using a single-stranded RNA ligase (T4 RNA ligase) (Figure 2D) (Wang and Ruffner, 2014, Nucleic Acids Res. 26:2502-2504). An example of a QRNA constructed according to the strategy shown in Figure 2D was implemented as shown in Figure 4A and Example 4 below. Alternatively, a click handle-containing transcript can be cyclized using a splint probe complementary to the sequences on its 5' / 3'-ends, enabling cyclization using a double-stranded RNA ligase such as T4 RNA ligase II (Figure 2E) (Chen et al., 2020, Nucleic Acids Res. 48:e54).Circular RNAs having click handles can also be generated through a tandem ligation process, where RNA transcripts containing 5’ / 3’-hydroxyl groups can be synthesized through in vitro transcription (IVT) and ligated to chemically synthesized oligonucleotides having click chemistry handles and phosphate groups on both the 5’ and 3’ ends. 5’-phosphate / 3’-hydroxyl ends can be ligated by T4 RNA ligase, and 5’-hydroxyl / 3’-phosphate ends can be ligated using RNA ligase RctB to obtain the desired circRNA. The ligation reaction can also be sprint-guided to improve the yield (Figure 2G). Alternatively, RNA transcripts can be made by in vitro transcription and have 5’-triphosphate and 3’-hydroxyl. Chemically synthesized oligonucleotides having 5’-phosphate, 3’-phosphate, and derivatized nucleotides are first ligated to the mRNA using T4 RNA ligase (binding the mRNA 3’-hydroxyl to the oligonucleotide 5’-phosphate). The mRNA 5’-triphosphate is then hydrolyzed to 5’-hydroxyl using calf intestinal alkaline phosphatase (CIAP). The mRNA is then circularized by ligating the 5’-hydroxyl and 3’-phosphate using RtcB ligase (Figure 2H). Instead of incorporating click handles before / during RNA cyclization, RNAs having a stem-loop motif can be synthesized through IVT and circularized through ribozyme-mediated back-splicing (Wesselhoeft et al., 2018, Nat Commun. 9:2629), and click handles can be introduced with hairpin modifying enzymes such as tRNA guanine trans-ferase (TGT) (Alexander et al., 2015, J. Am. Chem. Soc. 137:12756-12759) (Figure 2F).

[0074] Another way to combine cotranscriptional cyclization and click handle incorporation is to use synthetic oligonucleotides that are in vitro transcribed using engineered DNA polymerases that contain click handles as primers that anneal to a DNA template, synthesize RNA from the primers (Cozens et al., 2012, Proc. Natl. Acad. Sci. U.S.A. 109, 8067 - 8072), and are subsequently subjected to ligation / backsplicing-based cyclization (Figure 2I). Upon successful synthesis, the circRNA and capped oligo are crosslinked using click chemistry, resulting in type 1 circRNA. As an alternative to the aforementioned strategy, circular mRNA scaffolds can be constructed by chemical cyclization (e.g., RNA cyclized via non-natural / non-phosphodiester chemical linkages), e.g., by incorporation of click handles on the 5’ and 3’ ends of linear RNA. After successful chemical cyclization, this circular scaffold is then further modified by covalent addition of a 7-methylguanosine cap or a cap-containing oligonucleotide.

[0075] Example 2. Synthesis of Type 2 Capped Circular RNA For type 2 QRNA, 5'-capped mRNA transcripts containing an RNA hairpin in the 5'-UTR can be synthesized by IVT and co-transcriptional capping, and the first click chemistry handle (e.g., alkyne) can be introduced using a hairpin-labeling enzyme such as TGT. Subsequently, the second click chemistry is introduced by ligating a chemically synthesized oligo with a 3'-terminal azide handle to the 3'-end of the transcript, and the mRNA can be cyclized intramolecularly using click chemistry to yield type 2 QRNA (Figure 3A). Alternatively, a synthetic oligonucleotide containing a click handle can be used as a primer to anneal onto a DNA template and transcribed in vitro using an engineered DNA polymerase that synthesizes RNA from the primer (Cozens et al., 2012, Proc. Natl. Acad. Sci. U.S.A. 109:8067-8272, Freund et al., 2023, Nat. Chem. 15:91-100). The IVT product is then ligated to a 3'-azide / 5'-phosphate synthetic oligo to enable intramolecular chemical cyclization (Figure 3B).

[0076] Example 3. Synthesis of Type 3 Capped Circular RNA Description of the synthesis strategy: The synthesis scheme for type 3 QRNA is shown in Figures 1F - 1H. A short QRNA encoding the FLAG peptide (DYKDDDDK - SEQ ID NO: 1, D = aspartic acid, Y = tyrosine, and K = lysine) is prepared and tested. The 3xFLAG peptide (DYKDDDDKDYKDDDDKDYKDDDDK - SEQ ID NO: 2) having the sequence shown herein can be substituted or recoded with other reporter peptides (e.g., NanoBiT 11 - amino acid peptide, shown at www.promega.com / products / protein - interactions / live - cell - protein - interactions / nanobit - ppi - starter - systems / ?catNum = N2014) or therapeutic peptides (e.g., in the case of peptide - based vaccines).

[0077] Oligo 3.1 is generated by in vitro transcription and co-transcriptionally cyclized by the twister ribozyme array and RNA ligase RtcB (see Litke et al., 2019, Nature Biotechnol. 37:667-675) to generate circular RNA 3.2. Circular RNA 3.2 is then post-transcriptionally and site-specifically labeled using tRNA guanine transglycosylase (TGT) and a synthetic preQ1 cofactor analog (Figure 1G) to incorporate a 5-methyl-tetrazine handle in a tRNA-like hairpin structure (see Alexander et al., 2015, J. Amer. Chem. Soc. 137:12756-12759), generating circular RNA 3.3. Oligonucleotide 3.4 is prepared as described in Example 1 by replacing 3-azido-2,3-ddUTP with 5-TCO-PEG4-dUTP to generate a capped oligo with a 3’-TCO handle (Figure 1H). Oligo 3.4 is then ligated to circular RNA 3.3 via tetrazine-TCO cyclization, resulting in the desired type 1 QRNA 3.5 encoding 3xFlag. The sequences of oligos 3.1 and 3.4 are shown in Table 1.

[0078] (Table 1) Sequences of synthetic oligonucleotides used in the design of type 3 QRNA TIFF2025519910000002.tif72166 Note : The listed bases are all ribonucleotides unless otherwise specified. AG = m7G(5’)ppp(5’)(2’OMeA)pG (e.g., “CleanCap AG” 5’ cap analog from TriLink Biotechnologies), U = 5-TCO-PEG4-2-deoxyuridine, U = N 1 -methylpseudouridine or uridine.

[0079] Example 4. Capped circular mRNAs of type 1 have higher translation efficiency than uncapped circular mRNAs To facilitate synthesis, purification, and characterization, QRNA constructs using small molecule RNAs were used. The RNA template (Figure 4A) contained 5' and 3' complementary regions (shown as dashed lines) to facilitate annealing and subsequent cyclization (e.g., enzymatic ligation of the 5' and 3' ends). This RNA scaffold contained a short coding sequence encoding a 12 amino acid HiBit tag (shown in blue, MVSGWRLFKKIS - SEQ ID NO: 5), and a short poly(A) region following a coding sequence (CDS) that mimics the structure of linear mRNA. Either the 5' UTR or the 3' UTR contained a single C site for the installation of cytidine triphosphate (CTP)-azide during transcription. Incorporation of this single CTP-azide enabled clicking of the 5' 7-methylguanosine cap oligo onto the circular RNA scaffold.

[0080] The RNA scaffold containing the 5’ C-azide site had the following sequence (5’ to 3’): TIFF2025519910000003.tif18161.

[0081] The RNA scaffold containing the 3’ C-azide site had the following sequence (5’ to 3’): TIFF2025519910000004.tif18160.

[0082] The above sequences functioned as scaffolds for the "circular" coding portion of the tested mRNAs. The underlined 5’ and 3’ regions were complementary and promoted enzymatic cyclization of the RNA by enzymatic ligation. The bold type indicates the protein coding sequence (MVSGWRLFKKIS* - SEQ ID NO: 5) encoding the HiBit tag, where * is the stop codon. In addition, a single C site (shown as a bold uppercase letter) was encoded by this template and was located in either the 5’ or 3’ complementary region. Site-specific azide functionalization in the circular RNA scaffold was achieved only by encoding a single cytidine in these templates (Figure 4A).

[0083] The above RNA was synthesized by in vitro transcription (IVT) from the corresponding DNA template using the following nucleotide triphosphates as precursors: GTP instead of normal CTP, ATP, 5-azido-PEG4-CTP (Jena Bioscience, catalog number: CLK-0523), and N1-methylpseudo-UTP (Jena Bioscience, catalog number: NU-890) instead of UTP. To prevent non-specific azide incorporation within the coding region or other sites within these templates, all internal instances of cytidine were removed except for a single site (shown in bold uppercase).

[0084] The RNA generated by IVT contained a 5’ triphosphate and a 3’ hydroxyl group. After IVT synthesis, the RNA was treated with RNA 5’ pyrophosphohydrolase (RppH) (New England BioLabs, catalog number: M0356S) to generate a 5’ phosphate. These RNAs were then annealed and ligated together at their 5’ and 3’ ends using T4 RNA ligase 1 (New England BioLabs, catalog number: M0437M) to generate a circular “scaffold” that would function as the base of the QRNA structure (Figure 4A). The circularized RNA was separated from contaminants using high performance liquid chromatography (HPLC) purification (Figure 4B) and used as a subsequent scaffold for click functionalization.

[0085] Oligos containing a 5’ 7-methylguanosine cap and a 3’ alkyne group were synthesized (Figure 4A and Figure 4C). Synthetic RNA oligonucleotides with 5’-phosphate and 3’-alkyne were generated through solid-phase synthesis (ordered from IDT, sequence order code: / 5Phos / rArGrArArUrArA / 35OCTdU / ). Synthetic oligos with ammonia as the counterion were dissolved in DMSO and treated with m7GDP-imidazolide in 4% (v / v) 1-methyl-imidazole in DMSO at 100 equivalents for 3 hours at 55 °C and purified by HPLC. This 5’ cap oligo was covalently attached to the circular RNA scaffold using copper-catalyzed azide-alkyne click chemistry (Figure 4D).

[0086] For the expression assay, QRNA expression was compared with various precursors, including a linear template or a cap-oligo covalently attached to a non-capped circular template (Figure 4D). QRNA and related synthetic precursors (shown as 1 - 6 in Figure 4D) were transfected into HeLa cells using Lipofectamine (ThermoFisher Scientific, catalog number: LMRNA001), and bioluminescence was measured 8 hours after transfection using the Hibit lysis assay (Promega, catalog number: N3030). A significant enhancement of translation was observed when the linear precursor was conjugated to an oligo capped on the 5’-end rather than the 3’-end, indicating that the triazole-linked cap induced an enhancement of translation efficiency (TE) (1, 3, 4 shown in Figure 4F). Similarly, increased luminescence was achieved when the circular RNA was conjugated to a capped oligo, where the more upstream click handle in the original 3’-UTR was superior to that in the more proximal 5’-UTR (2, 5, 6 shown in Figure 4E), and QRNA was identified as a viable strategy for improving circRNA translation.

[0087] Example 5. Cross-linking reaction contemplated for the synthesis of capped circular RNAs The experiments described in this disclosure demonstrate the synthesis of branched poly(A) oligonucleotide mRNAs. Similar conditions described herein can be applied to chemical conjugation and intramolecular conjugation in the synthesis of type 1 and type 2 QRNAs, respectively. Chemically modified poly(A) oligonucleotides were obtained from Integrated DNA Technologies and suspended in RNase-free water to a final concentration of 100 uM.

[0088] For thiol-ene / incoupling conjugation (an organic reaction between a thiol (R-SH) and an alkene (R2C=CR2) or alkyne that forms a thioether (R-S-R’) with the chemical structure demonstrated in Figure 5A), the disulfide-protected thiol-oligo was deprotected with 100 molar excess of TCEP (tris(2-carboxyethyl)phosphine) and immediately mixed with an equimolar alkene / alkyne-modified oligo and incubated at 37 °C for 30 minutes to 1 hour. When radical conditions were used, a sub-stoichiometric amount of 2,2-dimethoxy-2-phenylacetophenone was added and incubated at room temperature under 370 nm irradiation (Kessil, catalog number: KSPR160L370). The sizes of the products and precursors of the crude thiol-ene / ino oligonucleotide conjugation of a 15 nt model substrate containing only one conjugation handle are shown in Figure 5C.

[0089] For amine-phosphate conjugation (upper panel, Figure 5B), the oligo was mixed with 1 - 2 equivalents of EDC as an additive in an excess amount of imidazole. The reaction mixture was incubated at 37 °C for 30 minutes to 1 hour.

[0090] Regarding IEDDA (Inverse-demand Diels-Alder reaction) conjugation, methyltetrazine (Me-Tz) and trans-cyclooctene (TCO)-labeled oligos were obtained from the corresponding amine-modified oligos with tetrazine-PEG5-NHS ester (Click Chemistry Tools, catalog number: 1143) or TCO-PEG4-TFP ester (Click Chemistry Tools, catalog number: 1198) at a molar ratio of 500:1 (small molecule: oligonucleotide) in 100 mM NaHCO3 at 4 °C overnight. The NHS-labeled products were purified using ethanol precipitation. The Me-Tz / TCO-labeled oligos were suspended in RNase-free water and incubated at 55 °C for 30 minutes.

[0091] In the CuAAC (copper-catalyzed azide-alkyne conjugation) conjugation, azide / alkyne-containing oligonucleotides were mixed at the indicated molar ratios (1:1 for 1-branched oligos, 2:1 for 2-branched oligos, 3:1 for 3-branched oligos). The sizes of the products and precursors of CuAAC and IEDDA 30 nt oligonucleotides having three EU / TCO handles reacting with 30 nt N3 / Tz-modified oligos are shown in Figure 5D. The branched oligo structures are shown in Figure 6. The oligonucleotide mixture was diluted in modified 1.5× click chemistry buffer (Lumiprobe, catalog number: 61150, containing 5% SUPERase inhibitor, 5% DMSO, and 5% 10 mM dNTP mixture [ThermoFisher Scientific, catalog number: 18427089]) that was briefly degassed by argon purging for 20 minutes before the reaction. For a typical 100 μL reaction, 33 μL of the oligonucleotide solution was mixed immediately before the reaction with 66 μL of click chemistry buffer and 2 μL of 100 mM L-ascorbic acid solution (Sigma Aldrich, catalog number: A5960). The mixture was incubated at 37 °C for 1 hour and the reaction was stopped by the addition of 1 μL of 500 mM EDTA (pH 8.0). The reaction was first purified using the Monarch RNA Cleanup Kit (NEB, catalog number: T2040), and the crude product was repurified using RNase-free HPLC on an Agilent 1260 Infinity II HPLC with acetonitrile [Sigma Aldrich, 34851] and 100 mM hexylamine / acetic acid (pH 7.0, containing 10% urea w / v) as the mobile phase. The HPLC fractions were analyzed by Novex TBE Urea gel, stained with 1× SYBR Gold (ThermoFisher Scientific, catalog number: S11494), and visualized using a BioRad ChemiDoc MP Imaging System (catalog number: 12003154). Next, the desired fractions were pooled, desalted, and concentrated using the Monarch RNA cleanup kit for small-scale preparations or ethanol precipitation for large-scale preparations.

[0092] Example 6. Covalently linked internal caps drive robust translation in linear and circular RNAs Systematic investigation of 5’ modifications for mRNA translation Conventional approaches for preparing capped therapeutic mRNAs include enzymatic or co-transcriptional capping. For enzymatic capping, mRNA transcripts are treated with capping enzymes and methyltransferases after in vitro transcription (IVT) (Ramanthan et al., 2016, Nucleic Acids Res. 44:7511-7526). Co-transcriptional capping is achieved by spiking a synthetic cap analog into the IVT reaction. The first-generation dinucleotide cap analog m 7 G(5’)ppp(5’)G results in an unintended “reverse” orientation of the m 7 G cap, and this problem was solved by the adoption of anti-reverse cap analogs (ARCA) (Grudzien-Nogalska et al., in Methods in Enzymology (Academic Press, 2007), 431:203-227, Stepinski et al., 2001, RNA 7:1486-1495). The recent development of trinucleotide / tetranucleotide cap analogs for the direct incorporation of cap-1 / cap-2 structures and most cap modifications has been screened using tri / tetranucleotides (Ishikawa et al., 2009, Nucleic Acids Symp Ser. 53:129-130, Sikorski et al., 2020, Nucleic Acids Res. 48:1607-1626, Jurga et al., Messenger RNA Therapeutics (Springer Nature, 2022)). However, neither method provides access to modifications beyond the first two bases due to the different incorporation efficiencies of various cap structures, introducing potential bias in screening. In addition, purification of capped mRNA from uncapped mRNA has not been easily achieved considering the similar physicochemical properties of both species.

[0093] To address these issues, the capping step was separated from mRNA synthesis. Oligonucleotides with defined chemical modifications were readily synthesized on solid phase and were subsequently chemically capped using guanosine diphosphate imidazolide (mGDP-Im) derivatives (Abe et al., 2022, ACS Chem. Biol. 17:1308-1314). Modifying the oligonucleotide counterion to ammonium enabled robust capping without any divalent ion additives, and fine-tuning of the reverse-phase high-performance liquid chromatography (RP-HPLC) gradient with more hydrophobic hexylammonium ions enabled isolation of large-scale 100% capped products (Figs. 7A-7D). These capped oligos were then ligated to 5'-monophosphorylated mRNA containing N-methylpseudouridine (mΨ) generated from IVT, followed by treatment with bacterial RNA 5'-pyrophosphohydrolase (RppH). 7 G guanosine diphosphate imidazolide (m 7 GDP-Im) derivatives (Abe et al., 2022, ACS Chem. Biol. 17:1308-1314). Modifying the oligonucleotide counterion to ammonium enabled robust capping without any divalent ion additives, and fine-tuning of the reverse-phase high-performance liquid chromatography (RP-HPLC) gradient with more hydrophobic hexylammonium ions enabled isolation of large-scale 100% capped products (Figs. 7A-7D). These capped oligos were then ligated to 5'-monophosphorylated mRNA containing N-methylpseudouridine (m 1 -methylpseudouridine (m 1 Ψ) generated from IVT, followed by treatment with bacterial RNA 5'-pyrophosphohydrolase (RppH).

[0094] The modularity of this workflow enables the construction and evaluation of a diverse array of mRNAs with a series of cap and 5' UTR modifications (Fig. 8A). mRNA structures were classified into four dimensions: (1) first base identity, (2) phosphodiester bond, (3) sugar backbone, and (4) cap modification. First, the effect of nucleotide identity at the “+1 position” on translation was evaluated, where protein yields varied in the order A>G~C~U as reported in the literature (Sikorski et al., 2020, Nucleic Acids Res. 48:1607-1626). Changing A to m 6 A further enhanced the expected total protein production, and interestingly, incorporation of non-canonical inosine (I) showed a similar effect (Fig. 8B). Subsequently, m 7 G-G, the “wild-type” base identity in ARCA-capped synthetic mRNA, was used as a benchmark for subsequent screening, and m 7 G-A was used as a control construct for other aspects of mRNA modification.

[0095] Regarding the phosphodiester bond, the introduction of phosphorothioate (PS) into the cap triphosphate bridge has been previously reported to improve protein yield (Kawaguchi et al., 2020, Angew. Chem. Int. Ed Engl. 59:17403 - 17407), and the introduction of PS between the +1 and +2 positions decreased translation. Further introduction of PS at positions +1 to +7 rescued translation to normal levels but was still not beneficial (Figure 8C).

[0096] Regarding modifications on the ribose sugar backbone, substituting adenosine 2'-hydroxyl with 2'-deoxyfluoro (2FA) inhibited translation. Switching to the chirality-inverted L-adenosine (LA) or 2'-deoxyadenosine (dA) resulted in non-significant changes in expression. The introduction of locked nucleic acid (LNA), 2'-methoxy (2OMe), and 2-methoxyethoxy (2MOE) significantly increased mRNA translation, and the introduction of a single LNA base resulted in a 4.8-fold increase. Extending the dA backbone to positions +1 to +6 did not significantly modify expression, but increasing the number of 2OMe and 2MOE modified bases resulted in 6.9-fold and 5.4-fold increases, respectively, at 24 hours. However, LNA increased expression only at the +1 position, and modifying the position from +1 to +6 resulted in a decrease in activity. (Figure 8D)

[0097] For the evaluation of cap modifications, the inventors synthesized the m 7 GDP-Im analog. The cap structure was modified by substituting the m 7 G methyl group with benzyl (Bn 7 G) and chlorobenzyl (ClBn 7 G), or by the LNA sugar backbone (m 7modified by having (Kore et al., 2009, J. Am. Chem. Soc. 131: 6364 - 6365, Wojcik et al., 2021, Pharmaceutics 13(11): 1941). The inventors also used a structure previously developed as a high - affinity eIF4E inhibitor rather than as an mRNA cap, chlorobenzyl - O - ethoxy (ClBnOEt 7 G) included (Chen et al., 2012, J. Med. Chem. 55: 3837 - 3851). In contrast to previous reports, all aromatic substitutions did not show performance superior to m 7 G, but all successfully induced translation compared to uncapped mRNA. This discrepancy may be due to the fact that these hydrophobic modifications result in better isolation of capped mRNA during purification after co - transcriptional capping, and the inventors also observed a larger retention time shift in HPLC according to recent reports (Inagaki et al., 2023, Nat. Commun. 14: 2657). m 7 G - LNA actually succeeded in enhancing translation 4.5 - fold in 24 hours (Figure 8E). The inventors then demonstrated that these modifications from different dimensions could be combined to further amplify the effect, and the simultaneous introduction of m 7 G - LNA + LNA at the +1 base or 2OMe on +1 to +6 further enhanced translation 8.6 - fold and 7.5 - fold, respectively (Figure 8F). Furthermore, chemical modifications of the 5′ cap and downstream nucleotides can be combinatorially optimized to potentially modify mRNA translation by maximizing the affinity for eIF4E or increasing the resistance to decapping by hDcp2 (Figure 8G - H).

[0098] Internal capping drives robust translation on circRNA Since the additional internal cap structure enhanced translation on linear transcripts, the inventors sought to apply this approach to drive the translation of circRNAs. Conventionally, circRNAs lack a cap and poly(A) tail and require an IRES for translation initiation (Figure 10A). However, the initiation rate by IRES is known to be slower than that of the canonical cap-dependent mechanism (Koch et al., 2020, Nat. Struct. Mol. Biol. 27:1095 - 1104). The branched cap was unable to prevent exonuclease degradation of the uncapped mRNA “stem” (Figure 9B), while circRNAs have previously been reported to have enhanced exonuclease resistance and stability in vivo (Wesselhoeft et al., 2018, Nat. Commun. 9:2629, Chen et al., 2022, Nat. Biotechnol. 41(2):262 - 272). Thus, the internal capping strategy can simultaneously maintain the high stability of circRNAs while hijacking the cap-dependent translation initiation mechanism to enhance its translatability (Figure 10B and Figure 10C). The inventors named such capped circular mRNAs QRNAs due to their similarity to the letter “Q” of the construct.

[0099] Regarding the synthesis of QRNAs, simultaneously achieving RNA cyclization and incorporating site-specific click chemistry handles is a major challenge. As a first proof-of-concept, the inventors synthesized the minimal RNA encoding HiBiT, engineered its sequence to contain only a single cytosine in its UTR, and enabled the introduction of an azide handle by substituting CTP with azide-labeled CTP (5-azido-PEG4-CTP) during IVT. This minimal mRNA was then cyclized by T4 RNA ligase with the assistance of homology regions in the 5’ and 3’ UTRs, resulting in an azide-labeled circRNA, which was purified by HPLC and confirmed to be RNase R resistant. Then, m 7G-capped OU-labeled oligos were conjugated to azido-circRNA to yield minimal QRNA (Figure 4A–C). Encouragingly, enhanced translation was observed in QRNA compared to its circRNA precursor (Figure 4D–4F).

[0100] To generalize QRNA synthesis to longer transcripts, alternative workflows for nucleotide depletion are needed. For this purpose, we synthesized Nano luciferase (Nluc) encoding mRNA and cyclized it through intron backsplicing, a standard approach for circRNA preparation. For the incorporation of click chemistry handles, pre-queuosine 1 (preQ1) was used to introduce a minimal hairpin sequence upstream of the CDS that can be site-specifically recognized and labeled by tRNA guanine transglycosylase (TGT) (Ehret et al., 2018, Mol. Pharm. 15:737–742). Utilizing TGT and synthetic preQ1-azide, we introduced a single azide handle into the circRNA and conjugated it with a 5′-capped oligo containing a single 5-octadiynyl dU site (OU) (Figure 10D). To enrich the QRNA abundance, a hydrophobic Bn 7 G-cap analog was used to enhance its interaction on RP-HPLC and the enriched product was characterized by duplex RNase H assay (Figure 10E). Still not as efficient as normal m 7 G-capped linear mRNA, QRNA translated more effectively than its circRNA counterparts (with and without IRES) (Figure 10F). On the other hand, the synthesis of circRNA precursors still 1 depends on ribozyme splicing, which is incompatible with Ψ, so QRNA was also only compatible with uridine, while linear mRNA expression 1Significantly improved after Ψ replacement (Figure 10G). Such differences are not due to the intrinsic translatability of the QRNA constructs, but the toxicity of uridine as the expression of co-transfected control Fluc mRNA was also significantly reduced when uridine-containing linear / QRNA was transfected (Figure 10G). As a result, only the multi-capped linear mRNA 1 Due to its compatibility with Ψ and to retain higher therapeutic value, it was continued up to animal experiments.

[0101] Materials and Methods Plasmid cloning, characterization, and purification (linear + circ): mRNA expression vectors were generated as described above. Briefly, the sequence encoding the protein of interest (CDS) was inserted into an optimized backbone containing (in order) the T7 promoter sequence, 5’ human alpha-globin UTR, CDS, 3’ human alpha-globin UTR, 100×A template-encoded poly(A) tail, and Esp3I linearization site. The CDS-containing plasmid / gene block was PCR amplified, gel purified, assembled into the optimized backbone using NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621S), transformed into stable cells, and sequence verified by full plasmid sequencing.

[0102] The firefly luciferase construct was obtained from the pmirGLO dual-luciferase miRNA target expression vector (Promega, E1330). The Renilla luciferase construct was obtained from pmirGLO without cloning into an optimized vector. The nanoluciferase construct was obtained by gene synthesis from Genewiz.

[0103] Linear mRNA Synthesis and Characterization: The DNA plasmid was obtained as described above and linearized by Esp3I (NEB, R0734S). The linearized plasmid was purified using the DNA Clean&Concentrator-25 kit from Zymo Research (D4033) and characterized by agarose gel electrophoresis. The mRNA construct was synthesized by in vitro transcription (IVT) using the HiScribe T7 High Yield RNA Synthesis Kit [NEB, E2040S] (for T7 promoter constructs), with 100% substitution of UTP with N1-methylpseudouridine-5'-triphosphate [Trilink, N-1081-1] and addition of 1:50 SUPERase-In RNase inhibitor [ThermoFisher Scientific, AM2694], following the manufacturer's protocol. After the IVT reaction, the DNA template was digested with TURBO DNase and purified using the Monarch RNA Cleanup kit [NEB, T2040L]. The mRNA concentration was quantified using the Qubit RNA HS Assay [ThermoFisher Scientific, Q32852] or Qubit RNA BR Assay [ThermoFisher Scientific, Q10210]. Unless otherwise specified, the mRNA product was suspended in RNase inhibitor-containing RNase-free water (hereinafter referred to as RNase-free water) at 1:50 (v / v) and stored at -80°C.

[0104] General Conditions for RP-HPLC Purification: All purifications were performed on an Agilent 1260 Infinity II HPLC. Acetonitrile (solvent A) [Sigma Aldrich, 34851], 100 mM hexylamine / acetic acid in water (pH 7.0, containing 20% acetonitrile w / v) (solvent B), 50 mM diethylamine / acetic acid + 50 mM ammonium acetate in water (pH 7.0) (solvent C) were used as the mobile phase, and a PLRP-S column was used as the stationary phase.

[0105] Method 1: Using a 100A pore size, 0%A + 100%B (0 - 5 minutes, hold), 10%A + 90%B (5 - 10 minutes, linear increase), 25%A + 75%B (10 - 55 minutes, linear increase).

[0106] Method 2: Using a 4000A pore size, 0%A + 100%B (0 minutes), 20%A + 80%B (0 - 2 minutes, linear increase), 70%A + 30%B (2 - 30 minutes, linear increase).

[0107] Method 3: Using a 4000A pore size, 0%A + 100%C (0 minutes), 25%A + 75%B (0 - 25 minutes, linear increase).

[0108] Capped oligonucleotide synthesis: 12 nmol of solid-phase synthesized oligonucleotide (containing ammonium as counterion) was dissolved in a solution of 40 mM m7GDP-Im (or corresponding cap analog) in 42 μL of anhydrous DMSO, and 8 μL of 1-methyl-imidazole was added. The reaction mixture was mixed well and heated at 55 °C for 3 hours. Then, the reaction was quenched by the addition of 50 μL of water and directly subjected to HPLC purification using Method 1. The fractions containing the capped product were pooled, lyophilized, resuspended in RNase-free water, and stored at -80 °C until use. The concentration of the capped oligo was quantified using the Qubit microRNA assay kit [Invitrogen, Q32880] and a NanoDrop.

[0109] Enzymatic Ligation of Modified Oligonucleotides to mRNA: 5’-Triphosphorylated mRNA was first treated with RppH [NEB, M0356S] according to the manufacturer's protocol to generate 5P-mRNA, which was purified using the Monarch RNA Cleanup Kit. Synthetic oligo and 5P-mRNA were mixed at a molar ratio of 25:1 and diluted with 2×50% PEG-8000, 10×T4 RNA Ligase Buffer, 10×T4 RNA Ligase [Promega, M1051], and RNase-free water. The reaction was incubated at 37 °C for 30 min and inactivated by the addition of 50×500 mM EDTA (pH 8.0). The product was purified first by the Monarch RNA Cleanup Kit and then by RNase-free HPLC (Method 2). The purified fractions were eluted using the Monarch RNA Cleanup Kit, desalted, and the ligation efficiency was characterized using the RNase H assay as described above. (17) In the case of incomplete ligation, a second round of the reaction was performed.

[0110] Modification Screening in Time-Course Dual Luciferase Assay: HeLa cells [ATCC, CCL-2] were maintained in DMEM medium [ThermoFisher Scientific, 119951] containing 10% FBS and 1% penicillin-streptomycin [ThermoFisher Scientific, 15070063] in a 37°C incubator with 5% CO2 and passaged at a 1:10 ratio every 3 days. One day prior to mRNA transfection, HeLa cells were seeded at 90% confluence in individual wells on a 24-well plate. The next day, 50 ng of Renilla luciferase (internal control) mRNA and 50 ng of modified firefly luciferase mRNA were transfected using Lipofectamine MessengerMAX Transfection Reagent [ThermoFisher Scientific, LMRNA003] according to the manufacturer's protocol. Additional controls containing Renilla luciferase mRNA only or lipofection reagent only were included. Three individual transfections were performed for each condition. Six hours after transfection, the transfection medium was removed, the cells were trypsinized, and re-seeded into three white clear-bottom 96-well plates [Corning, 3610] in phenol red-free medium. At 8 / 24 / 48 hours after transfection, the cell culture medium was removed, and the cells were rinsed with DPBS. The cells were lysed, and luciferase activity was measured using the Promega Dual Glo Luciferase Assay System [Promega, E2920]. Briefly, 50 μL of PBS and 50 μL of firefly luciferase working solution (prepared according to the manufacturer's protocol) were added to each well using a multi-channel pipette and mixed by pipetting. After a 10-minute incubation using gentle shaking and light protection at room temperature, firefly luciferase luminescence was measured using a microplate reader. Then, 50 μL of freshly prepared Renilla luciferase Stop&Glow working solution (prepared according to the manufacturer's protocol) was added. Renilla luciferase luminescence was measured similarly after a 10-minute incubation.For both firefly and Renilla luciferase assays, the background was measured in cells treated with Lipofectamine reagent alone and subtracted. The firefly luciferase / Renilla luciferase in each well was used as the mRNA activity readout. When Nluc and Fluc were used, the protocol was carried out similarly using the Nano-Glo Dual-Luciferase Reporter Assay System [Promega, N1610].

[0111] Circular mRNA synthesis and characterization: The DNA template was cloned, PCR amplified, and gel purified as mentioned in the previous section for use as the IVT template. CircRNA was synthesized as described in the literature by using the HiScribe T7 High Yield RNA Synthesis Kit [NEB, E2040S]. After IVT, the DNA template was digested with Turbo DNase [ThermoFisher, AM2238]. The reaction mixture was heated to 70 °C for 5 min and then immediately cooled on ice for 3 min, after which GTP was added to a final concentration of 2 mM and the reaction mixture was incubated at 55 °C for 15 min. CircRNA was enriched by treatment with RNase R [Lucigen Corporation, RNR07250] for 1.5 h and the product was column purified. The CircRNA product was characterized by gel electrophoresis.

[0112] QRNA synthesis and characterization: CircRNA with a TGT hairpin was synthesized as described in the previous section. The TGT enzyme was expressed in E. coli as described in the literature. (18) circRNA was labeled with preQ1-azide by incubating 1 μM of circRNA, 100 μM of preQ1-azide, 10 μM of TGT, 10 μL of SUPERase-In RNase inhibitor in 1× TGT reaction buffer (100 mM HEPES, pH 7.3, 5 mM DTT, and 20 mM MgCl2) in a total reaction of 100 μL at 37 °C for 2 h. The labeled circRNA was purified and clicked using general conditions for click reactions for 30 min with Bn7 It was subjected to a click reaction with a G-capped alkyne-labeled oligo. The circRNA was then subjected to RP-HPLC purification to remove the linearized portion (Method 2), pooled and desalted, and subjected to another round of RP-HPLC purification to isolate the QRNA product. The QRNA product was characterized by RNase H assay with two primers upstream / downstream of the TGT site.

[0113] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0114] (Table 2) Sequences described in this application TIFF2025519910000005.tif240170TIFF2025519910000006.tif252170TIFF2025519910000007.tif252170TIFF2025519910000008.tif252170TIFF2025519910000009.tif252170TIFF2025519910000010.tif252170TIFF2025519910000011.tif134170

Claims

1. mRNA encoding a peptide or polypeptide, The 5' end contains a cap structure, A derivatized nucleotide located between the cap molecule and the mRNA region encoding the polypeptide, The 3' end covalently bonded to the aforementioned derivatized nucleotide and A type 2 capped circular RNA molecule containing [the specified element].

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

3. An RNA oligonucleotide comprising a 5' end containing a cap structure and a 3' end, A cyclic RNA molecule comprising a twister ribozyme, mRNA encoding a peptide or polypeptide, an oligonucleotide portion forming a hairpin, and a derivatized nucleotide located within the hairpin, Includes, The 3' end portion of the oligonucleotide is covalently bonded to the derivatized nucleotide within the hairpin of the cyclic RNA molecule. A type 3 capped circular RNA molecule.

4. The capped circular RNA molecule according to any one of claims 1 to 3, wherein the derivatized nucleotide includes a portion that can react with the 3' terminal portion by bioconjugation chemistry.

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

6. The aforementioned cap structure is 7-methylguanosine (m ７ G), 7-benzylguanosine (Bn ７ G), 7-Chlorobenzylguanosine (ClBn ７ G), Chlorobenzyl-O-ethoxyguanosine (ClBnOEt ７ A capped cyclic RNA molecule according to any one of claims 1 to 3, comprising G), or any derivative thereof.

7. The aforementioned cap structure, A 7-methylguanosine cap further comprising one or more locked nucleic acids (LNAs), or one or more 2'-methoxy (2OMe), or any derivative thereof. A capped circular RNA molecule according to any one of claims 1 to 3, comprising:

8. A capped circular RNA molecule according to claim 1 or 2, further comprising one or more modified nucleotides.

9. The modified nucleotide is pseudouridine, N １ - Methylpseudolidine (m １ Ψ), 6-methyladenosine (m ６ A) The capped cyclic RNA molecule according to claim 8, comprising 5-methylcytidine, inosine, or any derivative thereof.

10. The capped cyclic RNA molecule according to claim 8, wherein the modified nucleotide comprises locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethoxy (2-MOE) sugar backbone, or any derivative thereof.

11. The capped circular RNA molecule according to claim 3, further comprising one or more modified nucleotides.

12. The modified nucleotide is 6-methyladenosine (m ６ A) The capped cyclic RNA molecule according to claim 11, comprising 5-methylcytidine, inosine, or any derivative thereof.

13. The capped cyclic RNA molecule according to claim 8, wherein the modified nucleotide comprises locked nucleic acid (LNA), 2'-methoxyribose (2-OMe), 2-methoxyethoxy (2-MOE) sugar backbone, or any derivative thereof.

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

15. A plurality of RNA oligonucleotides, each containing a 5' terminal and a 3' terminal portion with the aforementioned cap structure, Multiple derivatized nucleotides at the 5' position relative to each of the mRNA regions encoding a peptide or polypeptide in the cyclic RNA, It further includes, Each of the 3' ends of the plurality of RNA oligonucleotides is covalently bonded to each of the plurality of derivatized nucleotides. The capped circular RNA molecule according to claim 14.

16. The capped circular RNA molecule according to claim 15, wherein each mRNA region encoding the peptide or polypeptide includes a 3' polyA sequence.

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

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

19. A pharmaceutical composition comprising the capped circular RNA described in claim 17 and a pharmaceutically acceptable carrier.

20. (a) A step of synthesizing an RNA oligonucleotide comprising a 5' end containing a cap structure, mRNA encoding a peptide or polypeptide, a derivatized nucleotide located between the cap structure and the mRNA region encoding the polypeptide, and a 3' end containing a portion, (b) The step of reacting the derivatized nucleotide with the 3' end to form a covalently capped circular RNA molecule. A method for producing a capped circular RNA molecule according to claim 1, comprising:

21. The synthesis of the aforementioned RNA oligonucleotide (a) A step of synthesizing a first RNA oligonucleotide comprising a 5' end containing a cap structure, the mRNA encoding a peptide or polypeptide, and a hairpin structure between the capped 5' end and the mRNA encoding the peptide or polypeptide, (b) A step of derivatizing the nucleotides in the hairpin structure of the first RNA, (c) A step of synthesizing a second RNA oligonucleotide containing a 3' terminal portion that is reactive with the derivatized nucleotide, (d) The step of ligating the 3' end of the first RNA molecule with the 5' end of the second RNA molecule. The method according to claim 20, including the method described in claim 20.

22. The synthesis of the aforementioned RNA oligonucleotide (a) A step of synthesizing a first RNA oligonucleotide primer comprising the 5' end containing a cap structure, the derivatized nucleotide, and a complementary sequence to a DNA template encoding a peptide or polypeptide. (b) A step of transcribing the first RNA oligonucleotide from the primer along the DNA template to generate mRNA encoding a peptide or polypeptide, (c) A step of synthesizing a second RNA oligonucleotide containing a 3' end containing one portion, (d) Ligate the 3' end of the first RNA oligonucleotide encoding a peptide or polypeptide sequence to the 5' end of the second RNA molecule. A method for producing a capped circular RNA molecule according to claim 3, comprising:

23. (a) A step of generating a cyclized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region, (b) A step of synthesizing an RNA oligonucleotide having a 5' end containing a cap structure and a 3' end containing a portion that is reactive with the derivatized nucleotide, and (c) The step of reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the cyclic RNA. A method for producing a capped circular RNA molecule according to claim 2, comprising:

24. The synthesis of circular RNA oligonucleotides (a) A step of synthesizing an RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide and complementary sequences on the 5' and 3' ends for promoting cyclization, wherein the derivatized nucleotide is located within the complementary sequences, (b) Step of cyclizing the RNA oligonucleotide. The method according to claim 23, including the method described in claim 23.

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

26. The synthesis of circular RNA oligonucleotides (a) A step of synthesizing an RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide and a hairpin structure containing an enzyme recognition site for introducing the derivatized oligonucleotide into the RNA oligonucleotide, (b) A step of reacting the RNA oligonucleotide with the enzyme to produce the derivatized nucleotide within the hairpin structure, (c) Step of cyclizing the RNA oligonucleotide. The method according to claim 23, including the method described in claim 23.

27. The synthesis of circular RNA oligonucleotides (a) A step of synthesizing a first RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide and hydroxyl groups at both the 5' and 3' ends, (b) A step of synthesizing a second RNA oligonucleotide comprising the derivatized nucleotide and phosphates at both the 5' and 3' ends, (c) A step of generating a cyclized RNA oligonucleotide by ligating the 5' phosphate terminus and the 3' hydroxyl terminus, and ligating the 5' hydroxyl terminus of the first oligonucleotide and the 3' phosphate terminus of the second oligonucleotide. The method according to claim 23, including the method described in claim 23.

28. The synthesis of circular RNA oligonucleotides (a) A step of synthesizing a first RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide, a 5' end containing triphosphate, and a 3' end containing hydroxyl, (b) A step of synthesizing a second RNA oligonucleotide comprising the derivatized nucleotide and phosphates at both the 5' and 3' ends, (c) A step of generating a third oligonucleotide by ligating the 3' end of the first oligonucleotide to the 5' end of the second oligonucleotide, (d) The step of hydrolyzing the triphosphate at the 5' end of the third oligonucleotide, (e) The step of ligating the 5' end of the third oligonucleotide to the 3' end to generate a cyclized RNA oligonucleotide. The method according to claim 23, including the method described in claim 23.

29. The synthesis of circular RNA oligonucleotides (a) A step of synthesizing an RNA oligonucleotide primer comprising the derivatized nucleotide and a complementary sequence to a DNA template encoding a peptide or polypeptide, (b) A step of transcribing the RNA oligonucleotide to further include mRNA encoding a peptide or polypeptide, (c) Step of cyclizing the RNA oligonucleotide. The method according to claim 23, including the method described in claim 23.

30. (a) A step of generating a cyclized RNA molecule comprising an mRNA region encoding a peptide or polypeptide and a derivatized nucleotide outside the mRNA region, (b) A step of synthesizing an RNA oligonucleotide having a 5' end containing a cap structure and a 3' end containing a portion that is reactive with the derivatized nucleotide, and (c) The step of reacting the derivatized nucleotide with the 3' end portion of the RNA oligonucleotide to form a covalent bond between the RNA oligonucleotide and the cyclic RNA. Includes, The synthesis of circular RNA oligonucleotides i. A step of synthesizing an RNA oligonucleotide comprising the mRNA region encoding a peptide or polypeptide, a hairpin structure containing an enzyme recognition site, and twisted ribozyme sequences at both the 5' and 3' ends. ii. A step of reacting the RNA oligonucleotide with the enzyme to produce the derivatized nucleotide within the hairpin structure. iii. The step of cyclizing the RNA oligonucleotide using the twister ribozyme sequence. A method for producing a capped circular RNA molecule according to claim 3, comprising:

31. The method according to claim 23, wherein the derivatized nucleotide includes a portion that can react with the 3' terminal portion by bioconjugation chemistry.

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

33. The method according to claim 23, wherein the cyclized RNA is produced by ribozyme-mediated splicing, enzymatic ligation, or click chemistry-mediated cyclization.

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